WO2021102094A1

WO2021102094A1 - Parallel purification of biomolecules over a range of process scales

Info

Publication number: WO2021102094A1
Application number: PCT/US2020/061203
Authority: WO
Inventors: Cristopher HOLLANDER; Thomas Edward MCDONAGH
Original assignee: Bristol-Myers Squibb Company
Priority date: 2019-11-20
Filing date: 2020-11-19
Publication date: 2021-05-27

Abstract

Provided herein are systems and methods for separation of analytes from a plurality of samples using parallel chromatography. In some embodiments, the systems and methods include two or three parallel flowpaths and may further include an autosampler. In some embodiments, the systems and methods are applicable to mesoscale purification of biological samples, for example, proteins and antibodies.

Description

PARALLEL PURIFICATION OF BIOMOLECULES OVER A RANGE OF

PROCESS SCALES

REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to U.S. Provisional Application Ser. No. 62/937,990, filed Nov. 20, 2019, which is incorporated by reference herein in its entirety.

BACKGROUND

[0002] Biologies are making up an increasing proportion of the global drug discovery pipeline. Supporting the expansion of biologies drug discovery requires higher throughput techniques for the expression, purification and characterization of both therapeutic candidates and reagents. Target validation, lead selection and in-vivo! in-vitro characterization are just a few of the many activities occurring during the drug discovery process that can require significant amounts of purified product.

[0003] Purification of recombinant protein plays a vital role in drug discovery and development. From target validation through lead selection and characterization, many discovery activities rely on the production of larger amounts of high quality product. These activities can include in vivo pharmacokinetics (pK), efficacy and toxicology studies, epitope mapping/crystallization, biochemical/biophysical characterization, formulation and stability studies among others. Each discovery program may have multiple lead candidates as well as variants engineered to achieve desired properties such as humanization, affinity maturation, pK, reduced immunogenicity, and target selectivity. Purification tends to be the biggest bottleneck in supplying recombinant protein in support of discovery programs. As multiple programs move through the discovery process the need for automated technologies that can process tens to hundreds of purifications per week over a range of scales will be useful as a means to overcome purification resource constraints.

[0004] For discovery activities requiring greater amounts of product, existing high- throughput technologies are limited. The lack of parallel purification platforms for production of tens to hundreds of milligrams of protein (mid-scale) limits drug discovery activities and slows drug development timelines.

SUMMARY

[0005] Described herein are systems and processes that accomplish automated parallel and multi-step chromatography over a range of scales. These configurations provide versatility beyond any off the shelf chromatography system allowing a user to perform complex operations and automate a range of processes. A 2.5x increase in throughput may be routinely accomplished with the systems and methods described herein. In some embodiments, the systems and methods described hereon provide true set up and walk away automation capability of multi-step processes combined with high-flow resin technologies result in additional increases in purification throughput and around the clock processing capability.

[0006] In some embodiments, the systems and methods described herein achieve parallel multi-step chromatography using a strategy employing three pumps ( e.g ., A, B, and Sample pump) for sample loading. Combining the three pump loading strategy with the flexibility to customize different flowpaths enables automated parallel multi-step purification over a range of scales. Parallel multi-step processes may be accomplished by directly loading the eluate from one purification step onto another column while retaining product within the plumbing of the system. Each column in a multi-step process may then be operated independently to achieve the desired outcome, e.g., concentration, further purification and/or buffer exchange. System clean-in-place (CIP) methods may be built into the programmed methods, and user interaction may be all but eliminated to minimize the potential for contamination of product. Full automation of multi-step purification processes may reduce labor costs, allow for multiple users with minimal training, and ensure consistency. In addition, the systems and methods described herein may be useful in alleviating mid-scale protein purification bottlenecks.

[0007] In one aspect, the disclosure provides a system for separation of analytes from a plurality of samples comprising: (a) a plurality of pumps in liquid communication with an injection valve; (b) a first versatile valve in liquid communication with one of the pumps; (c) a (i) column valve and (ii) a column or loop valve, each in liquid communication with the injection valve; (d) a column or loop valve in liquid communication with the first versatile valve; (e) a second versatile valve in liquid communication with: (i) a column or loop valve in liquid communication with the injection valve; and (ii) a column or loop valve in liquid communication with the first versatile valve; and (f) a detector for detection of analytes.

[0008] In some embodiments, the system further comprises an outlet valve in liquid communication with the detector to direct flow to: (i) the injection valve, (ii) the first versatile valve, (iii) a flowthrough collector, (iv) a fraction collector, and/or (v) a waste collector.

[0009] In some embodiments, the outlet valve is in a flowpath after the detector.

[0010] In some embodiments, the system further comprises a fraction collector connected to the outlet valve.

[0011] In some embodiments, a column, loop, and/or versatile valve is in liquid communication with a column.

[0012] In some embodiments, the system comprises three or more pumps.

[0013] In some embodiments, a plurality of pumps are in liquid communication with a plurality of intake lines.

[0014] In some embodiments, each pump is connected to an autosampler.

[0015] In some embodiments, the autosampler is configured to sample at least 2 or at least 3 samples at the same time.

[0016] In some embodiments, the detector comprises a UV, a conductivity and/or a pH detector.

[0017] In some embodiments, the system further comprises a mixer for blending intakes from a plurality of pumps or from a pump and a versatile valve, wherein the mixer is in liquid communication with the injection valve.

[0018] In some embodiments, the analytes are biomolecules. [0019] In some embodiments, each column is independently an affinity capture, ion exchange, size exclusion (e.g., gel filtration), hydrophobic interaction chromatography, and/or mixed mode chromatography column.

[0020] In some embodiments, the system further comprises a waste collector that accepts flow from the injection valve and/or a versatile valve.

[0021] In some embodiments, the waste flow is routed to the autosampler.

[0022] In some embodiments, each pump is in liquid communication with a sample container and/or a cleaning solution.

[0023] In some embodiments, the cleaning solution is an inorganic base (e.g., NaOH). [0024] In another aspect, the disclosure provides a system for separation of analytes in a plurality of samples comprising: (a) a first and a second parallel chromatographic flowpath capable of accepting jointly three or more samples; (b) a first junction integrating the first and the second parallel chromatographic flowpath to provide a joint chromatographic flowpath; (c) a detector for detecting analytes in the joint chromatographic flowpath; and (d) a second junction capable of directing the analytes back into the first and/or second chromatographic flowpath.

