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DE2652636A1 - Stabilising protein during separation, purification and storage - by adding amphiphilic cpds., esp. lipid(s), ionic and or nonionic detergents - Google Patents

Stabilising protein during separation, purification and storage - by adding amphiphilic cpds., esp. lipid(s), ionic and or nonionic detergents

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Publication number
DE2652636A1
DE2652636A1 DE19762652636 DE2652636A DE2652636A1 DE 2652636 A1 DE2652636 A1 DE 2652636A1 DE 19762652636 DE19762652636 DE 19762652636 DE 2652636 A DE2652636 A DE 2652636A DE 2652636 A1 DE2652636 A1 DE 2652636A1
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protective substances
separation
proteins
ionic
storage
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Hans Uwe Dr Rer Nat Wolf
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Wolf hans Uwe drrernat
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Wolf hans Uwe drrernat
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/24—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing atoms other than carbon, hydrogen, oxygen, halogen, nitrogen or sulfur, e.g. cyclomethicone or phospholipids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/36—Extraction; Separation; Purification by a combination of two or more processes of different types
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/96—Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates

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  • Analytical Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Microbiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
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  • Separation Using Semi-Permeable Membranes (AREA)

Abstract

Stabilisation of sensitive proteins and related cpds., esp. lipo-proteins and glyco-proteins, during their purification, sepn. and storage, is effected by adding protective substances, (I), having an amphiphilic structure, pref. lipids, partic. phospho-lipids, ionic and/or nonionic detergents. In an example, Ca2+-adenosine triphosphatase of human erythrocyte membrane was separated by gel chromatography in the presence of lecithin and "Tween 20" (RTM).

Description

Verfahren zur Staoilisierung von Proteinen und verwandtenProcess for the Staoilization of proteins and related

Verbindungen während ihrer Reinigung, Isolierung und Lagerung Die Erfindung betrifft ein Verfahren zur Stabillsierung empfindlicher Proteine und verwandter Verbindungen (insbesondere Lipoproteine und Glycoproteine) während ihrer Reinigung, Isolierung und Lagerung durch Zusatz stabilisierender Schutzsubstanzen mit amphiphiler Struktur.Compounds during their cleaning, isolation and storage The The invention relates to a method for stabilizing sensitive proteins and related ones Compounds (especially lipoproteins and glycoproteins) during their purification, Isolation and storage by adding stabilizing protective substances with amphiphilic Structure.

Die Reinigung vieler Proteine (incl. Proteide) erfolgt heute mit Hilfe weitverbreiteter Verfahren, wie z. B. The cleaning of many proteins (incl. Proteide) takes place today with The help of widely used procedures such as B.

der shromatographie oder der fraktionierten Fällung. Zahlreiche Proteine, besonders solche mit einem hydrophoben Oberflächenanteil £z. B. Membranproteine), lassen sich jedoch mit den herkömmlichen Methoden der Proteintrennung nicht isolieren, da sie während des Trenprozesses partiell oder total denatiert werden. Verschiedene Membranenzyme werden auf diese Weise inaktivert (vgl. dazu R. L. JULIANO, Biochim. Biophys.shromatography or fractionated precipitation. Numerous proteins, especially those with a hydrophobic surface portion £ z. B. membrane proteins), however, cannot be isolated using conventional methods of protein separation, as they are partially or totally denated during the cutting process. Different Membrane enzymes are inactivated in this way (cf. R. L. JULIANO, Biochim. Biophys.

Acta 300 (1973) 341 - 378).Acta 300 (1973) 341-378).

Die Ursache für die erwähnte Inaktivierung liebt in der spezifischen Struktur der Proteine und der sich hieraus ergebenden Verhaltensweisen in wäßrigen Medien Der Kontakt ihrer hydrophoben Oberfläche, die nach der Ablösung der Detreffenden Proteine von der Membran freigelegt wird, mit wäßrigen Medien führt zu einer häufig irreversiblen Strukturumwandlung (Konfortmationsänderung), in deren Verlauf die biologischen Aktivitäten der Proteine - also ihre wesentlichen. The cause of the mentioned inactivation loves in the specific Structure of proteins and the resulting behavior in aqueous Media The contact of its hydrophobic surface, which occurs after the detachment Proteins from the membrane being exposed, using aqueous media often leads to a irreversible structural change (change of confortmation), in the course of which the biological activities of proteins - that is, their essential ones.

