DE102008063592B4 - Multifunctional laboratory process bag for the pure production of biomolecules - Google Patents

Abstract

Method for providing subsets of purified biomolecules or biologicals of a totality of the same clonal origin, which can be repeated as often as desired, for quality tests or functional tests with the steps a) Introducing at least one divisible carrier matrix containing the biomolecules to be provided into a coherent, divisible volume that is formed by a laboratory bag or process bag , b) sterile closure under clean room conditions by closing, c) supplying storage liquid, cleaning solution or digestion solution to the carrier matrix, d) establishing the storability, cleaning and / or breaking down the biomolecules, whereby the biomolecules remain in the carrier matrix in a storable or cleaned form, e) storing or keeping the biomolecules in their carrier matrix within separable or detachable areas of the laboratory or process bag, the carrier matrix itself also being divided, f) sterile separation NEN of sub-areas of the laboratory or process bag containing the biomolecules to be provided in their carrier matrix, without the remaining material being contaminated.

Description

Millions of gene segments of the human genome and many animal genomes are available in redundant form as freezing cultures of E. coli clones (BACs, PACs, Shizuya et al., 1992, Ioannou et al., 1994) at resource centers. These mostly structurally and functionally largely uncharacterized gene segments are initially frozen only in the form of bacteria and can not be used in this form. Conventional DNA isolation methods (alkaline lysis, columns, etc.) provide very little DNA in long genomic clones (> 20 kb), which is intact only in a small fraction of a few percent maximum and degrades rapidly on storage. Such gene segments can be prepared according to a new DNA isolation method of the inventor (Schindelhauer and Cooke 1997, US 6331397 . DE 19720839B4 ) are linked to vectors for artificial chromosomes, such as the inventor's pTAT vectors, with descriptions for pTAT ( US 6331397 B1 / DE 197 20 839 B4 , Laner 2004), pTT (PhD thesis Laner 2004, online http://edoc.ub.uni-muenchen.de/4286, Laner et al., 2004) pTTE1 (Laner 2004, Laner et al., 2004, Laner et al. , 2005) and applications in primary cells and animals in PCT / EP2008 / 007422 , which are included here as references in their entirety. With the new DNA production process, a reliable, long-molecule gene construction material (> 20 kb, preferably> 100 kb) can be obtained and stored from E. coli clones with the highest degree of intactness, so that functional gene loci and multigene constructions can be used as artificial chromosomes and human artificial chromosomes artificial chromosomes with genetically relevant genes (genes of Mendelian hereditary diseases and non-Mendelian disease genes, protective genes in common diseases) can be provided with surrounding regulators and all required for a stable inheritance, self-replicating sequence elements as a reliable building material. This makes it possible to produce gene medications for medical internal (eg injection, inhalation, intraoperative) and external (eg primary cells, cell culture) somatic application forms from a large number of medically relevant gene molecules.

However, for clinically and animal clinically relevant functional analyzes, a quantity and quality is required that exceeds normal laboratory capacities in many cases. For long DNA molecules and artificial chromosomes, meaningful gene therapy studies with genomic gene segments can not be carried out because there is no adequate method for producing large quantities of drug-suitable DNA (> 20 kb) in practice. Structures for research are only limitedly possible because long, intact genetic material is not available in sufficient quantity and quantity and can not be produced routinely or only with enormous effort (see multigenic constructs for xenotransplantation).

The inventor named the new DNA quality "Grade 1 DNA", which was previously unattainable at lengths> 20 kb, which means that at lengths> 20 kb there is no single-strand break in the entirety of the molecules of a preparation. Grade 2 DNA additionally contains a single-stranded molecular fraction, and grade 3 DNA additionally contains a fraction with double-strand breaks, as with conventional DNA production methods> 20 kb, e.g. B. in alkaline lysis or when stored in aqueous solution or freezing or alcohol precipitation is the case.

For the production of the new grade 1 DNA quality, in which E. coli cloned gene molecules and genomic constructs for artificial chromosomes with lengths> 20 kb can be provided without strand breaks and stored loss-free for many years, an elaborate laboratory process is used, preferably enzymatic , chemical, and electrophysical methods for the purification and separation of E. coli residues and reject molecules (Schindelhauer and Cooke, 1997, DE 197 20 839 B4 and US 6331397 B1 of the inventor). In this case, prolonged incubation, diffusion, washing and purification steps are used, which in total exceed several weeks (predominantly incubation and waiting times at room temperature). Afterwards, genetic analyzes and functional tests for the functional composition of the molecule preparation from the long-established bacterial culture can be carried out. An urgent need for the underlying invention, which in the preferred embodiment enables a simple and stable storage of a manufacturing intermediate under clean room conditions, is the fact that only on this (first) storage stage, which then without further measures in the same bag for long-term storage of> 5 years and> 10 years and is easily carried out at room temperature, of many clones grown redundantly in E. coli, those clones with functionally flawless molecules are identified. Small samples of a gene pool for structural and functional analyzes are taken in a closed system. Parallel to storage, the right clones are identified. By creating larger gene stocks, the characterized molecules (ie, the proportion of structurally and functionally characterized molecules in the entirety of the molecules of a gene pool is characterized) are then available for further purification and isolation to the quality of the food grade in the process bag. Thus, after 7 years of storage, quantitative functioning of a 64 kb genomic PCR (very long PCR) of the human mucoviscidosis gene no degradation of single-stranded molecules compared to the storage of 1 year can be detected (Noerenberg 2006). Very long PCR made possible with grade 1 input DNA and in DE 10 2004 025 650 A1 of the inventor is incorporated herein by reference in its entirety. Very long PCR also serves to detect grade 1 quality from gene stores and provides cloning-capable, functional, genomic genes using a very low error rate (Kraner et al., 2007). After clone identity detection and knowledge of the functional fraction of all the molecules in the gene pool, the valuable material can be further purified from advanced purification procedures to isolation of pure grade 1 DNA molecules (i.e., virtually completely avoiding a fractured fraction). Broken molecules that are primarily present in each preparation of DNA or biomolecules are virtually completely removed from the support matrix in the purification process, so that the entirety of the remaining molecules is intact (in the preferred method, DNA molecules> 20 kb, but also> 50 kb). > 100 kb and longer). DNA molecules well below 20 kb can not be purified by this particular method and materials because they leave the agarose support matrix in electrophoresis along with the fraction fractured, even if they have grade 1 quality. Depending on requirements and storage size, after further multi-step procedures, part or all of the DNA material can be isolated from the process bag. The molecules in aqueous solution must be packaged sterile and stored at 0-4 ° C without freezing. In the present invention, it is considered that the filling for an application outside of the process bag takes place as a pure, aqueous solution and the further storage for a timely application is made directly from the manufacturing process in the bag and takes place in a closed system, with a suitable, shaking Packaging is used, which minimizes contamination and shear by shaking or pipetting and transport. Alternatively, shear forces can be completely avoided by further processing the grade 1 molecules by further incubations in the bag to a form suitable for application and packaging.

The chambers, solutions, pipettes, tubes, media changes to be used in the entire DNA purification process in the molecular laboratory and the high expenditure of time place an enormous requirement on the maintenance of continuous clean-room conditions. For the parallel production of multiple test construct lots with molecular numbers of> 10 ¹⁵ to 10 ¹⁸ molecules, as required for functional gene analysis and smaller clinical or animal clinical test applications, or larger productions> 10 ^t8 , for clinical and animal clinical applications and transfer / efficacy studies several centers / research facilities or to cover the total needs of a gene therapy drug, eg For example,> 10 ²⁰ functional molecules, there are currently no suitable methods under which a realistic clean room condition could be achieved. Thus, the process bag for the production of a DNA drug in sufficient quantity is a crucial step for gene therapy dar. Since these are very long DNA molecules, and genes are usually on longer chromosomal sections and work in chromatin context, with the Invention of the process bag also allows the production of DNA drugs for gene transfer research and further impact analysis with a variety of genes and constructs. The procedure in the process bag also allows for decentralized production of many different DNA sections in the new, unprecedented quality and quantity, so it can be expected that several specialty laboratories worldwide will supply a larger number of genes for research purposes in process bags and supplied, or on-site filled incubation kits and distributed apparatus to produce, store, characterize, and consume.

Thus, there are multiple levels of bag applications, from functional gene and gene effect research to multiple gene applications in artificial chromosomes (see xenotransplantation) by simply providing functional gene segments for functional constructions, clinically relevant studies with gene therapy constructs, and artificial chromosomes with corrected genomic ones self-replicating genes for the production of DNA drugs, but also distribution to user laboratories for decentralized gene storage (in the form of prefabricated process bags) for genome research centers from existing and further developed genomic libraries (PAC, BAC, pTAT vectors of artificial chromosomes), and the development of special bags for other drug developments from biomolecules and their production.

The methods used in the gene laboratory have so far mainly in open systems, which are not arbitrarily scalable and due to an enormous cost and space requirements practically not completely or only in small numbers in a classic clean room could be performed and in particular a required production increase not withstood. The conventional miniaturization and processing of biomolecules, such as for DNA analysis etc by pipetting robots, as used in DNA for analytical purposes, comes in the known form (multiwell, microtiter plates) practically out of the question, because significantly larger volumes must be processed and the bearing to be produced z. B. is not suitable for pipetting in the long-molecule grade 1 DNA in a protective agarose gel matrix.

In order to ensure clean room criteria cost-effectively even in the complex process of producing long DNA molecules, and to allow a flexible increase in production, the invention provides to include the manufacturing process in cost-effective, automated manufacturable modules / containers / bags / multi-chamber bags, so that clean room conditions the entire process can be created, an increase in production by increasing the number is easily possible, and equally the various material and sterilization and cleanliness requirements and storage times of the bag parts and Ansteckkammern and the process solutions / media can be considered.

The invention thus relates to the inclusion of a complex laboratory process in a multifunctional plastic container / bag in which virtually all the steps required for the process, movements, additions of solutions and enzymes, incubations, and storage done in a closed system. Only in the execution with the introduction of the bacterial mass, z. B. in the form of an agarose gel matrix in an external mold / screen frame one side is opened for subsequent welding. With internal mold, only one sterile port is opened. During electrophoretic cleaning, the bag system is temporarily opened under clean room conditions (filter boxes, laminar flow modules) at the electrode ports or via a port to vent gas and spent buffer. These or additional accesses also serve to escape electrophoresis gases and are then resealed (or welded) again.

Thus, even in a limited clean room area of a manufacturing facility, the production of a DNA drug can be performed even with large batch counts (different genes, multiple clinical trial constructs). If a larger amount of a DNA drug is needed, production can be ramped up easily and flexibly with larger numbers of bags.

The prerequisite for purification within a process bag with a support matrix is the selective binding of the biomolecules to be isolated (by entrapment in a gel lattice, by binding properties such as covalent bonding or receptors, antibodies, etc.) within the support matrix, during a series of purification steps to separate out all contaminants of others Components of the introduced biomass (eg bacteria, media, tissue digestions, cell cultures, etc.). After reaching the intended purity and dilution of unwanted components storage and testing of a sample takes place. Subsequently, the biomolecule is specifically treated to dissolve it out of the carrier matrix and pack it within the closed system.

As state of the art, it has been known from medical infusion solutions and mixed solutions for parenteral nutrition that through the use of multi-compartment bags (e.g. EP 0776649 A2 . US 4997083 A . DE 32 38 649 A1 . US 5296242 A . DE 94 01 288 U1 . EP 0790051 A2 . EP 0875231 A2 . US 5176634 A . US 5308334 A . US 6231559 A ) different substances with different sterilization processes or for the maintenance of stability can be mixed only shortly before application in a closed system (eg. DE 697 36 909 T2 . DE 600 30 682 T2 . US 4997083 A . US 5560403 A . US 6468259 A . DE 100 41 295 A1 ). Likewise, after removal within the bag, donor blood is roughly fractionated by centrifugation and plasma or cell fractions are transferred into connected bags by means of hoses, filtered and stored (various systems, eg. US 4425114 A . US 4098456 A . US 4915847 A . US 4637813 A . US 5382526 A ) or cells are cultivated (eg. DE 10 2006 026 179 A1 ).

So far, however, it is not known that multi-chamber systems are used with buffers for incubation and purification cascades of laboratory processes including electrophoresis for the molecular digestion, storage and purification of biomolecules in a process bag, including the through-blowing and sample extraction and packaging of a pure form of a biomolecule. The already existing bags and connection solutions and ports of nutritional, infusion and transfusion medicine result in several flexible solutions for the supply of buffers and media in a closed process bag. Thus, buffers and reaction solutions can be added by conventional infusion systems of autoclaved single chamber bags or multi-chamber bags. For the various versions exist automated, tailored to different materials, tested procedures for filling / manufacturing and sterilizing, autoclaving solutions and the subsequent application of drugs or nutrients through various port systems, as in multiple versions of infusion bags, blood bags, or multi-chamber bags known for parenteral nutrition. The concrete material requirements for process bags are varied and sometimes require further developments that can benefit from the know-how with bag materials of conventional systems.

However, the filling and subdivision of multi-chamber bags is technically still difficult, because today each chamber requires its own access to the filling, and thus spatially and technically automatable number of chambers is rapidly limited.

Peelnähte of multi-chamber bags are z. B. used when different content should be mixed just before use. Thus, according to the current state, the chambers are already separated from one another by means of peel-seam welding techniques prior to filling. In the invention to be attached to the process bag feed bags with solution cascades a much larger number of relatively small volumes is required, so that according to the current state of the art, a large number of hose pieces / filler neck would have to be welded, creating technically difficult space and increasing the Committee and the Costs would occur in an automated manufacturing process. An embodiment aspect of the processing bag described here, in particular one or more supply bags, therefore provides a new method for attaching peeled seams, in which a long tube chamber is first filled loosely with aqueous buffer via a port, and then the filled chamber at the points to be welded is pressed, and then the layers are welded together (through a filled bag). It is simply welded through the remaining aqueous traces / droplets in order to obtain a larger number of chambers filled with the same liquid in a multi-chamber bag ( 4 ). In one embodiment, the site to be welded is stored high above an edge surface / elevation, with the chamber contents running down to the right and left thereof. The area to be welded of the overlapping, suitable for a peel seam film (eg., Internal polyolefin) is preheated, if necessary, to obtain a substantial drying before setting the foil seam and only then the peel seam is welded. Likewise diverging rollers / pressure ridges in the area of the peel seam can be placed on and removed from each other prior to the soot-welding, so that liquid is forced outwards and subsequently the peel seam can be set therebetween. In this way, a low-cost inlet bag, with virtually unlimited switchable chamber number is generated with the same content, which can be used for the multi-stage reaction and dilution cascades. The loading but also the connection of the multi-chamber bag (for example, with 8 × 200 ml aqueous cleaning solution, or 6 × 200 ml catalysed surfactant solution for chemical digestion, or 8 × 400 ml electrolyte solution, etc.) to the process bag can now via a single filling and outlet nozzle take place, whereby the production and sterilization is initially greatly simplified as a long Einkammerschlauch and the filling. The subdivision also has a favorable effect on the transportability of the buffer cascades, because large volumes subdivided can withstand much higher acceleration / braking forces than a large common chamber. This also strain loads are lowered in the material, which indirectly a longer sterile storage can be guaranteed. The multi-chamber bag for reaction cascades thereby fulfills another important aspect of the invention, because at each stage of the carried over weeks reaction cascades, cleanings, incubations, an automatic control of the already emptied and still filled feed chambers by simply looking and counting is realized. Thus, in a further embodiment, in order to document all the enzyme / buffer / additions visible on the bag, additionally smaller dry / light protection / secondary chambers are introduced which contain storable enzymes (RNases, proteinases, nucleases, etc.) or buffer substances as powder and z. B. subsequently after sterilization attached to the bag and placed in the main buffer. Techniques for the protection and insertion of drugs in plastic containers exist (broken tubes, subsequent sterile insertion of Peelkammern, sterile bonding, applying gas and vapor protective layers, aluminum-coated films by pasting infusion bag films from the outside, etc.). Appropriate production of the clean room process bag avoids further sources of contamination in the supply of external substances, simplifies the application, simplifies the review / documentation of the process and reduces the risk of confusion or false documentation ( 5 ).