[0025] In yet another aspect, the disclosure provides a method for separation of analytes in a plurality of samples comprising: (a) loading a first and a second sample onto a first and second column or loop valve through the same injection valve; (b) loading a third sample onto a third column or loop valve through a versatile valve in parallel with step (a); (c) allowing the first, second and third samples pass through a chromatography column on a column or loop valve; (d) sequentially eluting the analytes of interest from the first, second, and third samples; and (e) allowing the eluted analytes from the samples to pass through a detector sequentially.

[0026] In some embodiments, the method further comprises allowing an outlet valve to direct flow to the: (i) injection valve, (ii) first versatile valve, (iii) flowthrough collector, (iv) fraction collector, and/or (v) waste collector.

[0027] In some embodiments, the outlet valve is in a flowpath after the detector.

[0028] In some embodiments, the method further comprises collecting fractions with a fraction collector connected to the outlet valve. [0029] In some embodiments, the method further comprises drawing a plurality of samples from a plurality of containers with an autosampler.

[0030] In some embodiments, the autosampler draws at least 2 or at least 3 samples at the same time.

[0031] In some embodiments, the loading in steps (a) and (b) is performed with 3 pumps.

[0032] In some embodiments, the detector comprises a UV, a conductivity, and/or a pH detector.

[0033] In some embodiments, the method further comprises blending intake from a plurality of pumps or from a pump and a versatile valve.

[0034] In some embodiments, the analytes are biomolecules.

[0035] In some embodiments, each column is independently an affinity capture, ion exchange, size exclusion (e.g., gel filtration), hydrophobic interaction chromatography, and/or mixed mode chromatography column.

[0036] In some embodiments, the method further comprises collecting waste from the injection valve, and/or a versatile valve.

[0037] In some embodiments, the method further comprises routing the waste flow to the autosampler.

[0038] In some embodiments, the method further comprises cleaning sample lines and/or autosampler with a cleaning solution.

[0039] In some embodiments, the cleaning solution is an inorganic base (e.g., NaOH).

[0040] In yet another aspect, the disclosure provides a system for separation of analytes from a plurality of samples comprising: (a) a plurality of pumps in liquid communication with an injection valve; (b) a first column valve in liquid communication with the injection valve; (c) a first versatile valve in liquid communication with the injection valve; (d) a column or loop valve in liquid communication with the first versatile valve; (e) a second versatile valve in liquid communication with the column or loop valve and the first versatile valve; and (f) a detector for detection of analytes in liquid communication with the second versatile valve. [0041] In some embodiments, the system further comprises a first outlet valve in liquid communication with the injection valve and the detector, wherein the first outlet valve is configured to direct flow to: (i) the injection valve, (ii) the first versatile valve; (iii) a fraction collector and/or (iv) a flowthrough collector.

[0042] In some embodiments, the first outlet valve is in a flowpath after the detector.

[0043] In some embodiments, the system further comprises a fraction collector connected to the outlet valve.

[0044] In some embodiments, a plurality of column and/or loop valves are in liquid communication with a plurality of columns.

[0045] In some embodiments, the system comprises 2 pumps.

[0046] In some embodiments, a plurality of pumps are in liquid communication with a plurality of intake lines.

[0047] In some embodiments, each pump is connected to an autosampler.

[0048] In some embodiments, the autosampler is configured to sample at least 2 samples at the same time.

[0049] In some embodiments, the detector comprises a UV, a conductivity and/or a pH detector.

[0050] In some embodiments, the system further comprises a second outlet valve in liquid communication with the injection valve.

[0051] In some embodiments, the second outlet valve is configured to direct flow to a flowthrough collector and/or a waste collector.

[0052] In some embodiments, the analytes are biomolecules.

[0053] In some embodiments, each column is independently an affinity capture, ion exchange, size exclusion (e.g., gel filtration), hydrophobic interaction chromatography and/or mixed mode chromatography column.

[0054] In some embodiments, the system further comprises a waste collector that accepts flow from the injection valve and/or a versatile valve.

[0055] In some embodiments, the waste flow is routed to the autosampler.

[0056] In some embodiments, each pump is in liquid communication with a sample container and/or a cleaning solution. [0057] In some embodiments, the cleaning solution is an inorganic base (e.g., NaOH).

[0058] In still another aspect, the disclosure provides a method for separation of analytes in a plurality of samples comprising: (a) loading three or more samples in parallel into a first and a second chromatographic flowpath; (b) conducting multi-step chromatography in the first and second chromatographic flowpaths; (c) integrating the first chromatographic flowpath into the second chromatographic flowpath to provide a joint flowpath; and (d) detecting analytes in the joint flowpath.

[0059] Other features and advantages of the instant disclosure will be apparent from the following detailed description and examples, which should be construed as limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

[0060] FIG. 1 illustrates an embodiment in which two loop valves (loop valve 1 and loop valve 2) and four versatile valves (VV1-VV4) are used to achieve parallel and multi- step chromatography. In FIGS. 1-5, “SyP” refers to system pump and “SaP” refers to sample pump.

[0061] FIG. 2 illustrates an embodiment with three parallel flowpaths, each with a dedicated column/loop valve, to be run simultaneously (the three parallel flowpaths, each originating from one of the three piston pumps are dashed). All three flowpaths can be independently directed to detection, outlet, and fractionation through versatile valve 2 (VV2).

[0062] FIG. 3 illustrates an embodiment in which the outlet valve has access to all column/loop valves in order to accomplish multi-step chromatography (available flowpaths originating from the outlet valve are dashed).

[0063] FIG. 4 illustrates an embodiment with two parallel flowpaths. With VV1 and VV2 in the default 1-3 positions, the loop valve is inline prior to the detector(s).

[0064] FIG. 5 illustrates an embodiment in which when versatile valves VV1 and VV2 are in the 1-4, 2-3 position, loop valve 1 is bypassed prior to the detector(s) and put inline post detection. This allows the elution from the column valve to be applied to a column on the loop valve post detection. [0065] FIG. 6 shows an exemplary elution profile comparison for three parallel flowpaths originating from the Sample Pump (S), A Pump (A) and B Pump (B). Chromatography scale shown is a 1.0ml HiTrap™ MabSelect™ SuRe™ followed by 2x 5.0ml HiTrap™ Desalt columns connected in series; load volume = 120ml clarified cell culture; first peak is the Protein A elution peak which is directed to the G-25 column; second peak is the G-25 desalting step which is directed to the fraction collector.

[0066] FIG. 7 shows an exemplary overlay of reversed phase HPLC (RP-HPLC) chromatograms for three antibodies (labeled Mab 1, Mab 2 and Mab 3) purified in parallel.

[0067] FIG. 8 shows an example of dynamic binding capacity of Protein A capture resins compared at 0.3 minute and 1 minute residence times.