Eigenschaften - meist verlorengehen.Properties - mostly lost.

Prinzipiell ist ein Schutz vor Inaktivierung dann möglich, wenn die empfindlicher Oberflächenanteile der betreffenden Proteine durch Zusatz spezieller Stabilisatoren vor dein Kontakt mit der wässrigen Phase geschützt werden. Hierfür kommen amphiphile Substanzen in Frage. In principle, protection against inactivation is possible if the sensitive surface parts of the proteins in question by adding special ones Stabilizers protected from contact with the aqueous phase will. Amphiphilic substances come into question for this.

deren Molekül einen hydrophoben und einen hydrophilen Toll besitzen, wie es bei vielen Detergentien der Fall ist.whose molecule has a hydrophobic and a hydrophilic toll, as is the case with many detergents.

Im wäßrigen Medium ist eine Stabilisierung von Membranproteinen durch den Zusatz von Phospholipiden möglich, die als wesentliche Bestandteile der Membranen die natürliche Umgebung dieser Proteinklasse darstellen. Dieser sehr wirksame stabilisierende Zusatz erlaubt jedoch wegen der Bildung relativ großer Protein-Lipid-Komplexe (Teilchengewicht > 2 x 106 Dalton) keine Auftrennung von Proteingemischen, d. l die Isolierung einzelner Proteine ist meistens nicht möglich (vgl. dazu Ch. TANFORD und J. Membrane proteins are stabilized in the aqueous medium the addition of phospholipids possible, which are essential components of the membranes represent the natural environment of this class of protein. This very effective stabilizing However, due to the formation of relatively large protein-lipid complexes (particle weight > 2 x 106 Dalton) no separation of protein mixtures, d. l the insulation individual proteins is usually not possible (see Ch.TANFORD and J.

A. REYNOLDS, Biochim. Biophys. Acta 457 (T976) 135 sowie H. U. WOLF, Habilitationsschrift, Mainz, 1976).A. REYNOLDS, Biochim. Biophys. Acta 457 (T976) 135 and H. U. WOLF, Habilitation thesis, Mainz, 1976).

Andererseits führt der Zusatz von Detergentien zu wäßrigen Medien, die als Aeguilibrierflüssigkeiten z. B. für Gelchromatographiesäulen eingesetzt werden, zwar in vielen Fällen zu guten Trennergebnissen, jedoch häufig auch zu irreversiblen Veränderungen (Inaktivierung) der nativen Enzyme (A. HELENIUS und K. SIMOMB, Biochim. On the other hand, the addition of detergents leads to aqueous media, as aeguilibrating liquids z. B. used for gel chromatography columns lead to good separation results in many cases, but often also to irreversible results Changes (inactivation) of the native enzymes (A. HELENIUS and K. SIMOMB, Biochim.

Biophys.- Acta 415 t1975) 29; H. U. WOLF, Habilitationsschrift, Mainz, 1976).Biophys. Acta 415 (1975) 29; H. U. WOLF, habilitation thesis, Mainz, 1976).

Der Erfindung liegt die Aufgabe zugrunde, die zu isolierenden Proteine durch den Zusatz spezieller Stabilisatoren vor partieller oder totaler Denaturierung und damit vor Verlust ihrer biologischen Eigenschaften zu schützen und gleichzeitig eine Auftrennung dieser Proteingemische zu ermöglichen. The invention is based on the object of the proteins to be isolated by adding special stabilizers before partial or total denaturation and thus to protect against loss of their biological properties and at the same time to enable these protein mixtures to be separated.

Diese Stabilisierung bei erhaltener Trennbarkeit der Proteine erreicht man dadurch, daß den für die Trennung der Proteine verwendeten Medien Gemische aus verschiedenen amphiphilen Substanzen zugesetzt werden, z. b. Gemische aus Phospholipiden und nichtonischen Detergentien. This stabilization is achieved while maintaining the separability of the proteins by making mixtures of the media used for the separation of the proteins various amphiphilic substances are added, e.g. b. Mixtures of phospholipids and non-ionic detergents.