Also, with chemical substances for buffer production prefilled bags are possible, which are filled in the form of empty bags as clean room process bag kits only on site with water and autoclaved.

An example of the execution size of a z. B. 30 × 40 cm process bag (see model 11 ) and a screen frame with 20 × 10 cm × 5 mm, the bottom of which flat, flat, torsionally, preferably between 0 ° C and 65 ° C deformation (in subsequently introduced into the process bag mold / carrier matrix), preferably stable in deformation during storage and autoclaving , deformation stable in steam autoclaving (when introduced into the Bag in open form before autoclaving the blank bag), preferably transparent.

For pouring the liquid agarose gel into the mold within the bag, via a small access for an external pipette or a port / hose adapter ( 1 . 2 ).

In this case, the presented in the bag casting frame ( 2A ) is centrally mounted on the bottom of the bag along a centerline of the screen frame and along a centerline of the bag bottom. The attachment is not over the entire surface, so that the bag can inflate and surrounding electrodes (3 or more) can be arranged spatially above and below the gel matrix ( 3 . 6 ), or unilaterally two electrodes in combination with other elution electrodes (1 or more) in a connected elution bag chamber, for the isolation of the grade 1 DNA and transfer into a closed-tube transport tube, can be spatially arranged ( 7 ).

An essential aspect of the storage of biomolecules in the process bag relates to the sterile removal of samples at different times of production and storage, to the removal of parts of the bearing. In one embodiment, the carrier matrix is easily breakable through the bag so that smaller or larger portions of the total area of the gene store are separated in the bag. The wash solution is removed via the spout for this step. The separated carrier matrix is now moved into a corner of the bag in which there are no connections. Between the sample in the corner (of, for example, 1 × 1 cm) and the remaining, large-area biomolecule storage are only small amounts of an aqueous washing solution, so that with the help of rods / rollers or slides or a flow increase a quasi-dry area can be formed, which is closed by a simple weld (possibly with previously done careful warming for more local drying), so that the bag corner is abgschweis with the sample / separated ( 6 ).

The traces of the aqueous, thin salt solution that could be in the area of the seam (precipitated salt crystals / vapor droplets etc) are simply in the generously set, re-solidifying seam or double seam (such as with a hand-held tweezers, or in an HF welding device, or with a Laser, depending on the material in a commercially available sealing device for welding with / without separating, optionally double, or with a specially adapted to the process bag films and preheating times welding tongs / table device for process bag) included. Thus, the stability of the remaining storage bag is retained, the sample removed is also in a closed bag and can be easily transported and opened for analysis. The printing of the corresponding bag corners and other intended for later withdrawals sections of the bag and the remaining bag with serial numbers and / or the information of the stored content gewehristet also a confusion-free sampling and easy handling. In this way, contamination sources can be avoided even when removing and sending out analysis sections of the batches and the valuable material sections. Throughout the process, conventional tubes (50 ml) and disposable pipettes (10 ml, 25 ml) are no longer needed for the large number of buffers, which up to now have been a considerable expense for each gene store in the normal laboratory procedure and a considerable source of contamination due to numerous opening / closing , In addition, numerous glass vessels and their cleaning before and after autoclaving can be dispensed with.

Other designs provide conventional plastic bags and adapters, such as those used for infusion solutions and in any size of z. For example, 100 ml or 5000 ml may be made of various film materials (eg, polypropylene bags for infusion). Each individual chamber can be filled, closed and sterilized independently and connected to the process bag as a closed system.

It is also known from such bags that an infusion solution can be subsequently added with another component which is dissolved in another container and added in the closed system by means of a plurality of possible valve / injection systems / ports. Other known applications use predetermined breaking points (rupture tubes, peel seams between bag compartments) between two or more compartments to consolidate components and thereby obtain the final application form (antibiotic solution, nutritional solution, etc.) for immediate use. Thus, powdered components such as enzymes or antibiotics can be welded into plastic and attached by applying oxygen barrier layers during film production or by subsequent adhesion. Optionally, such switchable capsules / chambers can also be introduced / welded into the plastic bag subsequently (for example after autoclaving a bag with another component / solution) and if appropriate automatically. Similarly, there are methods of retrofitting chambers that can be secondarily welded or bonded either via tubing and valve systems or by peel-seam or adapter assemblies.

Building on the prior art that sterile medical solutions can be prepared in plastic containers and bags and stored for> 1 year, and subsequent delivery of other components is possible, and the need for GMP DNA production for gene therapy, the invention has not only To perform a medical end product and its mixture for consumption of the application form in a low-cost and clean plastic bag, but to perform a complex molecular genetic and biochemical cleaning process in specially adapted to the process flow process bags made of plastics. By moving the multi-step process to obtaining highly purified, intact and functional biomolecules in a plastic bag, cost-effective mass production under clean room conditions is achieved and the long term storage requirement underlying the multi-gene storage process is created. Performing a complete laboratory procedure, including purification, storage, sample collection, functional characterization, electrophoretic purification, and elution to packaging in a multifunctional plastic bag, is novel and brings significant benefits, as demonstrated here for the production of gene stores with grade 1 DNA quality, for a long time To provide DNA sections for gene therapeutics.

However, laboratory process bags can also be applied to other similar methods for isolating biomolecules under clean conditions. Likewise, optionally certain sub-processes, in particular sub-processes of the process of the preferred application here in a process bag, can be performed better and cleaner than before. For example, the electrophoresis / pulse field electrophoretic purification performed in the bag is a novelty that suggests a number of similar electrochemical applications in other areas of biomolecule extraction. Likewise, the simple electroelution of biomolecules from carrier materials in the bag under clean room conditions is a powerful alternative to previous systems, which usually consist of open electrophoresis boxes with inserted elution chambers and dialysis membranes / filter membranes. Due to the low cost compared to a conventional clean room and the rapid increase in the production volume by using and storing multiple bags, as well as the rapid realization of automation for larger numbers, the cost-effective production of switchable buffer solutions, washing solutions, enzyme additives, enables the production process of gene stores in the process bag use of genes in a larger, cleaner form, thus creating the conditions for clinical trials and the development of gene drugs and their production.

Preferably, the newly developed method for producing a jump higher DNA quality (grade 1 DNA) for the production of DNA drugs in the process bag can be performed. As a result of the invention of the enclosed laboratory effluent in the bag, it is thus possible for the first time to realize a medically relevant DNA production under clean-room conditions, which is also suitable for increasing the production volume given rapidly growing demand. Thus, the procedure is a key procedure for the use of a new DNA technology with high-quality, long DNA molecules of whole genomes and the use of artificial chromosomes.

The amount of DNA that can be produced and stored in only a single bag (with, for example, 10 × 20 cm carrier material) already exceeds the amount that is usually available in a modern gene laboratory and enables a relevant storage, with significant cost reduction. The storage of the initially prepared DNA molecules as a gene storage is absolutely necessary, so that in addition to the high physical integrity and integrity of the DNA molecules produced, also the functionally intact fraction of the totality of the molecules in the current production unit can be determined. Since no other suitable propagation method than the cloning of DNA constructs in low copy vectors for the production of artificial chromosomes (PACs, BACs, pTAT vectors) in grade 1 DNA quality is available, as the cloning in bacteria (preferably E. coli or theoretically also as circular molecules in yeast cells, with orders of magnitude lower yields than in bacteria), is biologically caused by the many cell divisions during growth, in each individual preparation a real existing, new risk exists that single or many of the molecules of recombinations, Deletions, mutations of the DNA sequences could be affected.

To wear z. For example, the tandem repeat sequences of telomeres and centromeres and the repeating sections occurring in genomic sequences of an artificial chromosome risk recombination so that as a rule two to four single cell cultures of a suitable master culture of the same bacterial clone are usually performed in order to avoid prolonged growth in one Culture of 10 ¹⁵ and more cells is highly likely to obtain a high quality preparation. Some problematic genome sites may require higher culture numbers. In general, most of the sections are very well cloned in the mentioned low copy systems in E. coli, especially as redundant clone collections are often based on a more than 10-fold gene representation with randomly arranged sections, which are usually obtained individual functional sections and, through the preparation of high quality DNA, further methods for supplementing and further cloning incomplete clones are available, e.g. B. very long PCR of the inventor (eg Kraner et al., 2007).

When propagating the bacterial culture, after prolonged growth, in principle every molecule from each of the 10 ¹⁵ to> 10 ²⁰ E. coli cells has a potentially different mutation history. To put it bluntly, every DNA molecule could carry a different mutation. Since these would not be noticed when sequencing a sample of the whole molecule, and the sequence / function relationships for the much larger portions of a genomic gene section are not known (so only about 10% of the sequences to coding gene sections, spliceites and regulatory binding sites, while the other 90 % Although the gene functions can also significantly influence (chromatin context), but each potential variant can not be detected a priori at the sequence level), in addition to structural investigations for the evaluation of a gene storage, single molecule transfer / functional experiments must always be carried out (or sequencing of single molecule subcloning) In the case of a known sequence of a functional construct in order to detect the functional rate of the prepared DNA molecules of a preparation, for this reason, the functionality of a gene store can not be determined until after the preparation of the store, as a rule not determined before 1-3 months and / or after 1 year and later, with the functional proof with a small sample increasing drastically the value of the stored material and other gene stocks which do not correspond in a small sample of the required quality, discarded and discarded (or, at least largely structural integrity, eg. B. to very clean probe DNA for the production of labeled samples for fluorescence in situ hybridization (FISH) are processed). The success rate for a functional gene storage depends on the construct characteristics and E. coli growth conditions of the artificial chromosomes and on the intrinsic cloning stability of each contained gene segment.

This distinguishes two production phases of the process. Accordingly, during an initial phase for identifying suitable clones until the gene storage is deposited and deposited, different requirements are required of the materials than in the later, sometimes only years, proper purification for grade 1 DNA and the isolation of free DNA molecules for a targeted application.

The invention involves using different plastics according to the desired properties for the corresponding phase of the process. It should be noted that the invention is not based on the use or sequence of certain plastics, film materials, connector materials, etc., as they are present in the embodiments. With the availability of new plastics, plastic combinations or plastics with new additives, or the use of various multilayer films and laminates, as well as experience from the applications, it will be possible for those skilled in the art to produce bags in other finishes and sizes suitable for the particular requirements suitable for the respective laboratory process. In the exemplary embodiment, the materials currently used for infusion solutions or blood bags PVC, EVA and PP are used. It largely dispenses with PVC, which accounts for less than about 10% of the plastic mass used, but is used for the long-term storage of DNA. The properties of the sample materials provide specific advantages for the respective process phase and for the control and reduction of liberated substances (plasticizers, extenders, breakers, metals from stabilizers, process substances, UV absorbers, fillers, metals from heat stabilizers or catalysts).

Flexible PVC bags generally contain phthalate-containing plasticizers in large quantities, which are believed to have deleterious effects on humans in larger quantities received and in continuous contact. Especially due to the suspicion that DEHP and other phthalates, which have been used for many decades, could cause infertility in humans, the use of phtalate-containing PVC for children's toys and medical technology was increasingly abandoned, at least if the medically necessary supply of other materials was ensured could be. In addition, other non-phthalate-containing plasticizers for PVC in medical technology and other sensitive areas have been developed (DINCH and others). Thus, blood bags are preferably made of PVC because blood is stable therein. PVC is well suited to steam sterilization and has a very high long-term stability. In addition, it can be well welded in thicker foil layers (0.4 mm and more), which means that very long storage times of well over 5 years can be achieved. An example of the enormous long-term strength of PVC films is the use of pond liners in the open air, which are usually offered with a warranty of at least 10 years. PVC is a soft, adhesive, well printable, well storable even in the cold and oxygen / water vapor-tight or additionally lockable material, which ensures a long sterility. PVC thus fulfills the requirements for the long-term storage of biomolecules and for the different incubations within an agarose gel matrix at 0 ° C to 65 ° C Stability parameters with simultaneous temperature stability over the 121 ° C steam autoclaving addition, so that even empty bags without sticking the surfaces are autoclavable.

Since previously gene preparations / gene storage in a conventional manner in thick-walled 50 ml Falcon tube (glassy polypropylene or modified polystyrene available) were stored and long-term storage with chelating agents after 7 years storage at room temperature showed no noticeable quality deterioration, it is quite conceivable that the genetic stock in thicker Bags with a dense barrier function remain intact and sterile for well over 10 years, ensuring a time-delayed, cost-effective re-delivery of tested material and very large gene stocks, making DNA drugs mass-producible.

It should be remembered that in the very long storage that is sought, conventional experience with potentially leaking from the container wall materials can not be easily transferred to bag films. On the one hand, the contact surface is comparatively large, so that relatively high concentrations of the substances can occur quickly, and on the other hand, the total mass is relatively small when using a thinner foil (much smaller than in conventional preparations and containers and multiple container changes), so that in long-term storage in the thin-walled bag in which the entire process takes place, scarcely any replenishment from the material has to be reckoned with, especially since the washing processes initially take place with surfactants which absorb more than in the later, purely aqueous purification steps and electrophoresis, the fat-soluble plasticizers and out can flush out the innermost layers. Thus, net use of thin films (or combinations with thin films having different properties and combined film materials) could result in significantly less stress than using thick-walled containers of the same materials, especially in the multiple buffer changes that the actual isolation of the drug and wash out the vast majority of potentially accumulating substances.

Moreover, contacts with other materials, such as glass, electrophoresis chambers, cooling hoses, elution chamber, disposable pipettes, etc., are completely avoided, so that the entire amount of the container material coming into contact is minimized by the use of a process bag.

Meanwhile, plasticizer substitutes, eg. B. DINCH, which is sparingly soluble in water, has a lower migration rate, and typically contains no metal residues (Technical Bulletin January 2008 M6168d, BASF) used in PVC, of which no medical disadvantages for humans are known or at much higher test concentrations no fruit-damaging effects were demonstrable (Welle et al., 2005; Ref. Scientific Commettees 2008, SCENIHR Opinion,
http://ec.europa.eu/health/ph_risk/committees/04_scenihr/docs/scenihr_o_014.pdf).