[0068] FIG. 9 illustrates an exemplary timeframe for processing three antibodies using sequential vs. parallel purification.

[0069] FIG. 10 illustrates an embodiment with two parallel flow paths, one originating from the sample pump and one originating from the system pumps. The pump that has access to each flow path can be switched by changing the Injection valve position between sample pump load and direct inject.

[0070] FIG. 11 illustrates an embodiment with two parallel flow paths, in which when eluting a column plumbed into the column valve (dashed flow path), peaks can be directed to a second column on the loop valve through a dedicated outlet line plumbed into VV1 (dash-dotted line).

[0071] FIG. 12 illustrates an embodiment with two parallel flow paths, in which, when eluting a column plumbed into the loop valve (dashed flow path), peaks can be directed to a second column on the column valve through a dedicated outlet line plumbed into the manual load port (dash-dotted line). Loop E and F ports are plumbed with a short piece of tubing.

[0072] FIG. 13 illustrates an exemplary timeframes for staggered parallel vs. sequential two step purification. Time savings through parallel purification approaches 2x as more runs are performed. DETAILED DESCRIPTION

[0073] In order that the present description can be more readily understood, certain terms are first defined. Additional definitions are set forth throughout the detailed description.

[0074] It is to be noted that the term “a” or “an” entity refers to one or more of that entity; for example, “a nucleotide sequence,” is understood to represent one or more nucleotide sequences. As such, the terms “a” (or “an”), “one or more,” and “at least one” can be used interchangeably herein.

[0075] Furthermore, “and/or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term “and/or” as used in a phrase such as “A and/or B” herein is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone). Likewise, the term “and/or” as used in a phrase such as “A, B, and/or C” is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone);

B (alone); and C (alone).

[0076] It is understood that wherever aspects are described herein with the language “comprising,” otherwise analogous aspects described in terms of “consisting of’ and/or “consisting essentially of’ are also provided.

[0077] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure is related. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary Of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press, provide one of skill with a general dictionary of many of the terms used in this disclosure.

[0078] Units, prefixes, and symbols are denoted in their Systeme International de Unites (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, nucleotide sequences are written left to right in 5’ to 3’ orientation. Amino acid sequences are written left to right in amino to carboxy orientation. The headings provided herein are not limitations of the various aspects of the disclosure, which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification in its entirety.

[0079] The term “about” is used herein to mean approximately, roughly, around, or in the regions of. When the term “about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term “about” can modify a numerical value above and below the stated value by a variance of, e.g ., 10 percent, up or down (higher or lower).

[0080] As used herein, the terms “ug” and “uM” are used interchangeably with “pg” and “mM,” respectively.

[0081] As used herein, the recitation of a numerical range for a variable is intended to convey that the invention may be practiced with the variable equal to any of the values within that range. Thus, for a variable which is inherently discrete, the variable can be equal to any integer value within the numerical range, including the end-points of the range. Similarly, for a variable which is inherently continuous, the variable can be equal to any real value within the numerical range, including the end-points of the range. As an example, and without limitation, a variable which is described as having values between 0 and 2 can take the values 0, 1 or 2 if the variable is inherently discrete, and can take the values 0.0, 0.1, 0.01, 0.001, or any other real values > 0 and < 2 if the variable is inherently continuous.

[0082] All of the references cited herein are incorporated herein by reference in their entireties.

I. Parallel Multi-Step Chromatography Configurations

[0083] The instrument configuration and associated methods enable complete automation and consistent system performance so that any user can queue up samples for true walk-away purification. An automated sample loading method allows for air detection by the pumps, which allows the pumps to pause until the rest of the samples are fully loaded. Column cleaning and system sanitization are built into the methods so that contamination risk is minimized for all users. A. Configuration with Three Parallel Flowpaths

[0084] An exemplary instrument configuration to achieve automated parallel multi- step chromatography is shown in Fig. 1. This configuration has three independent parallel flowpaths, each originating from one of the three pumps (sample pump, system pump A, and system pump B). In some embodiments, the pumps are dual-head piston pumps. Each pump may be plumbed to an inlet valve with multiple positions (e.g., 2-10 positions, in some embodiments 7 positions), some of which can be used for sample application to accomplish parallel chromatography.

[0085] Programming the pumps to run in parallel may be accomplished with the Unicom software. Sample flow and system flow may be programmed to ran concurrently when the injection valve is in the sample pump load position. The A and B pumps may be ran at the same flow rate independently by programming the system pumps to ran a 50% gradient with double the intended flow rate.

[0086] In some embodiments, each parallel flowpath uses a dedicated column or loop valve. As shown in Fig. 1, such configuration may use two loop valves and one column valve (Fig. 1), although other configurations of loop and column valves can also be used. Columns are plumbed into the available ports on the column and loop valves. A loop valve (e.g., the second loop valve) may be configured as an X valve. An X valve may be an inlet valve that is added to the system in addition to A, B, and Sample inlets to increase the number of inlets in the system.

[0087] An independent flowpath from the sample pump is created by plumbing the existing column valve into the loop position of the injection valve. A loop valve is then plumbed to the column position of the injection valve for use with the pump A flowpath (Figs. 1 and 2). The ability of the column valve to reverse flow is beneficial for the sample pump flowpath. With the injection valve in the sample pump load position, sample flow is directed to the column valve while flow from pump A is directed to loop valve 1 (Fig. 2).

[0088] The third flowpath originating from pump B is created by integrating a loop valve (loop valve 2) flanked by two versatile valves (VV1 and VV2) between pump B and ultraviolet (UV) detector (Fig. 2). In the default position, VYl directs flow from pump B to the AB mixing T and the standard AB flowpath remains unchanged. In the 1- 4, 2-3 position flow is diverted from pump B to its own dedicated flowpath.

[0089] Versatile valve 2 (VV2) can direct either of the A, B and/or sample flowpaths (originating from pump A, pump B, or the sample pump, respectively) to detection, while the third flowpath is directed to a separate outlet valve (such as versatile valve 3 (VV3)). Versatile valves (VV3 and/or VV4) may be used as outlet valves, automating collection of column flow through/washes for each parallel flowpath. In Figs. 1-4, ultraviolet (UV), conductivity, and pH detectors are labeled “UV”, “Cond”, and “pH”, respectively. In some embodiments, using a combination of ultraviolet (UV), conductivity, and pH detectors for monitoring purification processes allows for precise monitoring of purification and/or separation processes.

[0090] To allow for multi-step chromatography, designated outlet lines may be plumbed from the existing outlet valve into the sample, A, and/or B flowpaths prior to the column valves. This allows flow to be directed from an outlet line to another column valve on the system. Plumbing an outlet line into the injection valve and versatile valve 1 (VV1) provides access to any column/loop valve from the outlet valve (Fig. 3).