Die für die Wirksamkeit dieser Gemische erforderliche homogene Dispersion wird durch Ultrabeschallung des Gemiscns erreicht.The homogeneous dispersion required for the effectiveness of these mixtures is achieved by ultrasound of the mixture.

Als Anwendungesbeispiel für die bes-chriebene Methode -sei die Isolierung und Stabilisierung der high-affinity 2+-Adenosintriphosphatase (Ca2+-ATPase)-der Human erythrocytenmembran gegeben (vgl. dazu lI U. WOLF, Habilitationsschrift, Mainz, 1976). Die Isolierung dieses Enzyms erfolgt mit Hilfe der Gelchromatographie auf Sepharose CL-GB oder CL-4B. As an application example for the method described, let it be insulation and stabilization of the high-affinity 2+ adenosine triphosphatase (Ca2 + -ATPase) -der Human erythrocyte membrane given (see lI U. WOLF, habilitation thesis, Mainz, 1976). The isolation of this enzyme takes place with the help of the gel chromatography Sepharose CL-GB or CL-4B.

Der Schutz dieses Membranenzyms vor Inaktivierung erfolgt während des chromatographischen Trennprozesses dadurcn, das die gesamte Trennsäule (6o - loo cm Länge, 2,6 cm Innendurchmesser) vor der Aufgabe des Trennguts mit einem Medium äquilibriert wird, das außer den für das Enzym essentiellen Ionen o.5 g Lecithin (aus Hühnereigelb) und 6.85 g Tween 20/Liter Aequilibrierlösung enthält. The protection of this membrane enzyme from inactivation takes place during of the chromatographic separation process, which takes the entire separation column (6o - loo cm length, 2.6 cm inner diameter) before the material to be separated is added with a medium is equilibrated, which in addition to the ions essential for the enzyme o.5 g of lecithin (from chicken egg yolk) and 6.85 g Tween 20 / liter equilibration solution.

Mit Hilfe einer 30 min. langen Ultrabescnallung dieses Mediums bei OOC unter Stickstoffbegasung werden aus dem Gemisch amphiphiler Substanzen gemischte Micellen erzeugt, deren Vorhandensein für den Erfolg der Trennung notwendig ist. Die Zusammensetzung des Elutionemedluins ist identisch mit derjenigen des Aequilibiermediums.With the help of a 30-minute ultra-strapping of this medium OOC under nitrogen gas are mixed from the mixture of amphiphilic substances Micelles generated, the presence of which is necessary for the success of the separation. The composition of the elution medium is identical to that of the equilibration medium.

Andere Bedingungen für eine erfolgreiche Isolierung unter gleichzeitiger Stabilisierung der oben erwähnten Ca2+-ATPase sind 0.5 g Lecithin (aus Hühnereigelb) und 1.0 g Triton X-100/Liter Lequilibrier- bzw. Elutionsmedium. Other conditions for successful isolation under simultaneous Stabilization of the above-mentioned Ca2 + -ATPase are 0.5 g lecithin (from chicken egg yolk) and 1.0 g Triton X-100 / liter le equilibration or elution medium.

Der mit Hilfe dieser Methode erzielbare Trennerfolg hängt im wesentlichen vom Verhältnis [Lipid]/[Detergens] ab. Das beruht auf der Tatsache, dalS durch-tie Aenderung dieses Verhältnisses die Elutionsvolumina der zu trennenden Proteine in unterschiedlich großem Ausmaß zu beeinflussen sind. Hierdurch besteht die Möglichkeit, daß Proteintrennungen, die in einem einzigen chromatographischen Schritt nicht durchführbar sind, weil die Differenzen zwischen den Elutionsvolumina einzelner Komponenten zu gering sind., durch die Wahl eines anderen Verhältnisses [Lipid]/[Detergens] doch noch erreichbar sind. Eine Aenderuhig der Trennbedingungen kann zusätzlich durch einen Wechsel des Detergens erfolgen, das zrr Bildung der genischten icellen eingesetzt wird. The success of the separation that can be achieved with the aid of this method essentially depends on the ratio [lipid] / [detergent]. That is due to the fact thatS by-tie This ratio changes the elution volumes of the proteins to be separated in to be influenced to different degrees. This makes it possible to that protein separations that are not feasible in a single chromatographic step because of the differences between the elution volumes of individual components to are low, but by choosing a different [lipid] / [detergent] ratio can still be reached. A calm change in the separation conditions can also be achieved by the detergent used for the formation of the mixed icelles must be changed will.

