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WO2019028172A1 - Capsules de filtration et de chromatographie et leurs procédés d'utilisation - Google Patents

Capsules de filtration et de chromatographie et leurs procédés d'utilisation Download PDF

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Publication number
WO2019028172A1
WO2019028172A1 PCT/US2018/044863 US2018044863W WO2019028172A1 WO 2019028172 A1 WO2019028172 A1 WO 2019028172A1 US 2018044863 W US2018044863 W US 2018044863W WO 2019028172 A1 WO2019028172 A1 WO 2019028172A1
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WO
WIPO (PCT)
Prior art keywords
pod
hours
minutes
kit
bottom end
Prior art date
Application number
PCT/US2018/044863
Other languages
English (en)
Inventor
William K. MCCONAUGHY
Stephen Robin HAMILTON
Original Assignee
Compass Therapeutics Llc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Compass Therapeutics Llc filed Critical Compass Therapeutics Llc
Priority to CN201880065040.0A priority Critical patent/CN111182951A/zh
Priority to JP2020505394A priority patent/JP2020529605A/ja
Publication of WO2019028172A1 publication Critical patent/WO2019028172A1/fr

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Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/10—Selective adsorption, e.g. chromatography characterised by constructional or operational features
    • B01D15/22—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to the construction of the column
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/10—Selective adsorption, e.g. chromatography characterised by constructional or operational features
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/10—Selective adsorption, e.g. chromatography characterised by constructional or operational features
    • B01D15/14—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to the introduction of the feed to the apparatus
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
    • 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
    • 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
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • 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
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
    • 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
    • C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
    • 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
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
    • C12M47/12—Purification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M99/00—Subject matter not otherwise provided for in other groups of this subclass
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/60—Construction of the column
    • G01N30/6004—Construction of the column end pieces