[0091] Programming and automation may be accomplished using suitable software, e.g ., Unicom 7.1 software. The Unicorn software uses phases which contain instructions for executing different operations, such as equilibration, wash, elution etc. The ability to customize instructions within a phase may be utilized to define the custom flowpath. Custom phases may be combined with available pre-defmed phases to allow for parallel chromatography.

[0092] Parallel purification was demonstrated by utilizing three pumps for sample loading and creating three independent flowpaths with the addition of four versatile valves and two loop valves as described above. A two-step antibody purification process consisting of Protein A capture to G-25 desalt was fully automated to ran in parallel resulting in a 2.5X time savings over sequential loading. Two chromatography scales were evaluated with this configuration for purification of 5mg to 120mg of protein where each flowpath was shown to have almost identical recovery. Automated system sanitization steps were built into the methods to ensure sample isolation and prevent contamination. Endotoxin content and cross-contamination were evaluated and shown to be well-controlled.

[0093] Midscale purification throughput was increased even more substantially using high flow Protein A capture resins and a custom robotic autosampler. Overall, a >4x increase in throughput was achieved over standard sequential purification. The custom robotic autosampler enabled automated line cleaning and conditioning, and the capability to queue up to 24 samples for parallel purification without user interaction. A user can place clarified cell culture on the robot deck, hit start and collect purified product from the refrigerated fraction collector. This results in significant time savings for purification scientists and minimizes training requirements for new users.

B. First Configuration with Two Parallel Flowpaths

[0094] In this embodiment, shown in Fig. 4, two pumps (A and B) are used to accomplish two-parallel-flowpath multi-step chromatography. A sample pump is not used in this configuration. In this configuration, one parallel flowpath originates from the A pump (A flowpath) and another parallel flowpath originates from the B pump (B flowpath). The flowpath originating from the B pump is constructed by plumbing the B pump into the sample port of the injection valve and plumbing the column valve into the loop of the injection valve, e.g ., as illustrated in Fig. 4. The A flowpath is modified using two versatile valves flanking a loop valve (shown as VV1 and VV2 in Fig. 4), put in the place of the standard column valve between the injection valve and the detector(s). The two versatile valves allow the loop valve to be bypassed pre-detection and put in-line post-detection to accomplish multi-step chromatography, e.g. , as illustrated in Fig. 4. The configuration allows the elution from the loop position of the injection valve (B flowpath) to bypass the loop valve, go through UV, pH and conductivity detection and then be applied to a column on the loop valve post-detection. An outlet line is plumbed into the manual injection port of the injection valve. This allows product from the loop valve to be applied to a second column in the loop of the injection valve. In some embodiments, five column positions are available on both the loop and column valves, enabling up to 5- step chromatography. [0095] Two additional X valves can be added to the A and B pumps to allow more buffers to be added to the system or more samples to be queued up for processing. For outlet collection on the B flowpath, an available sample inlet valve may be configured as a versatile valve. This allows 4 positions to be used for flowthrough collection. Any inlet valve can be configured in this manner for outlet collection. Multiple valves can be linked together for additional outlet lines if desired.

C. Configuration with Three Independent Flowpaths

[0096] The parallel chromatography systems and methods described herein may be used multi-step purification of a single sample. For example, all columns utilized in a multi-step process could be equilibrated in parallel. Each parallel flowpath may be run independently, meaning that run parameters such as columns, buffers, and flow rates can vary between the flowpaths. Flowthrough chromatography and in-line dilution may also be performed with this system, as described elsewhere herein.

D. Second Configuration with Two Parallel Flowpaths

[0097] In this embodiment, s parallel configuration was developed for with the intention of designing a cost effective, compact, modular unit where multiple systems could be combined to offer even more processing throughput. With this configuration the sample pump and system pump can load samples in parallel, or a staggered parallel operation can be performed using the sample pump with optional single injection autosampler. Multi-step processes can also be automated and the system retains the ability to form gradients with A and B system pumps.

[0098] Two parallel flow paths are created are created on the instrument with the addition of three versatile valves and a loop valve. In the default configuration, all flow paths remain unblocked to circumvent overpressure issues during startup. Sample lines can be used on both the sample and system inlet valves to accomplish parallel chromatography. Alternatively, the sample pump combined with an optional autosampler can access both the column valve and loop valve. This is accomplished by changing the injection valve position between sample pump load and direct inject respectively (Fig. 10). The A/B pumps can be run concurrently with the sample pump to operate the parallel flow path.

[0099] Multistep chromatography is accomplished using outlet lines (originating from the outlet valve) that can access both the column valve and loop valve. One outlet line is plumbed into the manual injection port of the injection valve and can access the column valve post detection (Fig. 11). Another outlet line is plumbed into VV1 and can access the loop valve (Fig. 12). A product peak from one column can be directed to a column on the opposing parallel flow path post detection. With 5 column positions available on both the column valve and loop valve up to 5 different column steps can be performed.

[0100] A timeline showing a staggered load strategy for the 2-step Protein A capture (5ml MabSelect PCC, 450cm/hr flow rate) to HiPrep Desalt, as described elsewhere herein, is shown in Fig. 13. Despite the elution step requiring access to both flow paths the method approaches a 2X increase in throughput by taking advantage of the systems parallel capabilities. For a 250ml Protein A load each two-step run can be completed in approximately 30 minutes. Combined with a Teledyne ASX-560 autosampler with 21x250ml sample capacity the system can process and formulate 21 antibodies at approximately 50mg scale in less than 11 hours without user interaction.

II. Product Manipulation/In-line Dilution

[0101] A convenient way to manipulate the product buffer matrix for a subsequent load is to use a desalting column. A desalting column may be added to each column/loop valve so that any column on the system can be loaded directly from a desalt column. The system has the capability to perform in-line dilution if desired. The B flowpath may be utilized in combination with pump A to perform an in-line dilution, pH adjustment, and the like. When versatile valve 1 is in position 1 -4/2-3 pump B is directed to its dedicated flowpath and an outlet line can be directed to the A/B mixing T. If loop 2 elution is directed to versatile valve 1 then turning on pump A will result in an in-line dilution/adjustment. The diluted product can then be directed to either loop 1 or column 1 depending on the injection valve position. III. Flowthrough Chromatography

[0102] Flowthrough chromatography may be accomplished between column valve 1 and loop valve 1. Column valve 1 and loop valve 1 may both be placed in-line where flow through from loop valve 1 is directed through the detection flowpath with fractionation capability. For example, a user could perform Protein A capture on loop valve 2 (B flowpath). The Protein A elution may be directed to a G-25 column on the column valve (injection loop). Flow from the G-25 column may then be directed to a Q membrane on loop valve 1 where the product goes through system detection and can be directed to another column or fraction collection.