Die Stabilität der oben erwähnten Ca2+-ATPase wird durch die genannten Zusätze an amphiphilen Substanzen entscheidend erhöht: während für das von der Membran abgelöste Enzym bei 0 - 20C Halbwertszeiten für die Inaktivierung von ca. 7 Stunden gemessen werden, liegt die Halbwert.sseit nach Zusatz der genannten amphiphilen Substanzen (d. h. nach dei gelchromatographischen Reinigung) bei mehr als 400 Stunden. Die Ausbeute an enzymatischer Aktivität beträgt nach der oben beschriebenen Gelchromatographie im allgemeinen 80 - loo % der zur Chromatographie eingesetzten Aktivität, während sie ohne amphiphile Substanzen oder bei Zusatz von Detergens allein auf 2 - 5 % abfällt (H. U. WODF, Habilitationsschrift, Mainz, 1976). The stability of the Ca2 + -ATPase mentioned above is ensured by the mentioned Additions of amphiphilic substances decisively increased: while for that of the membrane detached enzyme at 0-20C half-life for inactivation of approx. 7 hours are measured, the half-value is since after the addition of the amphiphilic Substances (i.e. after gel chromatographic purification) at more than 400 hours. The yield of enzymatic activity is according to the gel chromatography described above generally 80-10% of the activity used for chromatography, while without amphiphilic substances or with the addition of detergent only to 2 - 5% falls off (H. U. WODF, habilitation thesis, Mainz, 1976).

Die Anwendung amphiphiler Substanzen oder von Gemischen derselben zur Stabilisierung von Proteinen beschränkt sich nicht auf die Reinigung durch Gelchromatographie, sondern ist gleichermaßen auch bei anderen Proteiiitrenn- crua -isolierungsmethoden anwendbar, so z. B. The use of amphiphilic substances or mixtures thereof to stabilize proteins is not limited to purification by gel chromatography, It is also used in other protein separation methods applicable, e.g. B.

bei der Ionenaustauschchromatographie und der Fraktionie rung durch Ammoniumsulfat.in ion exchange chromatography and fractionation Ammonium sulfate.

Unter Anwendung aller Variationsmöglichkeiten, die diese Methode bietet, wie Variation des Verhältnisses [Lipid]/[Detergens], der Absolutkonzentration der amphiphilen Substanzen sowie der Wahl der geeigneten Lipid-und Detergenskomponente(n) sollte es möglich sein, für praktisch jedes Protein optimale Bedingungen sowohl hinsichtlich seiner Stabilisierung als auch seiner Isolierung zu finaen. Using all possible variations that this method offers, like variation of the ratio [lipid] / [detergent], the absolute concentration the amphiphilic substances and the choice of suitable lipid and detergent component (s) It should be possible for virtually any protein to have optimal conditions for both in terms of its stabilization as well as its isolation.

Claims (8)