Nevertheless, it is known that the plasticizers sometimes used in relatively large amounts in PVC can easily migrate in the plastic over a relatively long period of time and thus also diffuse into the substances to be stored during long-term storage, especially in the case of lipophilic filling (K. Inoue et al. 2005). Even if the surfactant-containing buffers in the process bag are renewed several times in the first few days and weeks of the cleaning process, plasticizer would certainly migrate back into the surfactant-containing storage solution after the subsequent long-term storage, even if initially a certain amount of the innermost layer had already been washed away. Based on the storability of blood cells in PVC but would not be expected that the bacteria and the resulting biomass and ultimately the contained, biologically inert DNA molecules would be damaged by the plasticizer. Plasticizer-free EVA also has a high resistance with a high water vapor barrier and is well weldable.

Since a certain storage must be carried out in the buffer multi-chamber bags to be connected, but this does not exceed the usual storage time of infusion solutions from months to a few years, and the bags do not have to withstand the many manipulations is preferred in one embodiment for all bags of refill solutions , which are used after storage, and for all short-term process bag (elution bag, elution chamber, transport hose, etc.) uses a plasticizer-free polyolefin plastic. PP bags are increasingly being used for infusion solutions and can be made by adding additional layers with a higher gas and vapor barrier. In addition, z. B. Spezialpolyolefinfolien with Peelnaht properties the possibility of producing multi-chamber bags with cascades of aqueous solutions according to the method proposed here.

Since gene therapy applications will increasingly be used on newborns and infants, the choice of materials must be made with particular caution and with a particularly favorable risk-benefit ratio. So is with regard to impurities in the to be applied DNA solution prefers to aim for a plasticizer-free material / bag system, such as only polyolefins, or to dispense with phthalate-containing plasticizers.

However, it should be noted that additives which can be used in polyolefin polymerization, such as catalysts and co-catalysts (or other additives in other plastics), are present in the bag produced (here, polypropylene) and may have little effect on the solutions. Although such would be harmless to humans (in the case of infusion, food packaging, etc.) owing to the at most trace amount and type of substances and the non-migratory behavior, they would hardly be detectable in conventional packaging and moderate storage times, but nevertheless could have an adverse effect during long-term storage contain contained inert, non-biological DNA molecules (directly chemical versus potentially genotoxic in the complex living system). So z. For example, the Ziegler-Natta catalysts used in polypropylene manufacture, which are usually organometallic compounds (cobalt, iron, nickel, etc.), or cocatalysts, pose a problem especially for the stability of the free DNA double helix, even if only Traces would be present for a long time in the storage solution, although such substances in these amounts would not have any detrimental or even beneficial effects on living biological systems, as is the case for iron and many metals and other trace elements.

An essential aspect of the invention in the execution of a process bag for the production of grade 1 DNA is the absolute avoidance of metal contact and the elimination of minute amounts of free metal ions / heavy metals, such as occur in clean tap water or mineral water or leak out of glass containers, as these in non-biological system can be damaging to free DNA molecules. Thus, the use of a plastic process bag by the new process and by the material metallic contact surfaces, as they occur in conventional cooling circuits of electrophoresis chambers, banned by dispensing with a cooling circuit. This also eliminates the need for conventional cooling circuits in glass vessels / heat exchangers / glass coils / cold traps / hoses, from which metal ions can additionally be washed out. To prepare the buffer solutions, only water from a Quarzglasbidistille is used. Platinum wire electrodes are replaced in the electrophoretic purification by carbon modifications, in particular glassy carbon or others, which decompose electrochemically virtually residue-free into escaping gas and not into solids and metal ions.

This also avoids the presence of other metals in the platinum. PVC with plasticizer has been virtually free from heavy metals since the absence of lead and the use of zinc and calcium for stabilizers. Thus, storage of the gene storage, regardless of the presence of plasticizers, in durable PVC or other geeigenten foil materials to make until after many years, the actual, near-consumption cleaning with buffer cascades of plasticizer-free materials. Since the multi-stage purification process is continued after the long-term storage of the gene stock (years), and a longer series of fresh buffer solutions is supplied, a complete complete depletion of all mobile substances present in the storage bag can be achieved, whereby the initial presence of plasticizers and other substances, as long as they serve the liability of the process bag and thus the long-term sterile storability of the DNA molecules are desirable or acceptable.

At the time of resuming the cleaning of the long-term storage to the medical end product, an advanced leaching of the plasticizer from the PVC bag has taken place, whereby the then existing only for a short time solutions (days), can absorb only negligible amounts of plasticizer. So that from the time of the continuation of the cleaning up to the final isolation of the free DNA molecules in the connected elution chamber no renewed softener supply takes place, a change of the material of the buffer bag to be connected to other autoclavable materials, eg. As polyolefins, preferably the polypropylene without plasticizer. With these relatively short residence times, no relevant DNA damage is to be feared from the solutions supplied with potentially trace-like metallic contaminants (determinable in the long-term test).

Thus, there are in principle subordinate requirements for the bag material during two different process phases. During Phase 1, from the introduction of the biomatrix to the storage of gene storage, it is first and foremost necessary to eliminate DNA-damaging contaminants, while for humans it is possible to accept substances that are critical but material-favorable because they are later washed out and heavily diluted. During Phase 2, pure Grade 1 DNA is prepared from the functionally characterized gene stocks in multi-level multi-week isolation, or high levels of functional DNA are isolated for drug application in aqueous solution. In particular, if the DNA is to be processed into a drug, during phase 2, the largest possible reduction of for the It is important to ensure sterility, freedom from particles and pyrogens, but also to avoid plasticizers in high concentrations, eg from transfer systems and hoses. For this reason, other materials are used for the supplied washing solutions in phase 2.

It should be noted here to the state of the art that it is not known whether metal traces of corresponding plastics ever measurably incurred in aqueous solutions and whether it could emanate a direct DNA damaging effect. It is known that polyolefins are particularly good, clean films for packaging, from which virtually no amounts of substance emerge compared to plasticizers. The material weighing described here is an example of how the best possible stability of the intact gene storage, and thus a high efficiency per molecule for a DNA drug can be achieved, and it can be ensured that the final DNA drug in an optimal , residue-free, health-safe medium is available.

A further measure for the reduction of enzyme-active metal ions are additions of chelating agents to the wash solutions, such as the frequently used EDTA (1-500 mM, in a specification which is as free as possible of heavy metals) or the one proposed here, especially for the additional complexation of divalent metal ions (eg. Zn, Ca from stabilizers) and heavy metals in small concentration throughout all stages of use of the EGTA (0.1 mM) compulsorily added in the process bag for the grade 1 DNA preparation.

In other versions of a clean room process bag, other and new materials are used selectively to meet the respective requirements. It is also conceivable that a> 5 years, preferably> 10 years storability of long DNA molecules could be demonstrated in a plasticizer-free sheet material that the process / storage bag and the feed bag can be made of a material.

Similar material considerations are made for the adapters, feed hoses, possibly adhesive, plastic inks and for the holding frame / gel mold. Approved materials for medical technology are used, whereby depending on the application special demands are placed on the strength, transparency, heat stability, adhesiveness or processability (eg injection molds made of polycarbonates, etc.) or valve closures especially designed for maintaining sterility over long periods of use and ports are used.

The use of a bagging laboratory can easily simplify conventional processes, buffers, and the use of electrophoretic cleaning processes, multiply yields, minimize waste, and adapt changing or advanced material parameters to the needs. The special feature is that the material to be cleaned is not introduced into an agarose gel as before, which is then introduced into a special pulse field gel electrophoresis chamber with buffer cooling system / circuit, but that only the gel material to be cleaned / the carrier matrix is present in the sealed bag and all steps (under clean room conditions) take place there (with drastic savings of agarose).

Sterile electrodes are inserted into the bag for the electrochemical cleaning steps, wherein the insertion accesses represent longer tubular protuberances which, after removal of the electrodes, can be re-welded with a simple, thermo-and / or time-regulated manual welding tongs. It works below the decomposition temperature of the material to avoid harmful reaction products. In one embodiment, during the current operation, the electrode channels of the tube-like Beutelaussackungen, are inserted over the electrodes with flexible electrode cables, obliquely upwardly angled open up operated ( 10 , Channels E1-4). This allows gas from the electrophoresis to escape from the buffer to the outside, while the gel matrix to be cleaned remains in the process bag.

Alternatively, the insertable electrode materials are inserted with an adapter in the bag and thus firmly connected ( 9 ). The electrodes are connected outside the adapter to the power cable (glued conductive, or screwed, clamped) and provided with an insulating layer (preferably shrunk thermoplastic coating that can be sterilized). The electrode rods are preferably sealed according to their diameter in the plug adapter against buffer leakage, so that only the electrode material itself, and not the cable or the cable and electrode connection touch the buffer indoors. In this solution with gas- and buffer-tight Elektrodeneinsteckung an additional buffer drain / gas outlet channel, which forms a pressure-regulating water column, opened so that the upwardly flowing gas and the upwardly derived buffer (warm, consumed) is discharged upwards, in which the Opening above the bag level is attached. For this, the bag may be specially designed, or skewed during electrophoresis, so that the interior area is inflated by buffer, and the drainage channel / gas drainage channel is the highest point of the bag, and the channel is further upwardly drained so that the buffer level In the discharge channel, the bag pressure is further increased before the buffer and the gas at the end of the channel escape ( 6 . 7 ).

The electrode materials can be varied depending on the requirements of the genetic stock. For example, low-cost graphite electrodes for a low-residue decomposition of the electrode into gas, a metal-free solution with a possible single use of the sterile electrode. However, in graphite there is a risk of carbon abrasion during insertion / insertion. Since graphite abrasion particles are biologically inert (compare cleanly applied Tatoo color) and the elution chamber, which is equipped to gain the free DNA molecules with a particle filter / filter membrane ( 7 . 8th ), no adverse effect is expected from the use of graphite and thus a low-cost disposable electrode material with simultaneous cost reduction and elimination of contamination sources used. The use of graphite electrodes and high-purity graphite electrodes and graphite composites free of impurities in the clean room process bag is the subject of the invention.

The invention of the method with a process bag includes in the execution of the grade 1 DNA preparation and application of gene stores for a DNA drug for internal use in humans, the use of glassy carbon electrodes, which allow a residue-free cleaning electrophoresis. Glassy carbon with fullerenartartigen structure represents an extremely hard, smooth, heat-resistant, autoclavable, and particle-free material that dissolves almost without residue in the electrolysis in gas. Thus, platinum secretions and secretions of other metallic platinum contaminants and electrochemical reaction products on the surface of conventional electrodes in pulsed field electrophoresis equipment could be avoided, which could counteract the stability of stored biomolecules. Thus, the subject of the invention is the use of glassy carbon electrodes and other carbon modifications in the clean room process bag, which can be manufactured depending on the starting material for the specific application and represent a very clean solution to all available electrophoretic purification.

Other electrode materials included in the invention for use in the process bag are carbon rods.

Another novel electrode material of the invention for use as an electrode in a clean room process bag, either integral / welded or secondarily employed, is carbon / graphite / pure carbon black / non-metallic inclusions in finely dispersed, bonded form as used in electrically conductive Plastic films is used. Manufacturing process (eg DE 695 25 902 T2 ) for similar films are known for use as antistatic packaging of electronic or explosive materials, or are used for powering compact electronic devices. The use as electrode material for molecular digestions and electrophoretic purification steps or electroelutions in clean room processes is not known. Thus, the subject of the invention is the use of electrically conductive plastic films as electrode material in a clean room process bag and the use of other mixed conductive electrode materials in the process bag, z. As silicates, ceramics and ligation or applied to the inside of the process bag conductive layers, films, laminates, paints.

In other embodiments, depending on the requirements of the expected residence time of the biomolecules in the subsequent buffer after electrophoretic cleaning or after electroelution other or changing electrode materials come into consideration.

Thus, optionally, platinum electrodes, graphite electrodes, glassy carbon electrodes, or carbon rods or other electrode materials may be used, and the electrodes may be provided for multiple sterilization / use or disposable.

Included in the invention are sterilizable, electrically nonconductive sheaths of the electrode terminals and connected electrical cables to allow the cables to be immersed in the operating buffer, whereby the feeding cable can dip into the buffer (open electrode channel design). Accordingly, the electrode ends and cables are sheathed with a nonconductive layer / lacquer / foil (eg shrinking property). The connection from cable to electrode is preferably reversible and is mounted in front of (ie below) the insulating sheath, plugged, clamped, screwed, or adhesively bonded.

Moreover, the pure space process bag creates a new, purer electrophoresis situation compared with the known pulsed field electrophoresis and electrophoretic chamber systems with buffer cooling circulation, comprising a continuous feed from a feed bag (for example a large-volume polypropylene infusion bag (eg, 3000 ml, 5000 ml), with sterilized pre-chilled (4 ° C) electrophoresis buffer (such as a Tris-acetate-EDTA buffer) and a separate continuous flow of heated, optionally rising, spent buffer under overflow pressure which avoids excessive mixing / circulation, and the bag is completely filled under moderate pressure and inflates by itself. In the inflated state, the necessary spatial electrode arrangement is ensured. In various embodiments, the electrodes are either fixed outside and the bag stored accordingly, or they are anchored in a slot opposite the guide in the bag, or they are z. B. inserted in guide devices on the screen frame of the carrier matrix.

Due to the one-way direction of the trailing buffer, the waste molecules transported into the buffer during the purification electrophoresis (E. coli residues, peptide residues from proteinase degradation, DNA and restriction enzyme degradation products, storage buffer fractions, residues from the precursors, residual soaps, etc.) are not fed into a cooling circuit, but instead are quickly removed from the bag. By this measure, the burden of the cleaning buffer with electrophoretic reaction products and with cleaned biostats and cleaning residues compared to a conventional, pulse-wave electrophoretic isolation is greatly reduced, so electrochemical reactions of circulating waste at the electrodes are reduced to a minimum. Furthermore, after the end point of the electrophoresis in which a complete purification of the gel has taken place, but there are still waste molecules in the buffer, a complete buffer change can be made and a second electrophoresis can be carried out, thereby effectively removing residual waste molecules ( 6 . 7 ). Cool storage of the process bag and cooled feed buffer eliminates the need for additional cooling with a pump circuit at a suitable applied voltage between 1.5-9 V / cm, eliminating the need for another significant source of contamination in conventional laboratory processes. Thus, no need to reuse multiple electrophoresis on, additional meter-long hoses, cooling lines and cooling units with fluid circulation and on pumps can be dispensed with, buffer chambers / dead spaces at hose joints are avoided, a costly sterilization of separate electrophoresis chambers, with sometimes difficult to access niches and dead space is complete avoided. Thus, the here invented cleanroom process bag with integrated electrophoresis and elution and the switchable Eluatkammer a fundamentally new, leaps and bounds better method for the entire process of biomolecule isolation, in particular the isolation of grade 1 DNA molecules for medical and veterinary use. In addition, a technical simplification Even in cases where clean room conditions are not absolutely necessary (availability of relevant quantities of high quality gene fragments for research), it enables a significant reduction in costs and a drastic increase in quality compared to conventional, open systems.