IV. Reverse Flow of Inlet Lines

[0103] Inlet lines that are routed through the robot may be longer than standard inlet lines and can air lock the pumps. In some embodiments, reverse flowing the lines for sanitization and conditioning was advantageous to priming the lines with the pumps. Reverse flow may be accomplished by integrating Y connectors between all three inlets (S, A and B) and their respective pumps. An outlet line originating from the outlet valve may then be introduced into each flowpath between the pumps and their respective inlet valves through the Y connector. Each flowpath may be blocked to accomplish reverse flow. This may be accomplished with the versatile valves that are introduced into the A and B flowpaths and for the Sample flowpath by blocking one position on the loop valve. Flow may then be directed to the blocked flowpath when a parallel flowpath is run through the outlet valve to the Y connector. When flow is directed through the Y connector, it is forced through the inlet valve rather than through the standard (blocked) flowpath and reverse flow is accomplished.

V. Parallel Autosampler

[0104] A robotic autosampler allows a user to queue up multiple samples and automate sample line cleaning and conditioning, significantly expanding purification throughput capability. The deck may be set up to load up to 12x500 ml samples with flow through collection, or 24 samples without flow through collection. If more capacity is required a robot with a larger deck can be constructed. The robot allows a user to place samples on the deck and queue up additional methods even when the system is in use. [0105] A parallel robotic autosampler may further increase the throughput capabilities of parallel systems. The parallel autosampler is a three-axis robot built with an aluminum bar stock frame. Stepper motors may be used to control movement in three directions: X, Y and Z. Three sippers (inlets) and three outlet lines were integrated into the Z axis head to allow for simultaneous loading and flow through collection. This capability allows for a single sample line and outlet line from each flowpath to be dedicated to sample loading and flow-through (FT) collection. The deck may be constructed of kaizen foam or 0.5 inch PVC with 2 ¼ diameter holes cut to hold 0.5L Nalgene^® wide mouth bottles (Thermo Fisher Scientific). Deck size, bottle size, and sipper head can be customized for each specific application. Although the exemplary configuration has three sippers for sample loading from a three-parallel system, a robot can be designed to work with multiple systems. Sanitization and conditioning of the sample lines associated with the robot may be performed by reverse flowing the lines with respective buffers. The autosampler may be connected to the system through a control unit ( e.g ., CU950 control unit from GE Healthcare). The stepper motors may be controlled with a microcontroller (such as an Arduino board from Arduino, LLC), programmed to recognize the digital signal from the chromatography system to advance the robot position.

VI. Column Loading

[0106] Column loading may be performed in parallel utilizing the air detection capabilities of the sample, A and B pumps. Typically, loading is automated with a watch command for air on the sample pump. Although it may be typical for this function to be used on the sample pump, each pump may have the same capability to watch for air detection. In some embodiments, the method uses separate blocks to watch for air on each pump simultaneously. When air is detected, the pump pauses and holds for the other pump/pumps to detect air. Once all pumps are triggered for air detection, the block continues and the method ends. VII. Elution

[0107] The parallel configuration described herein allows a user to perform three multi-step purifications in parallel as well as queue up multiple runs for increased throughput. In some embodiments, a single detector (or a single set of detectors) and a single fraction collector are utilized. Therefore only a single flowpath is directed to the detector(s) at any one time while the other flowpaths are directed to a dedicated outlet valve. For the methods described herein, steps such as conditioning, loading, washing, strip and final equilibration may be run in parallel, however the column elution steps are typically run sequentially. Elution peaks are directed to a HiPrep™ desalt column for buffer exchange through the outlet valve. In some embodiments, peak volumes are limited to the capacity of the column ( e.g ., a G-25 column) to provide complete buffer exchange. The column(s) are then eluted with peak detection, where peaks were directed to the fraction collector.

VIII. Recovery

[0108] A comparison of product recovery at two different chromatography scales was evaluated for each flowpath. The first scale was a 1ml HiTrap™ MabSelect™ SuRe™ capture followed by two 5ml HiTrap™ Desalt columns connected in series. The second scale was a 5ml HiTrap™ MabSelect™ SuRe™ capture followed by a 50ml HiTrap™ G- 25. A 5g to 25 g/L load was targeted on MabSelect™ SuRe™ at both scales. Clarified cell culture medium titer was measured at 0.196g/L using the Protein A HPLC titer method.

[0109] All scales and column loads evaluated resulted in low degree of flowpath variation. Difference in recovery between flowpaths was 4% or less for both 1ml and 5ml chromatography scales (Table 1). Peak shape and volume recovery was consistent between flowpaths (Fig. 6). Total holdup volume varied slightly between different flowpaths but minimal product volume and concentration differences were observed (<15%). Table 1. Comparison of flowpath recovery at different chromatography scales

[0110] A noticeably higher recovery was achieved with a 12.7g/L load on the 5.0ml HiTrap™ MabSelect™ SuRe™ column with a lmin residence time. With the 1.0ml HiTrap™ column, smaller elution volumes and small volume losses may result in a slightly lower recovery. The 5ml HiTrap™ MabSelect™ SuRe™ column was also run with a 26g/L load which showed an 81% recovery across parallel flowpaths. The MabSelect™ SuRe™ column has a dynamic binding capacity at 10% breakthrough of 22.5g/L for MabSelect™ SuRe™ with a lminute residence time and 10cm bed height. Lower recovery at this scale (81%) observed may have been due to overloading the column. These results demonstrate consistency between the three flowpaths.

[0111] Fig. 6 shows an elution profile comparison for the three-parallel flowpath configuration. Chromatography scale shown is a 1.0ml HiTrap™ MabSelect™ SuRe™ followed by 2x 5.0ml HiTrap™ Desalt columns connected in series. Load volume = 120ml clarified cell culture. First peak is the Protein A elution peak which is directed to the G-25 column. Second peak is the G-25 desalting step which is directed to the fraction collector.

IX. Flowpath Isolation and Sanitization

[0112] Since custom flowpaths are utilized to achieve parallel loading and multi-step chromatography, it is beneficial to ensure that all column loads remain isolated and that flowpaths common to different samples are sanitized between steps. A system clean-in- place (CIP) may be performed at the start of the method and precedes each elution step in order to prevent cross-contamination. The CIP may include a 0.2N NaOH sanitization and re-equilibration of the entire flowpath with appropriate buffer, including fraction collector and fraction collector accumulator as well as the outlet lines which loop to the column valves. Other inorganic bases (e.g., KOH) and other inorganic base concentrations can be used for sanitization, for example, 0.05 - 2N NaOH.