Patentansprüche 1. Verfahren zur Reinigung, Isolierung und Lagerung empfindlicher Proteine und verwandter Substanzen (insbes-ondere Lipoproteine und Glycoproteine), d a d u r c h g e k e n n z e i c h n e t, das zur Stabilisierung dieser Verbindungen Schutzsubstanzen mit amphiphiler Struktur verwendet werden.Claims 1. Process for cleaning, isolation and storage sensitive proteins and related substances (in particular lipoproteins and Glycoproteins), which are not used for stabilization of these compounds protective substances with an amphiphilic structure are used. 2. Verfahren nach Anspruch 1, d a d u r c h g e k e n n -z e i c h n e t, datS die verwendeten Schutzsubstanzen mit amphiphiler Struktur Lipide (insbesondere Phospholipide), ionische oder nichtionsche Detergentien oder Mischungen derselben sind.2. The method according to claim 1, d a d u r c h g e k e n n -z e i c h n e t, datS the protective substances used with amphiphilic structure lipids (in particular Phospholipids), ionic or non-ionic detergents or mixtures thereof are. 3. Verfahren nach den Ansprüchen 1 und 2, d a d u r c h g e k e n n z e i c h n e t, daß die verwendeten Schutzsubstanzen während mindestens einer Phase des Trenn- und Isolierungsvorgangs oder während des gesamten Trenn- und Isolierungsvorgangs in den bei den jeweiligen Prozessen eingesetzten Medien anwesend sind.3. The method according to claims 1 and 2, d a d u r c h g e k e n It is noted that the protective substances used during at least one Phase of the separation and isolation process or during the entire separation and isolation process are present in the media used in the respective processes. 4. Verfahren nach den Ansprüchen 1 - 3, d a d u r c h g e k e n n z e i c h n e t, daß die verwendeten Schutzsubstanzen in geeigneter VIelse (besonders mit Hilfe von Ultraschall) in den bei den jeweiligen Trenn- und Isolierungsprozessen eingesetzten Medien dispergiert werden.4. The method according to claims 1-3, d a d u r c h g e k e n n indicates that the protective substances used are suitable in a variety (especially with the help of ultrasound) in the respective separation and isolation processes media used are dispersed. 5. Verfahren nach den Ansprüchen 1 - 4, d a d u r c h g e k e n n z e i c h n e t, daß bei Anwendung chromatographischer Trenmethoden die entsprechenden Trennsäulen mit Medien äquilibriert werden, welche die Schutzsubstanzen enthalten.5. The method according to claims 1-4, d a d u r c h g e k e n n z e i c h n e t that when using chromatographic separation methods the appropriate Separating columns are equilibrated with media which contain the protective substances. b. Verfahren nach den Ansprüchen 1 - 4, d a d u r c h g e k e n n z e i c h n e t, daX bei Anwendung chromatographischer Methoden mit Hilfe von Medien eluiert wird, welche die Schutzsubstanzen enthalten.b. Method according to claims 1-4, d u r c h g e n n n z e i c h n e t, daX when using chromatographic methods with the help of media is eluted, which contain the protective substances. 7. Verfahren nach den Ansprüchen 1 - 4, d d a d u r c h g e k e n n z e i c h n e t, dalS bei Anwendung elektrophoretischer Trennmethoden dir verwendeten Schutzsubstanzen in den Elektrodenbädern sowie im Trägermaterial (z.7. The method according to claims 1-4, d d a d u r c h g e k e n Not to mention that you used electrophoretic separation methods Protective substances in the electrode baths and in the carrier material (e.g. B. in Gel) bzw. in der Trennkammer anwesend sind.B. in gel) or are present in the separation chamber. 8. Verfahren nach den Ansprüchen 1 - 4, d a d u r c h g e k e n n z e i c h n e t, daX die genannten Schutzsubstanzen in geeigneter Dispersion während der Lagerung der Proteine (bzw. Proteide) - besonders im flüssigen gefrorenen oder gefriergetrockneten Zustand - anwesend sind.8. The method according to claims 1-4, d a d u r c h g e k e n n it is noted that the protective substances mentioned are in a suitable dispersion during the storage of proteins (or proteids) - especially in liquid frozen or freeze-dried state - are present.
DE19762652636 1976-11-19 1976-11-19 Stabilising protein during separation, purification and storage - by adding amphiphilic cpds., esp. lipid(s), ionic and or nonionic detergents Pending DE2652636A1 (en)