During pulse field electrophoretic cleaning in the bag, unlike in known pulsed field electrophoreses, which serve to determine the length of long DNA fragments, no analytical properties are required. The desired travel widths are lower (eg <1 cm) than in conventional pulse field gel systems, which were also designed for a good selectivity and resolution for 10 cm travel distances and more, and z. For example, by computer-controlled regulation of the voltage of many (>> 4) electrodes ensure alignment of the separation paths and a uniform analysis image (see the prevailing CHEF technique, as in BioRad CHEF DR II, III, etc.). These properties required in analytical gel electrophoresis are not needed in purification electrophoresis. Here, only the principle is used that circular molecules with a length> 20 kb do not run in the pulsed field gel electrophoresis, if they are present without single and double strand breaks with plectonemic Supercoils. Damaged molecules or linearized E. coli genome fragments, on the other hand, leave the gel matrix and pass directly into the electrophoresis buffer. This does not require an analytical gel image, but only a registration of the voltage, current and time of current flow to ensure complete cleaning. It has been found that the retention of the intact molecules equally takes place at different voltage levels of 1.5-9 V in the cleaning buffer used, so that in one embodiment of the invention the electrophoretic cleaning in the process bag allows a simple, cooling-controlling temperature regulation via the voltage supply / amperage Use comes. Thus, an excessive heat development / cooling (eg desired between 8-20 ° C) can be counteracted, in one embodiment, the optimal cleaning condition (by 12 ° C) only via the resistance measurement and coupled voltage regulation without further temperature sensor takes place by in the course occurring too high increase in resistance to the initial value of a current pause or voltage reduction is switched. The switching current fields with an arrangement of the crossed poles of the parallel electrodes above and below the cleaning matrix are all in one Switching angle between 0 and 180 ° before, preferably 90-120 °, and are ensured by inserting four electrodes (minimum 3) in the process bag.

An embodiment of the Elektrodeneinsteckkanäle provides that for the electro-elution farther away, or in a weithalsig further bag (elution bag) located electrodes of the (net) Pluspole are used to the eluate in an intermediate filling chamber (transport tube, eluate) convict. This is sealed on one side with an electroelution membrane / dialysis membrane, stuck to the DNA molecules. On the other hand, it is provided with a DNA-permeable particle filter through which the DNA molecules eluted from the gel pass (see the prior art arrangement of two membranes in Biometra / Schleicher-Schuell open-system elution chambers) and carrier matrix / agarose residues and possibly depending on the material resulting electrode particles are stopped. Additional buffer bridges are added outside the range in which the eluted molecules move in the direction of the elution membrane / packaging container, in order to prevent electroosmotic effects (pH, buffer level shifts, etc.). By welding off the eluate chamber / transport tube from the elution bag and process bag, the clean room process bag has served its purpose ( 7 ). The removed electrodes are optionally cleaned and reused. Conveniently, the free DNA molecules in the aqueous solution, which is the filtered elution buffer without additives, are stored at 0-4 ° C without freezing, because Grade 1 DNA> 20 kb decays into Grade 2 and Grade 3 molecules upon freezing. In other embodiments, stabilizing additives can be added to the eluate chamber / transport tube (for increasing the viscosity, for DNA double helix stabilization, eg 100 mM NaCl, or with complexing substances, or with substances for the preparation of a transfection mixture (compare Lipofectamin, Lipoplexes, polyplexes, etc.) or for direct injection into organs, cell surfaces, vessels, etc. The transport tubes are suitably of a long, thin shape to maintain a high concentration of the eluate and not to transform shearing forces during transient vibrations into turbulent flows equipped with a non-contact tear / opening mechanism for sterile removal, in another version with a large-lumen, lateral screw cap ( 8th ), or with various donor sites, sterile ports attached to cannulas / infusion sets to allow transfer to a transfer solution or injection / dosage / remix. A side port in the transport tube also serves the potential / buffer equalization by building up an upwardly open buffer column against gravity.

The transport hoses are suitably light-protected, long, thin tubes, which allow a through-blowing and counteract by their shape during transport and when taking turbulent flows, such as a welded on both sides of a straw ( 8th ). The double-sided seal of the tube in which the eluate is located ensures contamination-free packaging. Before the container is welded off, by reversing the flow direction, the eluate is released from the dialysis membrane lying outside the weld seam and moved a few cm back into the tube (time pulse), whereupon the welding operation takes place. In this case, the membrane is removed on one side and separated on the other side of the remaining process bag, so that only the transport hose is present.

The present invention of the process bag method can provide replenishment of identical, functionally characterized DNA of the highest quality material and a smooth supply of genomic DNA drug. Thus, the method represents the necessary technological development leap for the clinical and animal clinical development of a gene therapy up to the coverage of the total requirement of a gene therapy construct.

Description of individual execution options and laboratory processes

• 1. Process bag for carrying out multi-stage processes in a closed system. Incorporation of a biomass in the form of a carrier matrix, purification of biomolecules, protected storage of biomolecules during process stages, recovery of samples in a closed system, identification of the functionality of the stored biomolecules from samples taken, separation of parts of a molecule storage in a closed system, isolation and clean preparation of the sterile stored biomolecules, separating and filling the desired fraction in a closed system.
• 2. Biomolecules purified in a process bag are generally defined molecular species, proteins, antibodies, coagulation factors, enzymes, carbohydrates, protein complexes, enzyme complexes, factors, metabolites, structural proteins (eg collagen) and molecules and molecular complexes of the nucleus, genetic material, chromosomes, Chromatin fractions, gene domains, genes, centromeric telomeres, DNA, RNA, genomic DNA, artificial DNA constructs, long DNA molecules, cloned DNA molecules, modified nucleic acids, molecules from cloned libraries, BACs, PACs, pTAT clones and banks, DNA molecules cloned in E. coli and products from cloned DNA molecules, and DNA molecules in the strict sense, long DNA molecules (> 20 kb) in grade 1 quality, ie no breaks in both strands of the DNA Double helix in the majority of the molecules of a preparation, preferably in> 50%, preferably in> 90%, in> 95%, in> 99% of the total number of molecules of a preparation / gene storage, physically intact DNA constructs, physically and functionally intact constructs for artificial chromosomes, PACs, BACs, pTAT clones in E. coli, intact gene stores, purified DNA preparations without E. coli DNA, intact preparations in which most of the molecules are functional, ie genes or chromosomal elements contained in more than 50% of the stored molecules, preferably in> 70% of the stored molecules, preferably in> 85% of the stored molecules, preferably in> 90% of the molecules present in the gene storage. According to the method, biomolecules are in particular pure forms of a molecular species defined in the biologically possible framework of molecular conformity, which originates from material of a living system, from an introduced biomass, animal, plant, human, microorganismal masses and liquids, living or dead, including environmental samples, sediments , Sludges, with the aim of production in the highest possible purity is isolated. In particular, pure forms of a defined, small number of 1 or 2 or a few individual species-related biomolecule species (eg genes, collagens) including the sometimes multiple biological variants of a biomolecule species (eg gene alleles, copy number variants, splice variants, chain length variants, collagen forms, etc.). ). There is virtually no limit to the variety of molecules to be isolated (eg thousands of medically relevant gene segments, thousands of medically relevant gene products, proteins, enzymes, antibodies, factors, biomolecules), especially in the form of banks containing hundreds of thousands of potentially relevant biomolecules can contain. - Excludes manufactured biomolecules in the form of unpurified biomasses, coarse sediments, centrifugates and centrifugation supernatants, filtrates, column fractions, in which no further isolation or significant enrichment of individual specific molecular species. The focus is on pure molecular form, pre-existing, concentrated mixtures of many molecules, simple cell digestions, cell cultures, simple cell lysates, cell disruptions or plasma fractions with a complex molecular composition (eg plasma fractions of transfusion medicine containing various coagulation factors), cell cultures, cell cultures containing stem cells , Cell fractions with stem cells (bone marrow, adult stem cells), blood and blood products, blood cells that are not purified to the pure form of a molecular species or closely circumscribed moieties, or moieties that are not processed into a pure form with the goal of substantial enrichment.
• 3. Sterile bag / container with provision for a cascade of closed-system biological, biochemical, electrochemical, biophysical, chemical and closed-system procedures to prevent contamination when performing complex laboratory procedures, for the safe storage of molecules, for the creation of stable gene stores , for the storage of long DNA molecules from clone banks, for the storage of clonal libraries at room temperature in a functionally characterizable form, for the characterization of a sterile gene storage for later isolation of molecules in grade 1 quality, for the provision of high quality, pure, functional DNA molecules for the provision of grade 1 DNA for human and / or animal use, for the production of artificial chromosomes, for the study of genomic genes and gene banks and gene dosage / activity relations, for the identification of disease genes / protective genes in gene Banks, for the creation of functionally characterized gene storage for the construction of artificial chromosomes, for the production of artificial chromosomes for gene therapy, for the production of a drug from a purified biomolecule fraction, for the production of a DNA drug, for gene transfer, for gene therapy.

The decisive factor is the spatially limited and closed container and the resulting simplification and cost-effective implementation of continuous clean room conditions throughout the entire molecular process, from the introduction of a biomass to the filling of the purified, characterized biomolecule.

A closed container system, preferably used as a disposable item, provides provisions for the different stages of the process and, during the process, the container material itself constitutes the packaging material for sampling, storage, and packaging of the recovered product under "consumption" of the container. The bag / container may be designed to be consumed, reduced, degraded, disassembled, or expanded again during the process. It should be noted here that the wall condition of the bag / container is not limited to the preferred, flexible, transparent "film and bag material", but may also provide partially solid or solid housing walls and closed chambers / Zones and connectivity may contain, in which the various processes can be carried out in a row, so that a processing of the molecular digestion in the closed system takes place. Thus, methods for lining solid container walls with a dense film or laminates for a clean room process bag are the subject of the invention.