[0113] In order to ensure sample isolation, parallel chromatography was performed with three different antibodies. The three samples produced by parallel purification were analyzed for cross-contamination using RP-LCMS. The intact molecular weights of samples 1, 2, and 3 were confirmed as 150,112 Da, 147,800 Da, and 147,792 Da, respectively. The lower abundance peaks that were considered to be possible cross contamination between the purification flowpaths were identified to be free cysteine and disulfide scrambling forms of the main peaks (Fig. 8 and Table 2). No detectable level of cross-contamination was observed.

Table 2. Deconvoluted mass of three different antibodies purified in parallel

*ND - No contaminating sample detected.

X. Endotoxin Measurement

[0114] Endotoxin contamination can be a problem for higher throughput systems handling multiple samples with complex flowpaths. System sanitization steps built into the purification methods typically help to reduce or eliminate the chance for sample contamination. These methods can provide multiple users confidence that the system is in a ready-to-use state and that the risk for endotoxin contamination is low. No contamination event after processing hundreds of samples has been observed using the systems and methods described herein. Endotoxin data for a set of 27 monoclonal antibodies (mAbs) consecutively purified using parallel chromatography are shown in Table 2. A majority of values fell significantly below 1 EU/mg product (Table 3). No correlation was observed between endotoxin level and expression titer or total amount of product recovered through purification.

Table 3. Endotoxin content of 27 antibodies purified using parallel chromatography

* For these expression lots, there was difficulty with DNA purification/expression and they exhibited slightly elevated endotoxin levels.

XI. High-Flow Protein A Capture

[0115] Mab Select™ SuRe™, MabSelect™ SuRe™ PCC, MabSelect™ PrismA, Poros™ MabCapture™ A Select and Purolite Praestro^® Jetted A50 were compared to determine which resin would have a favorable dynamic binding capacity at high flow rates and short residence times. Three of the resins evaluated showed dynamic binding capacity >20g/L at a 0.3 minute residence time (Fig. 8). Capture efficiency of these resins run at 450cm/hr was found to be equivalent to MabSelect SuRe run at 150cm/hr in the 1ml HiTrap™ format. MabSelect™ SuRe™ PCC was used for parallel chromatography, showing high binding capacities at both 0.3 and 1 minute residence times.

[0116] Utilizing the autosampler and MabSelect™ SuRe™ PCC allows for doubling the instrument capacity from 12 runs to 24+ runs over a 24 hour period. The combination of parallel loading, automation, and high flow capture resins provides sufficient capacity to accomplish over 100 midscale purifications in one week with a single system. The approach is also highly cost-effective utilizing and cycling only three columns required for the three parallel runs on the system. Fig. 8 shows dynamic binding capacity of Protein A capture resins compared at 0.3 minute and 1 minute residence times.

XII. Increase in Throughput

[0117] The herein-described parallel chromatography configuration achieves a 2.5X increase in throughput plus time savings accomplished through automation of multi-step chromatography processes. With the instrument configuration, two lines from each inlet valve are dedicated to sample application allowing for 6 runs to be completed prior to manual sample line cleaning. Automated sample loading is accomplished using a custom robotic autosampler allowing up to 24 samples to be queued up for purification. Combining the parallel chromatography system with high-flow resin technologies results in a > 4x increase in throughput over traditional sequential protein purification. Fig. 9 illustrates the timeframe for processing three antibodies using sequential vs. parallel purification.

XIII. Analytes

[0118] The systems and techniques described herein are useful for separation (e.g., purification) of analytes such as proteins, peptides, antibodies (including antibody fragments), nucleic acids, and other biomolecules.

XIV. Exemplary Protocols

A. Expression of monoclonal antibodies

[0119] Expi293™ cells (Thermo Fisher Scientific) were transfected with DNA encoding a human IgG and cultured according to the manufacturer’s recommendations. On the day of transfection, cells were adjusted to l-3xl0⁶ cells/ml with fresh medium in the volume appropriate for the flask size being used. Expifectamine™ 293 was added to Opti-MEM^® medium and incubated for 5 minutes at room temperature. Heavy and light chain DNA were then combined and mixed with the diluted Expifectamine™. The DNA- Expifectamine complex was incubated for 10-20 minutes at room temperature and then added to the Expi293™ cells at a final DNA concentration of 0.5-lmg/L. Transfected cells expressing IgG were grown at 37°C, 8% CO2 and 125 RPM. After 24 hours, the cells were fed with enhancers 1 and 2 (Thermo Fisher Scientific) at 5% of total volume. Culture was then harvested on day 6 or when viability drops below 80%. Clarification was accomplished by centrifugation or depth filtration followed by 0.2um filtration. Conditioned medium was stored at 4°C prior to purification.

B. Purification of recombinant monoclonal antibodies

[0120] An antibody purification process utilizing MabSelect™ SuRe™ capture followed by G-25 desalting (GE Healthcare Life Sciences) was carried out using the systems and methods described herein. Two different chromatography scales were automated. Small scale purifications between 5mg and 20mg were performed using a lml HiTrap™ MabSelect™ SuRe™ column followed by two 5ml HiTrap™ desalt columns connected in tandem. For scales in the range of lOmg to 120mg purification was performed with a 5ml HiTrap™ MabSelect™ SuRe™ column followed by a 50ml Hi Scale™ desalt column.

[0121] Protein A columns were equilibrated in parallel with 8 column volumes (CVs) of Dulbecco’s Phosphate Buffer Saline (lx) (-) calcium chloride, (-) magnesium chloride (Life Technologies) (PBS) followed by parallel equilibration of G-25 columns with 4 CVs of PBS. Conditioned medium was loaded in parallel at 150cm/hr and followed by a 10 CV wash with PBS. Elution of each column was performed in series with lOOmM glycine, 150mM NaCl pH 3.0. The MabSelect™ SuRe™ elution was monitored using a watch command for a specific 280nm absorbance dependent on the chromatography scale. When the watch criteria were met the product peak was directed to a G-25 column. Total G-25 load was restricted to 30% of the G-25 column volume to achieve efficient buffer exchange. Product was then eluted from the G-25 column with 1 CV of PBS.

Flow from the G-25 column was directed through the UV, conductivity, pH detectors and to the fraction collector. G-25 elution was monitored with a watch command for 280nm absorbance and the product peak was collected. Protein concentration was determined using an off-line absorbance measurement at 280nm and calculated using the molar extinction coefficient.