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Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE3240177A1 (en) * 1981-10-30 1983-05-11 Novo Industri A/S, 2880 Bagsvaerd STABILIZED INSULIN PREPARATIONS AND METHOD FOR THE PRODUCTION THEREOF
FR2532178A1 (en) * 1982-08-31 1984-03-02 Asahi Chemical Ind PROCESS FOR STABILIZING TUMOR NECROSIS FACTOR AND AQUEOUS SOLUTION OR STABLE POWDER CONTAINING THE SAME
EP0189838A2 (en) * 1985-01-31 1986-08-06 Solvay Enzyme Products, Inc. Thermal stabilization of alpha-amylase
EP0198388A2 (en) * 1985-04-08 1986-10-22 Research Corporation Enterally effective biologically active peptide or protein composition, method for the production thereof, and a pharmaceutical composition containing same
DE3723781A1 (en) * 1986-07-18 1988-01-21 Chugai Pharmaceutical Co Ltd MEDICINAL PRODUCTS CONTAINING STABILIZED G-CSF (GRANULOCYTE-COLONY-STIMULATING FACTOR) AND METHOD FOR THE PRODUCTION THEREOF
EP0268110A1 (en) * 1986-10-27 1988-05-25 Cetus Oncology Corporation Pharmaceutical compositions of recombinant interleukin-2 and formulation processes
US4925574A (en) * 1985-06-26 1990-05-15 Canadian Patents & Development Limited Purification of hemoglobin and modified hemoglobin by affinity chromatography
WO1993000443A1 (en) * 1991-06-26 1993-01-07 Bio-Technology General Corp. Purification of recombinant apolipoprotein e from bacteria
EP0610445A1 (en) * 1991-09-27 1994-08-17 Incyte Pharmaceuticals, Inc. Compositions comprising a bactericidal/permeability increasing protein and a lipid carrier, methods of making same, and uses thereof
EP0763100A1 (en) * 1995-03-31 1997-03-19 Lee Laboratories, Inc. Method of purification of clostridium difficile toxin a and production of mono-specific antibodies
EP3385870A1 (en) 2008-06-20 2018-10-10 Novartis AG Immunoglobulins with reduced aggregation

Cited By (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4614730A (en) * 1981-10-30 1986-09-30 Novo Industri A/S Stabilized insulin preparations and a process for preparation thereof
DE3240177A1 (en) * 1981-10-30 1983-05-11 Novo Industri A/S, 2880 Bagsvaerd STABILIZED INSULIN PREPARATIONS AND METHOD FOR THE PRODUCTION THEREOF
FR2532178A1 (en) * 1982-08-31 1984-03-02 Asahi Chemical Ind PROCESS FOR STABILIZING TUMOR NECROSIS FACTOR AND AQUEOUS SOLUTION OR STABLE POWDER CONTAINING THE SAME
EP0189838A2 (en) * 1985-01-31 1986-08-06 Solvay Enzyme Products, Inc. Thermal stabilization of alpha-amylase
EP0189838A3 (en) * 1985-01-31 1988-04-20 Miles Laboratories, Inc. Thermal stabilization of alpha-amylase
EP0198388A2 (en) * 1985-04-08 1986-10-22 Research Corporation Enterally effective biologically active peptide or protein composition, method for the production thereof, and a pharmaceutical composition containing same
EP0198388A3 (en) * 1985-04-08 1988-02-03 Research Corporation Enterally effective biologically active peptide or protein composition, method for the production thereof, and a pharmaceutical composition containing same
US4925574A (en) * 1985-06-26 1990-05-15 Canadian Patents & Development Limited Purification of hemoglobin and modified hemoglobin by affinity chromatography
DE3723781C2 (en) * 1986-07-18 1999-09-02 Chugai Pharmaceutical Co Ltd Use of polyoxyethylene sorbitan esters of an aliphatic acid to stabilize G-CSF (granulocyte colony stimulating factor)
DE3723781A1 (en) * 1986-07-18 1988-01-21 Chugai Pharmaceutical Co Ltd MEDICINAL PRODUCTS CONTAINING STABILIZED G-CSF (GRANULOCYTE-COLONY-STIMULATING FACTOR) AND METHOD FOR THE PRODUCTION THEREOF
EP0268110A1 (en) * 1986-10-27 1988-05-25 Cetus Oncology Corporation Pharmaceutical compositions of recombinant interleukin-2 and formulation processes
WO1993000443A1 (en) * 1991-06-26 1993-01-07 Bio-Technology General Corp. Purification of recombinant apolipoprotein e from bacteria
EP0610445A1 (en) * 1991-09-27 1994-08-17 Incyte Pharmaceuticals, Inc. Compositions comprising a bactericidal/permeability increasing protein and a lipid carrier, methods of making same, and uses thereof
EP0610445A4 (en) * 1991-09-27 1995-03-15 Incyte Pharma Inc Compositions comprising a bactericidal/permeability increasing protein and a lipid carrier, methods of making same, and uses thereof.
EP0763100A1 (en) * 1995-03-31 1997-03-19 Lee Laboratories, Inc. Method of purification of clostridium difficile toxin a and production of mono-specific antibodies
EP0763100B1 (en) * 1995-03-31 2008-05-14 Lee Laboratories, Inc. Method of purification of clostridium difficile toxin a and production of mono-specific antibodies
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