process bag

• a) with an access for the introduction of a carrier matrix with the biomolecule to be purified into the bag and sterile closure under clean room conditions by reversible closing, clamping, screwing, pressing, or irreversible closing, gluing, welding.
• b) with lockable / reopenable plug-in channels for electrodes for cleaning pulse field electrophoresis, electrophoretic purification, electroelution
• c) with electrode insertion channels and one or more buffer bridges for the electroelution of the purified biomolecules from the support matrix
• d) with Elektroelutionsvorrichtung and eluate chamber for the introduction / discharge of the purified molecules in a transport tube and closed off the eluate or the transport tube (eluate and transport tubes may be the same or different bag areas) by means of welding.
• e) with accesses for electrophoresis, which ensure the escape of electrophoretic gases and / or a directed buffer flow and are sterile weldable / resealable, alternatively with closable Elektrodeneinsteckadaptoren (screwed, pluggable) and ports for a gas outlet together with buffer outlet (directed upwards to Buffer level / pressure to regulate).
• f) with multiple sterile resealable ports / drains for the delivery and discharge of incubation buffers, enzymes, electrophoresis buffers, buffer bridges, electrodes. Of which at least three ports on the process bag (for the simultaneous discharge of electrophoresis buffer and gas, connecting a buffer bridge to the elution bag and connecting an eluate chamber and for other uses of the same ports at other times) and three ports on the elution bag (for the simultaneous inlet of electrophoresis buffer, connection a buffer bridge to the process bag and connection of the eluate chamber) and at least three Elektrodeneinsteckkanäle in the process bag (preferably 4 or more) and at least one Elektrodeneinsteckkanal (preferably 2 or more) in the elution bag. In this embodiment, the electrophoresis buffer runs from the elution bag portion over the buffer bridge and exits the system via the process bag. In order for the gas to pass through the buffer bridge into the process bag and exit via the process bag outlet, the elution bag portion is stored below and the process bag above with the process bag outlet attached to the upper pole of the process bag and the elution bag inlet at the lower pole of the elution bag and port of the elution bag to the buffer bridge at the upper pole of the elution bag is attached. During pulse field electrophoretic cleaning, the port of the process bag for the buffer bridge serves as the inlet of the electrophoresis buffer, and during the introduction of the gel matrix into the internal screen frame, this port (preferably large lumen) serves for gel casting. Another port in the transport tube / in the eluate chamber was not considered here (cf. 10 )
• g) with enema, preferably at the bottom (cold) for electrophoresis buffer and preferably at the top (warm), with preferably pressure-regulated puffing of the bag by buffer volume and regulation of the inlet / outlet water column difference or puffing of the bag by pump pressure or by rigid columns / wall, due to the design (welding seams, stresses, double layers)
• h) with drainage channel / port and hose and reclosable closure downwards for emptying the incubation buffer, preferably for rapid emptying by gravity
• i) with inlet / access points for the attachment of purification buffer cascades and the inflow of cooled electrophoresis buffer / elution buffer
• k) with access for the separate application of enzymes and enzyme solutions, alternatively with mutifunctional access / ports
• l) optionally the attachment of enzyme depots (eg, drying chambers for nucleases, RNase, lysozyme, proteinases, nucleases), preferably for sterile application after steam sterilization of the bag
• m) with insertion channels for electrodes / disposable electrodes, alternatively electrodes integrated in the process bag / electrode foil, adapted electrode materials in the process bag
• n) with at least one drain channel / hose up for gas / electrophoresis buffer drain
• o) with brackets, fixings, welds, forming zones, clamping zones, strapping inside or outside the bag for placement of the carrier matrix in the process bag
• p) with provisions for the use of different sized quantities, eg. B only filling a recessed well, eg. By storing the bag on a troughed surface so that only the carrier matrix is covered in the center of the bag to incubate with small volumes (eg 100 ml at Gene storage enzyme reactions, or 1 ml and less for miniature bags, or 1 liter and more for larger designs), or inflating the bag under inflation (eg, by gravity versus drain column pressure) to achieve large buffer volumes (e.g., 3000 ml for gene storage electrophoreses, or 30 ml and less for miniature bags, or 30 liters and more for larger versions).
• 4. Introduction and multi-stage processing of the molecules within a carrier matrix in a closed process bag in which the biomass in the form of a - Gels - colloids - gel matrix - agarose gel - LMP agarose (low melting point agarose) - Bacteria in gel matrix - E. coli clones in LMP agarose matrix BAC / PAC / pTAT clones in E. coli in LMP agarose matrix - bound to a binding matrix of other support materials - with a certain pore size - with a specific surface - silicates - sponge, tile - Ceramic - -Scree - lamellae, surfaces, threads, nets - Coated carrier materials with - Antibodies, binding molecules - receptor binding sites, receptors - Nucleic acids, DNA sequences - DNA binding proteins, histones, materials is introduced.
• 5. Introduction of a carrier matrix containing the biomolecule to be purified - By a reclosable filling channel, through which the liquid gel matrix is introduced before solidification in their mold located in the bag, by pipetting, by filling a tube, with a funnel, via a connected bacteria-gel mixer - By introducing the cast gel through a one-sided sterile reveal bag side / insertion opening.
• 6. Introduction of a carrier matrix and fixation within the trimmed, with welds (or walls) spatially limited bag chamber by - "loose fixation" in the bag in another permeable colloid / gel, in a permeable, three-dimensional net, in a three-dimensional fabric structure - in a permeable support frame (flexible screen frame) - in a permeable, dimensionally stable holding frame (eg plastic sieve frame) - In a one-piece or multi-part injection molding holding frame with hinged lid
• 7. To be introduced into the bag / permeable container insert, sieve cover, provision for fixing the carrier matrix in a mold permeable to enzymes, diffusion, and current, a) one-piece folded (preferably in the process with gel casting in process bag) or two-part mold, bottom with Sides and hinged lids or separate lids (preferred for applications with gel casting outside) b) permeable mold / frame with supports, frames with fold-out supports (spacers up and down) and c) mold / frame with devices for fixing / inserting electrodes , fold-out electrode holders d) Frame with separating / buckling / breaking device for removing parts of the carrier matrix e) Frame with a removable bottom foil / seal to prevent leakage through the sieve bottom during casting of the carrier matrix (subsequent removal of the foil / seal) Pulling out / removing or by dissolving ü at 37-55 ° C for days in aqueous, surfactant-containing medium, when running with a Low Melting Point Agarose support matrix, LMP agarose up to 55-60 ° C) f) Screen frame for stabilization of the support matrix while allowing accessibility to buffers , Enzymes, cleaning solutions, washing buffer, electrophoresis buffer, electroelution buffer, current, permeable to macromolecules, of solid plastic, of low-friction plastic, of plastic with low thermal deformation, low tension, with shock elasticity, preferably smoothed inside, low friction inner and outer surfaces of the screen frame , preferably transparent, preferably produced and collapsible as an injection molding mold, preferably can be plugged together by slight pressure through the welded bag g) sieve bottom dimensions only slightly larger than the carrier matrix. h) introduction of externally cast carrier matrices / gel matrices into the permeable screen frames i) alternatively direct introduction of the gel matrix without frame and obstruction by limited bag size, weld seam conditions for the internal fixation of the matrix in the center of the process bag. k) Alternatively, direct introduction of the gel matrix into the existing in the process bag, sterile screen frame, preferably one-piece folding, the Siebrahmendeckel is to lock after pouring the gel matrix in the bag l) Siebrahmendeckel in two-part design with clear orientation pluggable, several folding / dividing lines each bounded on both sides by parallel webs that form the new Seitenbegrenzung for breaking gel matrix after breaking and penetrate when folding / plugging the lid and bottom into the gel and thereby pinch or cut / cut the gel and divide it into several chamber surfaces, or prepare a subdivision. m) bottom sieve covered by foil, which has been inserted, was glued, preferably glued with a water-soluble adhesive, preferably a sealing foil of wasserlösslichem material n) cover / bottom webs in each sub-chamber, so small transport stable, the gel sheathing sections can be canceled (by kinking). Bending zones for different sized sections of the carrier matrix (for intermediate analyzes, analyzes, as package size, as storage sections etc.). o) Since the sections in the bag are moved apart, and then individual sections are sealed in a bag corner / a bag part, resulting from each section closed packaging sizes in the form of welded mini storage bags (closed system). p) The respective bag sectors to be welded are each printed with the serial numbers / barcodes.
• 8. Siebbodendichtungsfolie, sterilizable, water-soluble film which seals the bottom of the sieve frame for at least 1, preferably 10, preferably 20 minutes, so that the (about 37-40 ° C) warm gel matrix (gene storage) can be poured and the gel to to solidify after a few minutes (at about <25-30 ° C) can not leak. Thereafter, the gradual dissolution of the film begins in an aqueous environment so that the sieve pores of the soil for the subsequent cleaning procedures are permeable - or withdrawable film with externally graspable tab (insoluble)
• 9. Closed attachable feeding bags - Supply bag with ports suitable for the process bag for the sterile storage and application of cleaning solutions, buffers, washing solutions, for microbiological, biochemical, molecular genetic incubations, reactions, cleaning cascades by diffusion, cleaning cycles by electrophoresis. - Feed bag with preferably multiple chambers with solutions, buffers. - Division of the chambers by clamping or preferably by attaching a peel seam after filling by means of welding
• 10. Method for filling mutipler chambers by means of welding Filling the feed bag by filling via a port into a single-chamber bag, preferably a long tube bag, and subsequently setting the peel seams to create mutiple chambers with the same content, these are then individually successively by opening the peel seams over the outlet port attached to one side to empty, whereby the washing solutions / electrophoresis solutions are gradually supplied to the process bag. - Subdividing multiple chambers with an aqueous solution by blowing through after filling. - Subdividing the feed bags into multiple chambers for process control and documentation by visibility of already emptied chambers (incubation levels). Subdividing method by applying peeled seams after a loose filling by welding a peel seam through the filled bag by displacing the aqueous filling and welding through a suitable film, - Preferably by displacing the aqueous solution on a high web, through which the filling runs in the welding area to the left and right, wherein the Verteifung and the distance of the webs is a dosage in the chambers - Preferably by moderately preheating the welding zone for the most extensive drying before blowing through the peel seam. - Optionally, multi-compartment feeder bags without dry deposits with a Y port or other suitable ports / ports, for the subsequent delivery / application of enzymes to the wash solution / buffer solution in the closed system.
• 11. Feed bags with multiple chambers with washing solutions / buffers, attaching dry deposits (in suitable film, if necessary pasted over) with lyophilized enzymes, cleaning substances, powders after autoclaving the incubation buffer, which preferably occupy a fixed place in the cleaning cascade and "automatically" by opening the peel seams of the subsequent chambers are flushed in and released (process control, incubation, substance application also without further documentation immediately visible, verifiable).
• 12. Process bag with dummy bag chambers for closed sampling by means of welding / separating. Cutouts / corners in the process bag serve to receive portions of the carrier matrix. For this purpose, the support matrix / gel of the gene storage is bent in a holding frame along provided fracture lines, Alternatively, a gel fragment by pressure with an edge of Squeezed outside through the bag. The buffer volume is drained and the section is moved in the bag until it comes to rest in a bag corner / chamber printed with barcodes / numbers, etc. The bag section is cut off with a regulated manual or table-welding tongs (eg semi-automatic thermoplastic table-top welding machines with double-welded seam and separating knife). It is welded through small aqueous buffer residues between the bag layers. The amount of buffer / moisture is reduced for the setting of the weld in the seam by the bag (right) and the section (left) are stored over an edge, residual buffer so that both sides of the area of the weld, and possibly by moderate Preheating the weld seam a substantial drying of the inner surfaces takes place before the weld / separation is performed. For successive sampling and for the repeated provision of parts of the gene storage several dummy bag chambers are provided, which are wholly or gradually abgeschweisst as needed. The gel sections are without external contact in the bagged bag piece, which can be easily shipped and stored for analysis at another location, or after opening the contained DNA purification step to a purpose (use in another laboratory, research material, assessment function and quality, further processing into a gene transfer drug, DNA vaccine etc). If the sample is removed in the storage state before the purification to grade 1 DNA, the gel remaining in the gene storage bag is still available for the subsequent purification to grade 1 DNA. A sample may be taken at any time to perform quality assays, but may also be cut in the form of DNA purified to grade 1 DNA within the agarose gel (ie, pure, well storable, insensitive to shear forces), thus generating large quantities of characterized gene segments (e.g. B. 20-400 kb (and longer) molecular preparations of BACs / PACs, pTAT vectors (pTAT, pTAT-BS, pTT, pTTE1 and derivatives), artificial chromosomes, clones of banks for functional analysis for the study of gene effects, disease genes, protective genes A large part of the stored, functionally characterized DNA is thus already provided on this easily transportable and storable stage, which are also used directly in cloning, constructions as intact genetic material within the agarose gel matrix in biochemical reactions can., the quality thus obtained d functionally outstanding DNA material is used in all areas of biotechnology (plant genes, animal genes, human genes) and thus allows the widespread use of genetic material at a new technological level (functionally characterized gene storage in grade 1 quality as a reliable building material for artificial chromosomes). For direct medical or veterinary use, or further processing into the DNA drug, the DNA is further eluted in the closed system from the grade 1 DNA stock (agarose gel matrix) and provided in the form of an aqueous DNA solution. The invention is embodiments in which the aqueous DNA solution is further processed in the process bag by further connection of incubation solutions in a closed system to an application form (such as complexation with carriers, encapsulation, mixing with transfer agents, etc) (for example, by transfer to a connected Chamber from the eluate chamber or from the transport hose (see. 7 . 8th . 10 ).
• 13. elution bag with eluate chamber / transport tube with dialysis membrane, filter cartridges, Elektrodeneinsteckkanälen or integrated electrodes, welded electrodes, electrically conductive foil electrodes, inlet for electrophoresis buffer (bottom), drain electrophoresis buffer and gas outlet (above), preferably under inflation pressure by raised inlet buffer level (eg. by a raised compared to the drain level feed drop with electroelution buffer under gravity). Obtaining the electroeluate by keeping the DNA on a dialysis membrane at the left end of a thin transport tube, filtering the eluate on a DNA permeable membrane at the right end of the transport tube (retention of macrocomplexes, possibly electrode particles, if necessary plastic particles of the sieve frame, if necessary Agarosestückchen from connected process bag with the grade 1 DNA gene storage). For the electrode arrangement, the net positive poles are inserted to the left of the transport tube into a buffer bag, which is connected to the process bag via at least one further, open buffer bridge. The elution chamber thus consists of two bags with connections / buffer bridges, wherein one compound is the transport tube, at least one further compound is a preferably large-lumen buffer bridge, and the bags have one or more upwardly open channels for the buffer outlet / buffer overflow / gas outlet. The transferred from the gene storage molecules are detached by short Umpolen from the dialysis membrane and pipettierfrei returned to the transport tube (time / current controlled). The preferred thin, long, low-flow transport tube with a side port for sterile removal at the application site (and optionally for the balance of electro-osmotic pressure / buffer displacements in the transport tube during operation as an elution chamber by means of up-mounted equalization tube) is at both ends of the filters / membranes and attached bags (left elution bag, process bag on the right) separated by welding through the aqueous solution and thus recovered in a closed system. The long DNA molecules in the aqueous solution obtained from the Grade 1 gene stores are stored in the low-flow transport tubes at 4 ° C and used promptly (days to weeks). For the elution process of the grade 1 DNA molecules from the Gelmatrtix and transfer into a transport tube, a combination of the process bag with unilaterally inserted electrodes, a membrane-separated elution chamber, and an elution bag with electrodes is used. Process bags and elution bags are connected to each other via at least one additional buffer bridge. Process bag made of thermo-weldable, long-term stable film materials, characterized by Elektrodeneinsteckkanäle and buffer / Gasablasskanäle and connections for a transport hose and other buffer bridges (preferably großlumig). Elution bag made of heat-sealable film materials (plasticizer-free) characterized by Elektrodeneinsteckkanäle and buffer / gas drainage channels (or common drainage channel with process bag), connections for a transport hose / eluate chamber and for further buffer bridges (preferably großlumig).
• 14. Electrode materials for a process bag - Carbon modifications for the residue-free decomposition of the electrode in gas - Glassy carbon - Fullerenartiger glassy carbon - graphite (natural, artificial) - graphite composites - carbon - Carbon rods - Carbon composites (pure, bound carbon black in one material) - electrically conductive films - electrically conductive paints, film layers, adhesives, ceramics - Platinum, platinum composites, ligatures for use in the process bag - Cables and electrode materials firmly embedded in the bag wall, Conductive film layers, Conductive varnishes, adhesive adhesives, vapor-deposited metal / platinum, mixed materials on foil, ligation, glassy carbon, graphite, carbon, carbon dust, pure carbon black in the form of mixed materials, ceramics, film composites, laminates with conductive tracks or layers
• 15. Attach electrode adapters and cables to the process bag electrodes For the design with sealed electrode insertion channels, the sterile electrodes are plugged / screwed into the four (at least two, in other embodiments more than four) electrode accesses before filling the process bag with electrode buffer. Sealable Elektrodeneinsteck / Schraubadaptoren of hard, sterilizable material, preferably made of thermoplastic to the electrode material, attachable to the electrode rod, formable plastic sleeve, preferably glued, preferably provided with a seal, preferably with high fracture toughness and toughness, preferably with a high stiffness, preferably with a high heat stability, sterilisability, preferably splinter- and particle-free breaking, preferably steam sterilisable, preferably transparent, preferably with graphite, with glassy carbon, glued with platinum wire, sealable, moldable, preferably sterilizable with fitted electrode, lumen and inner electrode seal preferably adaptable to different electrode diameters, (eg 1 mm, 3 mm, 4 mm or larger depending on the process bag size). In one embodiment, the electrode material (eg, an electrode rod 30 cm in length and 3 mm in diameter) protrudes a few cm on the outside of the electrode bag adapter to attach a power cord plug to the electrode. The cable plug together with the adapter and a seal encloses the electrode dry and tight, preferably with a "click" or screwed tight, so that no liquid from the outside between the adapter and plug (eg, condenser water through the cooling, or waste buffer etc) can penetrate. In one embodiment, for. B. with reusable, autoclavable glassy carbon electrodes, the electrodes, once they have lost diameter, pushed within the adapter seal sticking, so that the Duchmesserverlust can be compensated, and a leakage of the adapter is counteracted. For this purpose, the area of the outer side of the electrode to be inserted must remain sterile, which can preferably be achieved by wide sealing rings or outer sheath, or can be performed with millimeter precision by means of a screw thread (external or electrode itself).
• 16. Making and Using Process Bags for the Production of Biomolecules
• 17. Create and use process bags for gene pool creation
• 18. Prepare and use process bags for the purification of grade 1 DNA
• 19. Making and using process bags to perform electrophoretic cleaning
• 20. Manufacturing and using process bags to perform pulse field gel cleaning
• 21. Making and using process bags to perform an electro-elution
• 22. Preparation and Use of Process Bags for Storage of Purified Biomolecules / Gene Bearing / Grade 1 DNA
• 23. Making and using process bags for product recovery by closed-system welding (through-blowing)
• 24. Welding in conjunction with clean room process bags for packaging, storage, and transport of durable material in a closed system
• 25. In the closed system from a process bag into an electro-elution chamber filled and closed severed, welded, clamped, unscrewed etc. transport tubes / eluate chambers and their preparation and use
• 26. Production and use of long, tubular transport containers made of film, which are filled from a closed process bag system and obtained by blowing through / welding. - Welded transport tubes with a small diameter and long length (similar to a straw) to minimize turbulent flow during transport shocks. - Pseudomehrkammriges transport holes with teilschliessenden Zwsichenwänden, foil layers to minimize currents and gravitational forces during transport. - Transport hose with one - Pseudogel, mesh, fleece, colloid to absorb aqueous DNA solutions up to large volumes without shaking (avoiding shear forces and turbulent flows).
• 27. Welding through, sealing through an aqueous solution in a moderately filled plastic bag as a new method of separation, separation, subdivision, separation by welding seams and cutting, temporary subdivision with peel seams, isolation of shares under clean room conditions with - thermoplastic - heating element - vibration - high frequency - Ultrasonic - laser - infrared - Process combinations and other welding processes.
• 28. Application of gene processing bags (genomic clones, BACs / PACs, pTAT vectors for artificial chromosomes) for: - the study of functional gene segments (screening, disease genes, protective genes, gene therapies, genomic construct-based vaccines and artificial chromosomes) - the application of multiple genes on artificial chromosomes - Construction of artificial chromosomes - Manufacture of a drug-suitable grade 1 DNA - for the production of a non-essential starting material for gene transfer research - Animal clinical and clinical studies (gene action, gene dose effect tests, gene transfer, gene therapy) - Further processing / mixing / complexing of the material for DNA / gene therapy Process Bag Applications - Processing / Mixing / Complexing of the DNA / Gene Grade 1 DNA Material from a Process Bag - Further Processing / Mixing / Complexing of the Material for DNA / Gene Therapy Applicati Grade 1 DNA from a Wrapped Transport Tube - Distribution of stored, characterized DNA to user laboratories - Distribution of empty bag kits and devices for disseminated gene storage and production of Grade 1 DNA for genome research, broad coverage of many research sites and medical centers new DNA quality and technology - cost-effective long-term storage of long DNA material from gene banks, banks of artificial chromosomes - back-cloning of isolated DNA molecules (eg. BACs, PACs, pTAT vectors in E. coli) from a small sample of a characterized functionalized genetic stock library, e.g. B. 1 year, 5 years, preferably up to> 10 years Storage of a gene storage bag at room temperature - Further cloning of a DNA construct from a small sample of a characterized gene storage with functional composition to another DNA component from another gene storage (eg Gene, gene border, replicator, gene variant, fragment to complement a gene locus, centromere) and to generate therefrom the next stored construct stage (eg an artificial chromosome with multiple gene segments for the generation of multitransgenic animals for xenotransplantation) - assembly of components various genetic libraries using biochemical methods made possible by grade 1 DNA (in gel site specific recombination, very long PCR) to a construct transfer of long intact DNA
• 29. Application of Grade 1 DNA from a process bag
• 30. Send / Expel Grade 1 DNA from a process bag
• 31. Send / Expel Grade 1 DNA in storable form in a process bag section
• 32. Perform very long PCR (very long PCR) reactions with template DNA (in the form of grade 1 within the agarose gel matrix) from a process bag or a shipped process bag section.
• 33. IGSSR Reactions of DNA Material of two components, of which at least one component is derived from a process bag, and used in the form of grade 1 DNA within the agarose gel matrix to quantitatively generate, without further cloning, long DNA molecules from characterized functional components.
• 34. Process bag for the functional development of genomic libraries, for the storage of testable DNA material from banks, and for the cost-effective storage of a genomic bank and backup copies (without cooling energy).
• 35. Implementation of complex, multi-step laboratory processes within a clean room process bag with the aim of purification and quantitative purification of specific biomolecules and corresponding bag designs, including large chamber bags, miniaturized bag designs, serial or parallel multi-bag arrangements, bag arrangements within fixed housings.
• 36. Implementation of complex, multi-step laboratory processes within a clean room process bag with the aim of purification, documentation and repeated sampling of biomolecules under clean room conditions for chemical and biochemical analysis or for the synthesis of biomolecules and corresponding bag designs.
• 37. Execution of complex, multi-step laboratory processes within a clean room process bag with the aim of purification, documentation and repeated sampling of biomolecules under clean room conditions for medical and veterinary identification, characterization, extraction, measurement of biomolecules and corresponding bag designs.
• 38. Performance of complex, multi-step laboratory processes within a clean room process bag with the goal of purification, documentation and repeated sampling of biomolecules under clean room conditions for the study of environmental samples and corresponding bag designs.
• 39. Execution of complex, multi-step laboratory processes within a clean room process bag with the aim of purification, documentation and repeated sampling of biomolecules under clean room conditions for biotechnology analysis, identification, characterization, extraction, measurement of biomolecules and corresponding bag designs.
• 40. Execution of complex, multi-step laboratory processes within a clean room process bag with the goal of purification, documentation and repeated sampling of biomolecules under clean room conditions for medical and veterinary diagnostics, identification, characterization, extraction, measurement of biomolecules and corresponding bag designs.
• 41. Manufacture, supply, distribution of welding apparatus (single units or combination apparatus and installations) for process bag use for Peel-weld welding (feed cascades with aqueous solutions)
• 42. Manufacturing, providing and distributing process bag welders in conjunction with process bag kits for - Sampling by welding Peel-weld welding (transport hose welding) - optionally with printer (barcodes) - as combination devices
• 43. Manufacture, supply, distribution of voltage devices and pulse field voltage devices that measure the resistance of the electrophoresis chamber at a test voltage across the electrodes, and switch to a large or sudden increase from the initial value, a voltage reduction or a current pause, or a gradual drop in resistance, a voltage increase switch to achieve a simple temperature regulation in the process bag. - which, as safety equipment in the event of severe fluctuations due to defective electrode material / plug connections, or leakage, terminate the power supply and give a warning signal. - Voltage devices that record the current flow to allow documentation - Voltage devices that can be operated simply in pulsed field electrophoresis mode, elution mode and in reversing mode (short pulse with acknowledgment tone).