C. Reversed-Phased Liquid Chromatography and Mass Spectromotry

[0122] Reversed-phase (RP) liquid chromatography of intact antibodies was performed on an Agilent 1290 HPLC system (Agilent Technologies, Santa Clara, CA) with a SePax Proteomix^® RP-1000 column (2.1 x 150mm) (SePax Technologies, Newark, DE). The column temperature was maintained at 75°C and the UV absorbance was monitored at 280nm. lOug of sample was injected onto the column equilibrated with a minimum 10 CVs of mobile phase A (0.1% trifluoroacetic acid in water). The flowrate was maintained at 0.25 mL/min. Separation was performed using a gradient from 70% mobile phase A:30% mobile phase B (0.1% trifluoroacetic acid in acetonitrile) to 50% mobile phase A:50 % mobile phase B over 30 minutes. The gradient was then increased from 50% mobile phase A:50% mobile phase B to 5 % mobile phase A:95% mobile phase B over 2 minutes and then held at 5% mobile phase A: 95% mobile phase B for an additional 3 minutes. Mass detection of the reversed-phase separated proteins was accomplished by directly introducing the output from the chromatographic column to the inlet of a Thermo Q-Exactive™ Mass Spectrometer (Thermo Fisher Scientific, Waltham, MA) equipped with an HESI source. The spectra were acquired using a Full MS method at a resolution setting of 17,500. Certain parameters were optimized as follows: spray voltage, 3.0kV; sheath gas flow rate, 25; S-lens RF level, 80; in-source CID, 80 ev; AGC target, le6; maximum injection time, 150 ms; microscans, 10. Full MS spectra were extracted from the total ion chromatograms (TICs) and deconvoluted using the ReSpect algorithm incorporated in Protein Deconvolution 3.0 software (Thermo Fisher Scientific, Bremen, Germany).

D. Endotoxin measurement

[0123] Endotoxin was measured using the Endosafe^® PTS reader (Charles River Laboratories) and Endoscan-V™ version 4.0 software. A PTS20 test cartridge with a 0.05-5 EU/ml sensitivity was used for the measurement. Each sample was diluted 1 : 10 in PBS giving a final sensitivity range of 0.5-50 EU/ml. The concentration of each diluted sample was recorded into the Endosafe^® software and 25 pL of the diluted sample was applied to each well of the test cartridge. Once prompted, testing was initiated and endotoxin levels in the sample were calculated and reported by the Endoscan-V™ software.

E. Protein A titer

[0124] The titer of mAb samples was determined by Protein A high performance liquid chromatography (ProA-HPLC) with UV detection at 280nm. Applied Biosystems™ POROS™ 20 micron Protein A Affinity Column, (Thermo Fisher Scientific, Waltham, MA) was used on an Agilent 1200 HPLC instrument, with mobile phase A as lOmM phosphate, 350mM NaCl, pH 7.0 and mobile phase B as lOmM phosphate, 350mM NaCl, pH 2.5. Harvested cell culture was centrifuged and filtered with a Pall 0.8/0.2 supor membrane syringe filter prior to loading onto the Protein A cartridge. After injection, the column was washed with 100% mobile phase A for 1.5 min at 2mL/min. Elution was performed with 100% mobile phase B over 2 minutes. A 5- point standard curve was generated by injecting increasing volumes of a known concentration mAb standard. The area under the curve was determined for each standard injection and plotted against the amount of standard injected. Linear regression analysis was performed on the plotted data with a resulting R² value >0.98. The injected amount of mAb sample was calculated from linear regression, y=mx+b, where: y is sample peak area, x is injected amount in pg, m is slope of the curve, and b is the y intercept of the curve. Concentration of sample was determined by the formula: concentration = (injected amount/injection volume).

F. Dynamic binding capacity of Protein A capture resins

[0125] Binding capacity experiments were performed using an IgGl monoclonal antibody expressed in recombinant Chinese Hamster Ovary (CHO) cells. HiTrap™ columns (1 ml) (GE Healthcare, bed height: 25 mm, diameter: 7 mm) packed with either MabSelect™ SuRe™, MabSelect™ PrismA, MabSelect™ PCC, Poros™ MabCapture™ A Select (Thermo Fisher Scientific), or Praestro^® Jetted A50 (Purolite Life Sciences) were equilibrated with 10 CV Dulbecco’s Phosphate Buffered Saline (DPBS, Thermo Fisher Scientific) washed with 10 CV DPBS and eluted with 100 mM glycine, 150 mM NaCl, pH 3.0. Neutralization was performed with a 1 :20 vokvol addition of 1M Tris pH 8. Column clean-in-place (CIP) was performed using 0.2M NaOH.

[0126] The dynamic binding capacity (DBC) at 10% breakthrough was determined for each Protein A resin. Conditioned IgGl medium was loaded onto the 1 ml columns at different flow rates to determine the impact of residence time on DBC. The DBC of each resin was determined at a residence time of 60 seconds and 20 seconds (1 and 0.3 ml/min respectively). Column flowthrough was collected in 0.5 ml fractions in a 96-well plate using the built-in fraction collector.

[0127] The DBC was determined by measuring the mAb concentration of the flowthrough fractions using a Protein A HPLC titer method with a standard curve generated with the purified protein of interest, and dividing by the titer of the load mAb determined using the same HPLC method. The percent breakthrough was determined for each fraction and graphed against the load density (mg mAb/ml resin).

Claims

What is claimed is:

1. A system for separation of analytes from a plurality of samples comprising:

(a) a plurality of pumps in liquid communication with an injection valve;

(b) a first versatile valve in liquid communication with one of the pumps;

(c) a (i) column valve and (ii) a column or loop valve, each in liquid communication with the injection valve;

(d) a column or loop valve in liquid communication with the first versatile valve;

(e) a second versatile valve in liquid communication with: (i) a column or loop valve in liquid communication with the injection valve; and (ii) a column or loop valve in liquid communication with the first versatile valve; and

(f) a detector for detection of analytes.

2. The system of claim 1, further comprising an outlet valve in liquid communication with the detector to direct flow to: (i) the injection valve, (ii) the first versatile valve, (iii) a flowthrough collector, (iv) a fraction collector, and/or (v) a waste collector.

3. The system of any one the preceding claims, wherein the outlet valve is in a flowpath after the detector.

4. The system of any one of the preceding claims, further comprising a fraction collector connected to the outlet valve.

5. The system of any one of the preceding claims, wherein a column, loop, and/or versatile valve is in liquid communication with a column.