Description of the figures

• A Siebrahmen mit Boden und Deckel aus fomstabilem Kunststoff und adichtender Bodenfolie (z. B. mit Stärke oder wasserlöslichem Kleber aufgebrachte Folie aus wasserlöslichem Polymer, z. B. Polyvinylalkohol). Gegen Verrutschen und Undichtigkeit ist die Folie eingepasst und mit dem Siebboden verbunden.
• B Der Siebrahmen wird mit geschmolzener Gelmatrix befüllt. Bis zum Erstarren des Gels und Aushärten dichtet die Folie den Boden gegen auslaufen ab. Anschliessend wird die Folie entfernt, bevorzugt in dem sie sich im Laufe des nächsten Tages bei 37°C im wässrigen, tensidhaltigen Inkubationspuffer auflöst, oder sie wird innerhalb eines Tages für Inkubationspuffer durchlässig und löst sich darin innerhalb mehrerer Tage bei z. B. 55°C vollständig auf.
• C Durch Zuklappen des Siebdeckels und Einrasten befindet sich das Agarosegel geschützt innerhalb des flachen Siebrahmens. Durch die zahlreichen Siebporen können Reinigungssubstanzen eindringen, frei in das Gel diffundieren und abtropfen. Ebenso kann ein Elektrophoresepuffer eindringen und Luft/Gasblasen entweichen, so dass ein elektrisches Feld ungehindert im Gel wirken kann. Durch Sollbruchlinien, die an unterteilenden Stegen entlang im Rahmen verlaufen, können kleine Teile des Gels durch Knicken abgebrochen werden. Die geschützten Gelstücke können vom Genlager wegbewegt werden. Das große Agarosegel des Genlagers befindet sich weiterhin geschützt in dem verbeleibenden Siebrahmen. Das Material des Siebrahmens hat bevorzugt ein größeres spezifisches Gewicht als Wasser, damit das Gel in wässrigen Puffern untertaucht.

4 Scheme Screen frame for a LMP agarose gel (with screen frame design).

• A Sieve frame with bottom and lid made of sturdy plastic and adhesive bottom foil (eg foil made of water-soluble polymer, eg polyvinyl alcohol, applied with starch or water-soluble glue). Against slipping and leaking the film is fitted and connected to the sieve bottom.
• B The screen frame is filled with molten gel matrix. Until the gel solidifies and hardens, the film seals the bottom against leakage. Subsequently, the film is removed, preferably in which it dissolves in the course of the next day at 37 ° C in aqueous, surfactant incubation buffer, or within a day for incubation buffer permeable and dissolves within several days at z. B. 55 ° C completely.
• C By closing the strainer lid and snapping into place, the agarose gel is protected inside the flat sieve frame. Due to the numerous sieve pores cleaning substances can penetrate, diffuse freely into the gel and drip off. Likewise, an electrophoresis buffer can penetrate and escape air / gas bubbles, so that an electric field can act unhindered in the gel. By predetermined breaking lines that run along dividing webs in the frame, small parts of the gel can be broken off by kinking. The protected gel pieces can be moved away from the gene storage. The large agarose gel of Genlagers is still protected in the fading screen frame. The material of the screen frame preferably has a greater specific gravity than water to allow the gel to submerge in aqueous buffers.

• A Bei der Ausführung mit dem im Prozessbeutel bereits steril verpackten, noch nicht zugeriegelten Siebrahmen dient ein weitlumiger Port der direkten Einbringung des geschmolzenen Agarosegels. Dabei wird das geschmolzene Gel entweder von Hand pipettiert, oder von einem Bakterien-Agarose-Misch- und Dosiergerät mit einem geeigneten Einfüllstutzen abgefüllt.
• B Bei der Ausführung mit externer Giessform wird diese nach dem Befüllen und Aushärten durch eine unverschlossene Seite des Prozessbeutels eingebracht. Die Beutelseite wird anschliessend durch eine Thermoschweissnaht verschlossen.

2 Scheme Filling the sieve frame with appropriate design.

• A In the version with the sieve frame, which is already sterile packed in the process bag, a wide-lumen port serves for the direct introduction of the molten agarose gel. The molten gel is either pipetted by hand, or filled by a bacterial agarose mixing and dosing with a suitable filler neck.
• B In the case of the version with external casting mold, this is introduced after filling and hardening through an unclosed side of the process bag. The bag side is then closed by a thermal welding seam.

3 Scheme purification procedure by incubation with buffer cascades

The gel enclosed in the bag is subjected to a multi-step treatment with cleaning buffers etc. Sufficient buffer quantities are used per incubation step, which just cover the gel (partial filling, if necessary by creating a bag well through an outer mold, so that the gel comes to rest at the lowest point and not the whole bag has to be filled, not shown). By means of a sterile sealable drain (by means of a downward-hanging tube by means of gravity and a second return valve), the spent buffer containing the substances obtained during the purification by diffusion is removed. By successively opening peeled seams, a next, fresh buffer volume for the next cleaning stage runs in each case. If necessary, a Y port allows the sterile delivery of further components (enzymes, active substances).

4 Scheme Welding method for setting peeled seams in a filled feed bag

• A Der Schlauchbeutel (z. B aus peelnahtfähiger Polyolefinfolie) wird über einen Port am Ende des Schlauchs mässig befüllt und autoklaviert.
• B Der Schlauch wird über einen oder mehrere Stege mit dem Schweisswerkzeug und der Gegenseite so gelagert, dass der Puffer rechts und links in die unteren Aussackungen abläuft und sich beide Folienlagen berühren. Restpuffer im Bereich der Schweissnähte wird durch Vorwärmen weitestgehend getrocknet, worauf die Peelnaht durch den befüllten Beutel hindurch geschweisst wird.
• C Das Ergebnis ist ein steriler Mehrkammerbeutel, der einfach und kostengünstig über einen einzigen Port befüllt wurde. Im Einsatz wird die jeweilige Reinigungsstufe, die sich im Prozessbeutel befindet, durch die Anzahl der entleerten und noch gefüllten Kammern einfach dokumentiert und kann jederzeit ohne weitere Dokumentation überprüft werden.

An aqueous buffer filled, autoclaved, tubular bag of peelable suitable film material is subdivided into a multi-chambered buffer cascade.

• A The tube bag (eg made of peelable polyolefin film) is filled moderately via a port at the end of the tube and autoclaved.
• B The hose is mounted via one or more webs with the welding tool and the opposite side so that the buffer runs off to the right and left into the lower pockets and contacts both film layers. Residual buffer in the area of the weld seams is largely dried by preheating, whereupon the peel seam is welded through the filled bag.
• C The result is a sterile multi-chamber bag that has been easily and inexpensively charged through a single port. In use, the respective cleaning stage, which is located in the process bag, is simply documented by the number of emptied and still filled chambers and can be checked at any time without further documentation.

5 Scheme buffer cascades, various examples of feed bags

Shown are three examples of feed bags with buffer cascades for different application levels. The left-hand cascade of cascades is used to create primary gene stocks from uncharacterized bacterial clones of certain E. coli clones (single colonies of a particular master clone for one application, or clone numbers from banks) and pre-confined to the storage stage. In the embodiment, the entire procedure is automatically documented by opening the peel seams of the individual stages. Enzymes and buffers can not be reversed and are in the closed system. The bag which preferably hangs on a stand of a mobile cart over the process bag is additionally printed with the appropriate incubation temperature for introduction into the corresponding oven / room. The given dilution factors (top right) represent only a guideline value for freely diffusing substances in a corresponding gel volume. The actual dilution levels differ depending on the decomposing effect of the cleaning solutions and the migration behavior and can differ greatly. The last buffer cascade is only needed for long-term storage and is used in the example after 180 days. After only 8-14 days, first samples can be taken to record the structural composition. Bearings that do not conform to the expected molecular shape are disposed of. In the course of storage further functional laboratory tests may be performed to further characterize a genetic stock. If functional molecules are detected, the middle buffer cascade is used. Depending on the requirements, sometimes 2-10 times less average cascades are needed than primary cascades. In the case of a gene bank, a slower rate of continuation is expected until the identification of less valuable clones from thousands of unknown clones. However, the gene storage represents a virtually indestructible, directly tapable form of molecules that become immediately available after characterization in identical functionality.

With the middle cascade a later enzyme treatment is achieved by prewashing with a neutral aqueous buffer. In the example, the concentration of EDTA drops to 1-2 mM, so that enzyme reactions in specific buffers can be carried out. After the enzyme treatment, the corresponding enzyme is removed by renewed protein degradation and then subjected to a second, in the example four-stage purification shown. The dilution series have meanwhile reduced the initially freely diffusible substances in the biomatrix to ppm concentrations.

In the right sample bag, an electrophoretic purification is prepared, with a neutral electrophoresis buffer replacing the storage buffer, which causes a substantial dilution of the EDTA (specified values), salt, and surfactant concentrations, so that subsequently by inserting the electrodes and connecting a single-chamber bag with z. B. 2 l electrophoresis buffer pulse field purification electrophoresis can be performed. (the volume and dilution values are only example sizes)

6 Scheme Pulsfeldreinigungselektrophorese in the process bag and welding a sample

Through a single-chamber feed bag with electrophoresis buffer with height-adjustable drip / port runs continuously into the filled process bag buffer. The cooled buffer enters the lower bag level and displaces the existing buffer upwards. The gel placed in the center of the bag is exposed to a pulsed electric field. For this purpose, stick electrodes are inserted into the process bag parallel to the gel surface via four ports and connected tightly to the bag by means of electrode adapters and cabled. In the embodiment shown, the gas produced during the electrolysis is discharged via an upper port together with the pressure-upflowing buffer. The cleaning products obtained in the buffer, which leave the gel matrix under the influence of current, accumulate in the buffer at the top and are separated off. In case of excessive heating, the voltage is reduced or interrupted, so that no cooling circuit is needed. By suitable electrode materials and avoiding the circulation in a cooling circuit, an immediate separation of the resulting substances is achieved, whereby unwanted electrolysis products are avoided and a high degree of purification is realized. The filling pressure of the bag and the cooling inlet are regulated by height-adjustable inlet and outlet levels, so that no pumps are used in this version.

A further advantage of the process bag made of a heat-sealable film material is the simple separation of a sample (shown at the top left of the process bag). In the state with deflated buffer, a piece of gel separated in the bag through the wall is moved into a corner or blind chamber of the bag. On the inside side of the sample, a small bag corner / bag is then separated by setting preferably two welding seams and separating cutters with a thermo-welding device. The process bag is still sterile closed. The bag portion with the sample is also sterile sealed and can be conveniently stored, shipped, or fed to functional analyzes.

By appropriate bag printing with barcodes and serial numbers of the intended locations of the process bag for the sampling and the remaining gene storage, the risk of a confusion of samples and gene storage can be effectively reduced.