6. The system of any one of the preceding claims, comprising three or more pumps.

7. The system of any one of the preceding claims, wherein a plurality of pumps are in liquid communication with a plurality of intake lines.

8. The system of any one of the preceding claims, wherein each pump is connected to an autosampler.

9. The system of any one of the preceding claims, wherein the autosampler is configured to sample at least 2 or at least 3 samples at the same time.

10. The system of any one of the preceding claims, wherein the detector comprises a UV, a conductivity and/or a pH detector.

11. The system of any one of the preceding claims, further comprising a mixer for blending intakes from a plurality of pumps or from a pump and a versatile valve, wherein the mixer is in liquid communication with the injection valve.

12. The system of any one of the preceding claims, wherein the analytes are biomolecules.

13. The system of any one of the preceding claims, wherein each column is independently an affinity capture, ion exchange, size exclusion (e.g., gel filtration), hydrophobic interaction chromatography, and/or mixed mode chromatography column.

14. The system of any one of the preceding claims, further comprising a waste collector that accepts flow from the injection valve and/or a versatile valve.

15. The system of any one of the preceding claims, wherein the waste flow is routed to the autosampler.

16. The system of any one of the preceding claims, wherein each pump is in liquid communication with a sample container and/or a cleaning solution.

17. The system of any one of the preceding claims, wherein the cleaning solution is an inorganic base (e.g., NaOH).

18. A system for separation of analytes in a plurality of samples comprising:

(a) a first and a second parallel chromatographic flowpath capable of accepting jointly three or more samples;

(b) a first junction integrating the first and the second parallel chromatographic flowpath to provide a joint chromatographic flowpath;

(c) a detector for detecting analytes in the joint chromatographic flowpath; and

(d) a second junction capable of directing the analytes back into the first and/or second chromatographic flowpath.

19. A method for separation of analytes in a plurality of samples comprising:

(a) loading a first and a second sample onto a first and second column or loop valve through the same injection valve;

(b) loading a third sample onto a third column or loop valve through a versatile valve in parallel with step (a);

(c) allowing the first, second and third samples pass through a chromatography column on a column or loop valve;

(d) sequentially eluting the analytes of interest from the first, second, and third samples; and

(e) allowing the eluted analytes from the samples to pass through a detector sequentially.

20. The method of claim 19, further comprising allowing an outlet valve to direct flow to the: (i) injection valve, (ii) first versatile valve, (iii) flowthrough collector, (iv) fraction collector, and/or (v) waste collector.

21. The method of any one of the preceding claims, wherein the outlet valve is in a flowpath after the detector.

22. The method of any one of the preceding claims, further comprising collecting fractions with a fraction collector connected to the outlet valve.

23. The method of any one of the preceding claims, further comprising drawing a plurality of samples from a plurality of containers with an autosampler.

24. The method of any one of the preceding claims, wherein the autosampler draws at least 2 or at least 3 samples at the same time.

25. The method of any one of the preceding claims, wherein the loading in steps (a) and (b) is performed with 3 pumps.

26. The method of any one of the preceding claims, wherein the detector comprises a UV, a conductivity, and/or a pH detector.

27. The method of any one of the preceding claims, further comprising blending intake from a plurality of pumps or from a pump and a versatile valve.

28. The method of any one of the preceding claims, wherein the analytes are biomolecules.

29. The method of any one of the preceding claims, wherein each column is independently an affinity capture, ion exchange, size exclusion (e.g., gel filtration), hydrophobic interaction chromatography, and/or mixed mode chromatography column.

30. The method of any one of the preceding claims, further comprising collecting waste from the injection valve, and/or a versatile valve.

31. The method of any one of the preceding claims, further comprising routing the waste flow to the autosampler.

32. The method of any one of the preceding claims, further comprising cleaning sample lines and/or autosampler with a cleaning solution.

33. The method of any one of the preceding claims, wherein the cleaning solution is an inorganic base (e.g., NaOH).

34. A system for separation of analytes from a plurality of samples comprising:

(a) a plurality of pumps in liquid communication with an injection valve;

(b) a first column valve in liquid communication with the injection valve;

(c) a first versatile valve in liquid communication with the injection valve;

(e) a second versatile valve in liquid communication with the column or loop valve and the first versatile valve; and

(f) a detector for detection of analytes in liquid communication with the second versatile valve.

35. The system of claim 34, further comprising a first outlet valve in liquid communication with the injection valve and the detector, wherein the first outlet valve is configured to direct flow to: (i) the injection valve, (ii) the first versatile valve; (iii) a fraction collector and/or (iv) a flowthrough collector.

36. The system of any one of the preceding claims, wherein the first outlet valve is in a flowpath after the detector.

37. The system of any one of the preceding claims, further comprising a fraction collector connected to the outlet valve.

38. The system of any one of the preceding claims, wherein a plurality of column and/or loop valves are in liquid communication with a plurality of columns.

39. The system of any one of the preceding claims, comprising 2 pumps.

40. The system of any one of the preceding claims, wherein a plurality of pumps are in liquid communication with a plurality of intake lines.

41. The system of any one of the preceding claims, wherein each pump is connected to an autosampler.

42. The system of any one of the preceding claims, wherein the autosampler is configured to sample at least 2 samples at the same time.

43. The system of any one of the preceding claims, wherein the detector comprises a UV, a conductivity and/or a pH detector.

44. The system of any one of the preceding claims, further comprising a second outlet valve in liquid communication with the injection valve.

45. The system of any one of the preceding claims, wherein the second outlet valve is configured to direct flow to a flowthrough collector and/or a waste collector.

46. The system of any one of the preceding claims, wherein the analytes are biomolecules.

47. The system of any one of the preceding claims, wherein each column is independently an affinity capture, ion exchange, size exclusion (e.g., gel filtration), hydrophobic interaction chromatography and/or mixed mode chromatography column.

48. The system of any one of the preceding claims, further comprising a waste collector that accepts flow from the injection valve and/or a versatile valve.

49. The system of any one of the preceding claims, wherein the waste flow is routed to the autosampler.

50. The system of any one of the preceding claims, wherein each pump is in liquid communication with a sample container and/or a cleaning solution.

51. The system of any one of the preceding claims, wherein the cleaning solution is an inorganic base (e.g., NaOH).

52. A method for separation of analytes in a plurality of samples comprising:

(a) loading three or more samples in parallel into a first and a second chromatographic flowpath;

(b) conducting multi-step chromatography in the first and second chromatographic flowpaths;

(c) integrating the first chromatographic flowpath into the second chromatographic flowpath to provide a joint flowpath; and

(d) detecting analytes in the joint flowpath.