7 Scheme Operation of the process bag with an electro-release bag, transfer into a transport hose

The process bag is connected to an elution bag by placing a port with a particulate filter and one behind it Eluatkammer and the eluate chamber is in turn connected via an interposed dialysis membrane with a port of the elution bag. The elution bag thus represents a small version of a process bag, which serves to expand the buffer-carrying area of the process bag. For this purpose, in addition to the connection via the eluate chamber, at least one large-lumen buffer bridge is connected. To compensate for the electro-osmotic buffer potential, there is additionally a side port located in the eluate tube (which can later serve as a sampling point), at which a tube is connected up during the elution (full power), which can allow a higher buffer level of the eluate chamber. Prior to the polarity reversal, a phase of low power is used for the return of the equalizing buffer, without losing eluate on the dialysis membrane. The eluate is transported by reverse polarity back into the free Eluatschlauch. Subsequently, the tube is closed and separated at both ends by means of the thermoplastic welding technique. In the illustrated embodiment, the sterile sealed side port is used for later removal of the eluate (see detail of transport hose / eluate chamber in FIG 8th ). Other embodiments pass the eluate electrically via the side port of the eluate chamber into further reaction vessels.

8th Scheme transport hose and eluate chamber with membranes

Screwable adapter halves with dimensionally stable sealing rings (black) clamp the DNA permeable filter membrane (white box) and the DNA retaining dialysis membrane (broken boxes) and connect the bag ports with the eluate chamber.

9 Scheme stick electrodes, electrode adapters, electrode plugs

Thin diameter glassy smooth surface glass electrodes are fixedly connected to an adapter cover and inserted through adapter ports equipped with a buffer gasket. By further screwing in case of prolonged operation, leakage can be counteracted by thinning of the electrodes in the region of the seal. Outside the adapter cover, a cable connector is fitted directly onto the protruding electrode, which is equipped with an insulating sleeve with sealing to the outside and shields the port, adapter and the electrode from penetrating liquid from the outside. A Sealed electrodes prevent leakage of the electrophoresis buffer. The electrode material of the stick electrodes projects out of the port / adapter. B On the dry side of the electrode, a plug with contact springs is mounted, which additionally protects the electrode against moisture penetrating from the outside.

10 Scheme cleanroom process bag with main components (without devices)

Representation of an embodiment of a cleanroom process bag with a (smallest possible) number of 6 ports Z1-6 (plus one port Z7 of the eluate chamber), Z1-3 at the process bag and Z4-6 at the eluate bag, and a small number of 4 electrode insertion channels and common gas / Buffer discharge during the electroelution operation (in the circuit on the right with access assignments).

During other cleaning stages, the elution bag and eluate chamber are disconnected and the ports are assigned differently (see labeling of the components on the left and top). By twisted bag storage during electrophoresis is in this process bag execution Z1 to the inlet below and Z2 to the gas / Pufferablauf above (not shown). Z3 is used to connect cleaning buffers and cascades. The sieve frame in the process bag is filled via port Z1.

11 Photo dry model process bag

Two-layer design made from a folded, 1 m wide tubular film. The process bag has 6 sampling points (three blind sacks in each level, left), 8 feed ports (Z1 large-lumen rear, Z2 large-lobed front, Z3-5, small lobed right upper level, Z6-8 small-lumen right lower level) and 4 electrode insertion channels (E1-4 ) with inserted graphite stick electrodes (simple design with open electrode plugging without seal adapter with a gas and buffer discharge via the upwardly directed electrode channels, which are made of bag material). A flat illustration, B supported spatial structure with electrode channels upwards.

12 Scheme Preparation of long intact DNA in an agarose matrix

Purification principle for long DNA molecules with grade 1 DNA quality and for permanent gene storage, from which a variety of gene applications can be performed much better, the subject of the invention is the bag method, whereby the complex laboratory process can be performed in a closed system. As a result, more and immediately unprecedentedly clean, functionally characterized molecules (GMP) can be provided in a cost-effective manner. The new technology can generate functional gene libraries with artificial chromosomes and cover the total demand for genomic DNA drugs.

references

• Schindelhauer D., Cooke H.J. (1997) Efficient combination of large DNA in vitro: in gel site specific recombination (IGSSR) of PAC fragments containing alpha satellite DNA and the human HPRT gene locus. Nucleic Acids Res. 25 (11): 2241-2243.
• Laner A. Development of DNA and expression markers for the detection of human artificial chromosomes and in vivo transfection analysis of improved HAC vectors. Doctoral thesis, Ludwigs-Maximilians University Munich (2004) (http://edoc.ub.unimuenchen.de/4286/)
• Laner A., Schwarz T., Christan S., Schindelhauer D. (2004). Suitability of a CMV / EGFP. Cassette to monitor expression of human artificial chromosomes but not transient tansfer in the cells forming viable clones. Cytogenet Genome Res. 107, 9-13.
• Laner A., Goussard S., Ramalho A.S., Schwarz T., Amaral M.D., Courvalin P., Schindelhauer D., Grillot-Courvalin C. (2005) Bacterial transfer of large functional genomic DNA into human cells. Gene Ther. 12, 1559-1572.
• Noerenberg M. Investigations on various aspects of intactness of long DNA molecules for the construction of human artificial chromosomes. Diploma thesis, Life Sciences Center Weihenstephan, Technical University Munich (2006).
• Kraner S., Christan S., Zoller M., Jung C., Schindelhauer D. (2007) Reconstructing functional gene loci using PCR. Eur J Hum Gen 15 suppl. 1 / C19: 20.
• Welle et al. (2005) PVC softener migration Hoses into enteral feeding solutions. Pharma International 3/2005: 17-21.
• K. Inoue et al. 2005 Clin Chem Acta 358: 159-166.
• Scientific Commettees 2008, SCENIHR Opinion, http://ec.europa.eu/health/ph_risk/committees/04_scenihr/docs/scenihr_o_014.pdf
• DINCH, Technical Information Sheet January 2008 M6168e, BASF

Claims (49)

A method of randomly providing replicas of purified biomolecules or biologicals of a whole of the same clonal source for quality testing or functional testing with the steps a) introducing at least one splittable carrier matrix containing the biomolecules to be provided into a coherent, subdividable volume which is formed by a laboratory bag or process bag, b) sterile closure under clean room conditions by closing, c) supplying storage liquid, cleaning solution or digestion solution to the carrier matrix, d) production of the storage, purification and / or digestion of the biomolecules, wherein the biomolecules remain in the carrier matrix in a storable or purified form, e) storing or storing the biomolecules in their carrier matrix within separable or separable regions of the laboratory or process bag, whereby the carrier matrix itself is divided, f) sterile separation of portions of the laboratory or process bag containing the biomolecules to be provided in their carrier matrix without contaminating the remaining material. A method according to claim 1, wherein as step 9) a gain of the biomolecules, optionally by elution, followed. Device for carrying out the method according to one of the preceding claims, consisting of at least one laboratory bag or process bag, combinable via ports or as a unit connected to at least one a) sieve frame for producing the splittable carrier matrix in the laboratory or process bag, b) feed bag or feed bag for supplying storage liquid, cleaning solution or digestion solution, c) elution bags, optionally connected to an eluate chamber, a tubular transport container or a transport tube, d) electrodes. Method according to one of claims 1 or 2, wherein closing means clamping, screwing, pushing, reversible or irreversible closing, gluing, welding, The method of any of claims 1, 2 or 4, wherein the biomolecules or biologicals are selected from defined molecular species, proteins, antibodies, coagulation factors, enzymes, carbohydrates, protein complexes, enzyme complexes, factors, metabolites, structural proteins (eg collagen) and molecules and Molecular Complexes, Genetics, Chromosomes, Chromatin Infractions, Gene Domain, Gene, Centromeric Telomeres, DNA, RNA, Genomic DNA, Artificial DNA Constructs, Long DNA Molecules, Cloned DNA Molecules, Modified Nucleic Acids, Molecules of Clone Banks, BACs, PACs, pTAT Clones and Banks, DNA molecules cloned into E. coli and products from cloned DNA molecules, and in the narrower sense, DNA molecules, long DNA molecules (> 20 kb) in grade 1 quality, ie no breaks in both strands of the double helix in the majority of the molecules a preparation, preferably in> 50%, preferably in> 90%, in> 95%, in> 99% of the total number of molecules of a preparation / gene storage, physically intact DNA constructs, physically and functionally intact constructs for artificial chromosomes, PACs, BACs, pTAT clones in E. coli, intact gene storage, purified DNA preparations without E. coli DNA, intact preparations in which the majority of the molecules are functional, ie contained genes or chromosomal elements function in> 50% of the stored molecules, preferably in> 70% of the stored molecules, preferably in> 85% of the stored molecules, preferably in> 90% of the molecules present in the gene storage. Method according to one of claims 1, 2 or 4 to 5, wherein the biomolecules represent in particular pure forms of a molecular species defined in the biologically possible framework of the molecular conformity, which originate from material of a living system, from an introduced biomass, animal, vegetable, human, microorganismale masses and liquids, live or dead, including environmental samples, sediments, sludges. Method according to one of claims 1, 2 or 4 to 6, wherein the biomolecules in particular pure forms a defined, small number of 1 or 2 or a few individual related biomolecule species (eg genes, collagens) including the multiple biological variants of a Biomolekülspezies (z B. genallele, copy number variants, splice variants, chain length variants, collagen forms, etc.), and wherein the molecules to be isolated (eg, thousands of medically relevant gene segments, thousands of medically relevant gene products, proteins, enzymes, antibodies, factors, biomolecules) in large Diversity, especially in the form of banks, which may contain hundreds of thousands of potentially relevant biomolecules. Method according to one of claims 1, 2 or 4 to 7, wherein excluded are prepared biomolecules in the form of unpurified biomasses, coarse sediments, centrifugates and Zentrifugationsüberstände, filtrates, column fractions, in which no further isolation or significant enrichment of individual specific molecular species to a molecular clean form in the foreground stands, pre-existing, only concentrated mixtures of many molecules, simple cell digestions, cell cultures, simple cell lysates, cell digests or plasma fractions with a complex molecular composition (eg plasma fractions of transfusion medicine containing various coagulation factors), cell cultures, cell cultures containing stem cells, cell fractions with stem cells (Bone marrow, adult stem cells), blood and blood products, blood cells that are not purified to the pure form of a molecular species or closely circumscribed moieties, or moieties that are not with the goal of essential enrichment are processed into a pure form. Method according to one of claims 1, 2 or 4 to 8, wherein a carrier matrix in the form of a - Gels - colloids - gel matrix - agarose gel - LMP agarose (low melting point agarose) - Bacteria in gel matrix - E. coli clones in LMP agarose matrix BAC / PAC / pTAT clones in E. coli in LMP agarose matrix - bound to a binding matrix of other support materials - with a certain pore size - with a specific surface - silicates - sponge, tile - Ceramic - -Scree - lamellae, surfaces, threads, nets - Coated carrier materials with - Antibodies, binding molecules - receptor binding sites, receptors - Nucleic acids, DNA sequences - DNA binding proteins, histones, materials is present. A method according to any one of claims 1, 2 or 4 to 9, wherein the incorporation comprises a carrier matrix containing the biomolecule to be purified - The use of a reclosable filling channel, through which the liquid gel matrix is introduced before solidification in their mold located in the bag, by pipetting, by filling a tube, with a funnel, via a connected bacteria-gel mixer - The introduction of the cast gel through a one-sided sterile openable bag side / insertion opening. Method according to one of claims 1, 2 or 4 to 10, wherein the introduction of a carrier matrix and optionally the fixation within the trimmed, with welds (or walls) spatially limited bag chamber by - "loose fixation" in the bag in another permeable colloid In a translucent, three-dimensional net, in a three-dimensional fabric structure, - in a permeable support frame (flexible screen frame), - in a permeable, dimensionally stable support frame (eg plastic screen frame), in a one-piece or multi-part injection molding support frame hinged lid. A method according to any one of claims 1, 2 or 4 to 11, wherein the feeding of claim 1c) comprises filling from multiple chamber feed bins and buffer cascades. Apparatus according to claim 3 wherein a feed bag is made according to claim 3b) Filling of the feed bag by filling via a port into a single-chamber bag, preferably a long tube bag, and subsequent setting of the peel seams to produce multiple chambers with the same content. These are then to be individually discharged one after the other by opening the peel seams over the discharge port attached to one side, whereby the washing solutions / electrophoresis solutions are gradually supplied to the process bag. - Subdividing multiple chambers with an aqueous solution by blowing through after filling. - Subdividing the feed bags into multiple chambers for process control and documentation by visibility of already emptied chambers (incubation levels). Subdividing method by applying peeled seams after a loose filling by welding a peel seam through the filled bag by displacing the aqueous filling and welding through a suitable film, - Preferably by displacing the aqueous solution on a high web, through which the filling runs in the welding area to the left and right, with a dosage in the chambers takes place through the recess and the distance of the webs - Preferably by moderately preheating the welding zone for the most extensive drying before blowing through the peel seam. - Optionally, multi-compartment feeder bags without dry deposits with a Y port or other suitable ports / ports, for the subsequent delivery / application of enzymes to the wash solution / buffer solution in the closed system. Device according to one of claims 3 or 13, wherein the feed bag with multiple chambers with washing solutions / buffers, attached dry deposits (in suitable film, possibly pasted over) with lyophilized enzymes, cleaning substances, powders after autoclaving the incubation buffer, preferably in the cleaning cascade a occupy a firm place and are "automatically" flushed and opened by opening the peel seams of the following chambers (process control, incubation done, substance application). Device according to one of claims 3 or 13 to 14, wherein the process bag is equipped with blind bag chambers for closed sampling by means of Abschweiß / Abtrennen, wherein Aussackungen / corners in the process bag to receive portions of the carrier matrix serve, wherein the carrier matrix or the gel of Genlagers in a holding frame along provided fracture lines is bent. Device according to one of claims 3 or 13 to 15, wherein supply bags are shot over the laboratory bag or process bag suitable ports for the sterile storage and application of cleaning solutions, buffers, washing solutions, for microbiological, biochemical, molecular genetic incubations, reactions, cleaning cascades by diffusion , Purification cycles by electrophoresis, wherein Feed bag with preferably multiple chambers with solutions, buffers, - A division of the chambers by clamping or preferably by attaching a peel seam after filling by means of welding are provided. Device according to one of claims 3 or 13 to 16, wherein process bags are provided for carrying out multi-stage processes in a closed system, comprising introducing a biomass in the form of a carrier matrix, purification of biomolecules, protected storage of biomolecules during process stages, recovery of samples in a closed system , Identification of the functionality of the stored biomolecules on the basis of samples taken, separation of parts of a molecular store in a closed system, isolation and pure preparation of sterile stored biomolecules, separation and filling of the desired fraction in a closed system. A device according to any one of claims 3 or 13 to 17, wherein a sterile bag / container provides for a cascade of closed-system biological, biochemical, electrochemical, biophysical, chemical incubations and procedures to prevent contamination in carrying out complex laboratory procedures, for secure storage of molecules, for the creation of stable gene stores, for the storage of long DNA molecules from clone banks, for the storage of clones at room temperature in a functionally characterizable form, for the characterization of a sterile gene storage for later isolation of molecules in grade 1 quality, for the provision of high-quality, pure, functional DNA molecules, for the provision of grade 1 DNA for human and / or animal use, for the production of artificial chromosomes, for the investigation of genomic genes and gene banks and gene dosage / activity relations, for the identification of disease genes / protective genes in gene banks, for the creation of functionally characterized gene stores for the construction of artificial chromosomes, for the production of artificial chromosomes for gene therapy, for the manufacture of a medicament from a purified biomolecule fraction, for the manufacture of a DNA drug, for gene transfer, for gene therapy. Device according to one of claims 3 or 13 to 18, wherein the process bag a) with an access for introducing a carrier matrix with the biomolecule to be purified into the bag and sterile closure under clean room conditions by reversible closing, clamping, screwing, pressing, or irreversible closing, gluing, welding, b) with lockable / reopenable plug-in channels for electrodes for cleaning pulse field electrophoreses, electrophoretic purification, electroelution, c) with electrode insertion channels and one or more buffer bridges for the electroelution of the purified biomolecules from the support matrix, d) with electroelution device and eluate chamber for the introduction / discharge of the purified molecules into a transport tube and closed welding of the eluate chamber or the transport tube (eluate chamber and transport tube may be the same or different bag regions) by means of through-welding, e) with accesses for electrophoresis, which ensure the escape of electrophoretic gases and / or a directed buffer flow and are sterile weldable / resealable, alternatively with closable Elektrodeneinsteckadaptoren (screwed, pluggable) and ports for a gas outlet together with buffer outlet (directed upwards to To regulate buffer level / pressure), f) with multiple sterile resealable ports / drains for the supply and discharge of incubation buffers, enzymes, electrophoresis buffers, buffer bridges, electrodes, g) with enema, preferably at the bottom (cold) for electrophoresis buffer and preferably at the top (warm), with preferably pressure-regulated puffing of the bag by buffer volume and regulation of the inlet / outlet water column difference or puffing of the bag by pump pressure or by rigid columns / wall, due to the design (setting of welds, tensions, double layers), h) with drainage channel / port and hose and reclosable closure downwards for emptying the incubation buffer, preferably for rapid emptying by gravity, i) with inlet / entrances for the attachment of purification buffer cascades and the inflow of cooled electrophoresis buffer / elution buffer, k) with accesses for the separate application of enzymes and enzyme solutions, alternatively with mutifunction accesses / ports, l) optionally the attachment of enzyme depots (eg, drying chambers for nucleases, RNase, lysozyme, proteinases, nucleases), preferably for sterile application after steam sterilization of the bag, m) with insertion channels for electrodes / disposable electrodes, alternatively electrodes integrated in the process bag / electrode foil, adapted electrode materials in the process bag, n) with at least one drain channel / hose up for gas / electrophoresis buffer drain, o) with brackets, fixings, welds, mold zones, clamping zones, strapping inside or outside the bag for the placement of the carrier matrix in the process bag, p) with provisions for the use of different sized quantities, eg. B only filling a recessed well, eg. By storing the bag on a troughed surface so that only the carrier matrix is covered in the middle of the bag to incubate with small volumes (eg 100 ml for gene storage enzyme reactions, or 1 ml and less for miniature bags, or 1 l and more in larger designs), or inflating the bag under inflation (eg, by gravity against down-plume pressure) to achieve large buffer volumes (eg, 3000 ml for gene storage electrophoreses, or 30 ml and less for miniature bags). or 30 l and more for larger versions). Is provided. The device of any one of claims 3 or 13 to 19, wherein at least three ports on the process bag (for the simultaneous discharge of electrophoresis buffer and gas, connecting one buffer bridge to the elution bag and connecting one eluate chamber and for other uses of the same ports at other times) and three ports on the elution bag (for the simultaneous admission of electrophoresis buffer, connection of a buffer bridge to the process bag and connection of the eluate chamber) and at least three Elektrodeneinsteckkanäle in the process bag (preferably 4 or more) and at least one Elektrodeneinsteckkanal (preferably 2 or more) are provided in the elution bag. Device according to one of claims 3 or 13 to 20, wherein a frame / permeable container insert, sieve wrapper, means for attaching the carrier matrix in a mold permeable to enzymes, diffusion, and electricity is provided in the following manner: a) one-piece hinged ( preferably in the case of the embodiment with gel casting in the process bag) or two-part mold, bottom with sides and hinged lid or separate lid (preferably in the case of design with gel casting outside), b) permeable mold / frame with supports, frame with fold-out supports (spacers up and down ) and c) hollow mold / frame with devices for fixing / inserting electrodes, fold-out electrode holders, d) frame with separating / buckling / breaking device for removing parts of the carrier matrix, e) frame with a removable bottom foil / seal to prevent leakage through the sieve bottom during casting of the carrier matrix (subsequent removal of the foil / seal by pulling / Removal or dissolution over days in an aqueous, surfactant-containing medium at 37-55 ° C, when running with a support matrix of low melting point agarose, LMP agarose up to 55-60 ° C), f) screen frame for the stabilization of the carrier matrix with simultaneous accessibility for buffers, enzymes, cleaning solutions, washing buffer, electrophoresis buffer, electroelution buffer, current, permeable to macromolecules, made of solid plastic, low-friction plastic, plastic with low thermal deformation, low stress, with shock elasticity, preferably smoothed inside, low-friction interior and outer surfaces of the screen frame, preferably transparent, preferably g) screen mold dimensions only slightly larger than the carrier matrix, h) introduction of externally cast carrier matrices / gel matrices into the permeable screen frames, i) alternatively direct insertion k) alternatively, direct introduction of the gel matrix into the sterile sieve frame existing in the process bag, preferably in one piece, the sieve cover is after pouring the gel matrix into the gel matrix without frame and obstruction by limited bag size, weld seam conditions for the internal fixation of the matrix in the center of the process bag L) Closing lid for two-part design with clear orientation pluggable, several folding / dividing lines bounded on both sides parallel by bars, which form the new lateral boundary for the gel matrix contained therein after breaking off and at m collapse / collapse of lid and bottom penetrate into the gel and pinch or cut / cut the gel and divide into several chamber surfaces, or prepare a subdivision, m) bottom sieve covered by foil that has been inserted, glued, preferably with a water-soluble Adhesive was glued, preferably a sealing foil of wasserlösslichem material, n) cover / bottom webs in each sub-chamber, so small transport-stable, the gel sheathing sections can be canceled (by kinking). Buckling zones for different sized sections of the carrier matrix (for intermediate analyzes, analyzes, as package size, as storage sections etc.), o) Since the sections in the bag are moved apart and then individual sections in a bag corner / a bag part are welded off, from each section closed, Packaging sizes in the form of shrink-wrapped mini storage bags (closed system), p) The respective bag sectors to be welded are each printed with the serial numbers / barcodes. Device according to one of claims 3 or 13 to 21, wherein a Siebbodendichtungsfolie, sterilizable, water-soluble film which seals the bottom of the screen frame for at least 1, preferably 10, preferably 20 minutes, so that the (about 37-40 ° C) warm gel matrix (in Genlagern) can be poured in and the gel is allowed to solidify after a few minutes (at ca <25-30 ° C) can not leak, and then begins the gradual dissolution of the film in an aqueous environment, so that the sieve pores of Soil are permeable to the downstream cleaning procedures, - Or a pull-out film is provided with externally tangible tab (insoluble). Device according to one of claims 3 or 13 to 22, wherein the attachable supply bag - Supply bag with ports suitable for the process bag for the sterile storage and application of cleaning solutions, buffers, washing solutions, for microbiological, biochemical, molecular genetic incubations, reactions, cleaning cascades by diffusion, cleaning cycles by electrophoresis, Feed bag with preferably multiple chambers with solutions, buffers, - Subdivision of the chambers is formed by clamping or preferably by attaching a peel seam after filling by means of welding through. Device according to one of claims 3 or 13 to 23, wherein the elution bag with eluate / transport tube with dialysis membrane, filter cartridges, Elektrodeneinsteckkanälen or integrated electrodes, welded electrodes, electrically conductive foil electrodes, inlet for electrophoresis buffer (bottom), drain electrophoresis buffer and gas outlet (above), preferably under filling pressure by raised intake baffle level (eg by a relative to the drain level raised inlet drip with electroelution buffer under gravity) is equipped. Device according to one of claims 3 or 13 to 24, wherein the electrode materials of the electrodes according to claim 3d) are selected from - carbon modifications for residue - free decomposition of the electrode into gas - glassy carbon - fullerene type glassy carbon graphite (natural, artificial) - graphite composite - carbon - Carbon rods - Carbon composites (pure bonded carbon black in one material) - Electrically conductive films - Electrically conductive paints, film layers, adhesives, ceramics - Platinum, platinum composites, alloys for use in process bags - Cables and electrode materials firmly embedded in the bag wall, Conductive film layers , conductive lacquers, conductive adhesives, evaporated metal / platinum, mixed materials on foil, alloys, glassy carbon, graphite, carbon, coal dust, pure carbon black in the form of mixed materials, ceramics, film composites, laminates with conductive sheets or layers Use of a device according to any of claims 3 or 13 to 25 for providing biomolecules. Use of a device according to one of claims 3 or 13 to 25 for applying genetic stores. Use of a device according to any one of claims 3 or 13 to 25 for the purification of grade 1 DNA. Use of a device according to one of claims 3 or 13 to 25 for performing an electrophoretic cleaning. Use of a device according to one of claims 3 or 13 to 25 for carrying out a pulsfeldgelelektrischen cleaning. Use of a device according to one of claims 3 or 13 to 25 for carrying out an electroelution. Use of a device according to one of claims 3 or 13 to 25 for the recovery of products by welding in a closed system (through-blowing). Welding using a device according to one of claims 3 or 13 to 25 in connection with cleanroom process bags for the packaging, storage, and transport of durable material in the closed system. Tubular transport container, transport tube or eluate chamber prepared according to claim 3 for carrying out the method according to one of claims 1 or 2. Foil-shaped transport containers made of film according to claim 34, which are filled from a closed process bag system and obtained by blow-through / welding, - Welded transport tubes with a small diameter and long length (similar to a straw) to minimize turbulent flows during transport shocks, - Pseudomehrkammriges transport tube with teilschliessenden partitions, foil layers to minimize currents and gravitational forces during transport, - Transport hose with one - Pseudogel, mesh, fleece, colloid to absorb aqueous DNA solutions up to large volumes without shaking (avoiding shear forces and turbulent flows). Welding or sealing through to carry out the method according to one of claims 1 or 2 by an aqueous solution in a moderately filled plastic bag as a method for separation, separation, subdivision, separation by welding seams and cutting, temporary subdivision with Peelnähten, isolation of shares under clean room conditions with - thermoplastic - heating element - vibration - high frequency - Ultrasonic - laser - infrared - Process combinations and other welding processes. Use of a device according to one of claims 3 or 13 to 25 in the gene area (genomic clones, BACs / PACs, pTAT vectors for artificial chromosomes) for the - determination of functional gene segments (screening, disease genes, protective genes, gene therapies, vaccines based on genomic constructs and artificial chromosomes) - application of multiple genes to artificial chromosomes - construction of artificial chromosomes - production of drug grade 1 DNA - for the production of a reliable starting material for gene transfer research - animal clinical and clinical studies (gene action, gene dose effect tests, gene transfer, gene therapy) - processing / mixing / complexing of the material for DNA / gene therapy applications in the process bag - Further processing / mixing / complexing of the material for DNA / gene therapy applications of the grade 1 DNA from a process bag - Further processing / mixing / complexing of the Mater DNA / Gene Grade Applications of Grade 1 DNA from a Wrapped Transport Tube - Distribution of stored, characterized DNA to user laboratories - Distribution of empty bag kits and devices for the disseminated gene docking and production of grade 1 DNA for genome research, broad coverage of many research sites and medical centers with the new DNA quality and technology - cost effective long term storage of long DNA material from gene libraries, banks of artificial chromosomes - recloning of isolated DNA molecules (eg BACs, PACs, pTAT vectors in E. coli) from a small sample of a characterized gene storage cell with functional composition, eg. B. 1 year, 5 years, preferably up to> 10 years Storage of a gene storage bag at room temperature - Further cloning of a DNA construct from a small sample of a characterized gene storage with functional composition to another DNA component from another gene storage (eg Gene, gene border, replicator, gene variant, fragment to complement a gene locus, centromere) and to generate therefrom the next stored construct stage (eg an artificial chromosome with multiple gene segments for the generation of multitransgenic animals for xenotransplantation) - assembly of components various genetic libraries by means of biochemical methods, which are made possible by grade 1 DNA (in gel site specific recombination, very long PCR) to a construct - transfer of long intact DNA. Use of a purified grade 1 DNA according to claim 28 for carrying out functional or quality tests according to claim 1. Use according to claim 38 for performing very long PCRs, or for reactions with template DNA (in the form of grade 1 within the agarose gel matrix). Use according to claim 38 in IGSSR reactions of DNA material of two components, at least one component of which is from a process bag, and used in the form of grade 1 DNA within the agarose gel matrix to quantitatively generate, without further cloning, long DNA molecules from characterized functional components , Use of a device according to any one of claims 3 or 13 to 25, wherein process bags are used for the functional opening of genomic banks, for the storage of testable DNA material from banks, and for the cost-effective storage of a genomic library and security copies (without cooling energy). A method according to any one of claims 1, 2 or 4 to 12, wherein performing complex, multi-step laboratory processes within a clean room process bag with the goal of purification and quantitative purification of specific biomolecules and corresponding bag designs, including large capacity bags, miniaturized bag designs, serial or parallel multi-bag arrangements, bag assemblies within solid housing is included. Method according to one of claims 1, 2 or 4 to 12, wherein performing complex, multi-step laboratory processes within a clean room process bag with the aim of purification, documentation and repeated sampling of biomolecules under clean room conditions for chemical and biochemical analysis or for the synthesis of biomolecules and appropriate bag designs is included. Method according to one of claims 1, 2 or 4 to 12, wherein performing complex, multi-step laboratory processes within a clean room process bag with the goal of purification, documentation and repeated sampling of biomolecules under clean room conditions for medical and veterinary identification, characterization, extraction, measurement of Biomolecules and corresponding bag designs is included. A method according to any one of claims 1, 2 or 4 to 12, including performing complex, multi-step laboratory processes within a clean room process bag with the goal of purification, documentation and repeated sampling of biomolecules under clean room conditions for the study of environmental samples and corresponding bag designs. Method according to one of claims 1, 2 or 4 to 12, wherein performing complex, multi-step laboratory processes within a clean room process bag with the goal of purification, documentation and repeated sampling of biomolecules under clean room conditions for biotechnology analysis, identification, characterization, extraction, measurement of Biomolecules and corresponding bag designs is included. Method according to one of claims 1, 2 or 4 to 12, wherein carrying out complex, multi-step laboratory processes within a clean room process bag with the aim of purification, documentation and repeated sampling of biomolecules under clean room conditions for medical and veterinary diagnostics, identification, characterization, extraction, Measurement of biomolecules and corresponding bag designs is included. Device according to one of claims 3 or 13 to 25, wherein pass-through devices (individual devices or combination devices and systems) for process bag purposes for Peelnahtdurchschweißen (feed cascades with aqueous solutions) are included. Device according to one of claims 3 or 13 to 25, wherein voltage devices and pulse field voltage devices are formed, which measure the resistance of the electrophoresis chamber via the electrodes at a test voltage, and switch to a large or sudden increase from the initial value, a reduction of the voltage or a current pause, or switch on a gradual drop in resistance, a voltage increase to achieve a simple temperature regulation in the process bag, wherein - the safety equipment in case of strong fluctuations due to defective electrode material / connectors, or leakage, which terminates the power supply and gives a warning signal, - Voltage devices are provided which record the current flow to allow documentation - Voltage devices are provided, which can be operated either simply in Pulsfeldelektrophoresemodus, elution mode and Umpolmodus (short pulse with acknowledgment tone).

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