Definitions

  • the present disclosure relates to the field of biotechnology, and more specifically, to the capture and purification of recombinant proteins.
  • the present disclosure is based, at least in part, on the generation of novel filtration and chromatography pods for capturing and purifying recombinant proteins from a liquid cell culture.
  • the filtration and chromatography pods offer several benefits over previous methods of purifying recombinant proteins, including, e.g., speed, ease of use, enhanced protein stability, eliminating the need for multiple culture vessels and other consumables, which allows for rapid capture and purification of the recombinant protein of interest.
  • Other advantages of the presently claimed invention are described herein.
  • filtration and chromatography pods that include a container being formed by a wall and having a top end and a bottom end, where the wall forms a seal with the bottom end, and one or more of the wall, the top end, and the bottom end include at least one area covered by a semi-porous or porous material that allows for a liquid to reversibly flow from the exterior of the pod into the interior of the pod.
  • the bottom end includes at least one area covered by the semi-porous or porous material.
  • the semi-porous or porous material is characterized by one or both of: (a) not allowing a cell to flow from the exterior of the pod into the interior of the pod and (b) retaining or substantially retaining the resin within the interior of the pod. In some embodiments, the semi- porous or porous material does not allow a cell to flow from the exterior of the pod into the interior of the pod. In some embodiments, the semi-porous or porous material retaining or substantially retaining the resin within the interior of the pod.
  • the semi-porous or porous material is characterized by not allowing a cell to flow from the exterior of the pod into the interior of the pod and retaining or substantially retaining the resin within the interior of the pod.
  • the cell is a mammalian cell.
  • the cell is an insect cell or a yeast cell.
  • the cell is a bacterial cell (e.g., an E. coli cell).
  • the cell is not a bacterial cell.
  • the semi-porous or porous material retains or substantially retains the resin within the interior of pod structure.
  • the material is a semi-permeable or porous membrane. In some embodiments of any of the pods described herein, the material is a semi-permeable or porous mesh. In some embodiments, the mesh is coated with a hydrophilic material or a hydrophobic material. In some embodiments of any of the pods described herein, the mesh is made of nylon or polyester. In some embodiments of any of the pods described herein, the material has a pore size of about 50 nm to about 100 ⁇ m. In some embodiments of any of the pods described herein, the material has a pore size of about 50 nm to about 50 ⁇ m.
  • the material has a pore size of about 100 nm to about 50 ⁇ m. In some embodiments of any of the pods described herein, the material has a pore size of about 10 ⁇ m to about 50 ⁇ m. In some embodiments of any of the pods described herein, the material has a pore size of about 20 ⁇ m to about 50 ⁇ m. In some embodiments of any of the pods described herein, the material has a pore size of about 100 nm to about 30 ⁇ m. In some embodiments of any of the pods described herein, the material has a pore size of about 500 nm to about 20 ⁇ m.
  • the material has a pore size of about 1 ⁇ m to about 10 ⁇ m. In some embodiments of any of the pods described herein, the material has a pore size of about 2 ⁇ m to about 5 ⁇ m.
  • the container has an interior volume of about 0.1 mL to about 500 mL. In some embodiments of any of the pods described herein, the container has an interior volume of about 0.1 mL to about 250 mL. In some embodiments of any of the pods described herein, the container has an interior volume of about 0.1 mL to about 100 mL. In some embodiments of any of the pods described herein, the bottom end includes at least one area covered by the material. In some embodiments of any of the pods described herein, the wall includes at least one area covered by the material. In some embodiments of any of the pods described herein, the bottom end and the wall each include at least one area covered by the material.
  • the top end includes at least one area covered by the material. In some embodiments of any of the pods described herein, the top end and the bottom end each include at least one area covered by the material. In some embodiments of any of the pods described herein, each of the at least one area has a surface area of about 1 mm 2 to about 25 cm 2 . In some embodiments of any of the pods described herein, each of the at least one area has a surface area of about 1 mm 2 to about 20 cm 2 . In some embodiments of any of the pods described herein, each of the at least one area has a surface area of about 1 mm 2 to about 10 cm 2 .
  • each of the at least one area has a surface area of about 1 mm 2 to about 5 cm 2 . In some embodiments of any of the pods described herein, each of the at least one area has a surface area of about 1 mm 2 and 2.5 cm 2 . In some embodiments of any of the pods described herein, each of the at least one area has a surface area of about 1 mm 2 and about 1 cm 2 .
  • the bottom end is flat. In some embodiments, the bottom end has a hemispherical shape. In some embodiments of any of the pods described herein, the bottom end has a conical shape. In some embodiments of any of the pods described herein, the wall has a cylindrical shape. In some embodiments of any of the pods described herein, the wall has a conical shape. In some embodiments of any of the pods described herein, the wall has a spherical shape. In some embodiments of any of the pods described herein, the wall has a hemispherical shape. In some embodiments of any of the pods described herein, the wall has an hourglass shape.
  • the pod further includes a chromatography resin disposed in the interior of the container.
  • the chromatography resin is a commercial chromatography resin.
  • the chromatography resin is an affinity chromatography resin.
  • the affinity chromatography resin is a protein A resin, a protein G resin, a protein L resin, a nickel resin, or a magnetic bead resin.
  • the chromatography resin has a volume of about 0.1 mL to about 500 mL.
  • the chromatography resin has a volume of about 0.1 mL to about 250 mL. In some embodiments of any of the pods described herein, the chromatography resin has a volume of about 0.1 mL to about 100 mL. In some embodiments of any of the pods described herein, the
  • the chromatography resin has a volume of about 0.1 mL to about 50 mL. In some embodiments of any of the pods described herein, the chromatography resin has a sterility assurance level of about 1 x 10 -6 to about 1 x 10 -10 .
  • kits that include any of the pods described herein. Some embodiments of these kits can further include instructions for using any of the pods described herein. Some embodiments of any of the kits provided herein can further include a means for stably positioning the pod in the interior of a culture vessel.
  • the culture vessel is shake flask, a shake tube, a bioreactor, or a multi-well plate.
  • the bioreactor is a perfusion bioreactor. In some embodiments of any of the kits described herein, the bioreactor is a feed batch bioreactor.
  • kits that include: (a) any of the pods described herein; and (b) one or more panels that can be secured over each of the one or more areas in the wall, such that leakage of a fluid from the interior of the pod to the exterior of the pod through the wall is prevented.
  • kits that include: (a) any of the pods described herein; and; (b) an outer sleeve that can be secured over the pod in order to cover the at least one area in the wall, such that leakage of a fluid from the interior of the pod to the exterior of the pod through the at least one area in the wall is prevented.
  • the sleeve is an empty chromatography column.
  • kits that include: (a) a pod of any of the pods described herein; and (b) one or both of: an elution outlet that can be stably fixed onto the bottom end; and a buffer reservoir that can be stably fixed onto the top end.
  • the kit includes the pod and the elution outlet. In some embodiments of any of the kits described herein, the kit includes the pod and the buffer reservoir. In some embodiments of any of the kits described herein, the kit includes the pod, the elution outlet, and the buffer reservoir. In some embodiments of any of the kits provided herein, the elution outlet is a funnel. In some embodiments of any of the kits provided herein, the elution outlet includes a flat surface having a port. In some embodiments of any of the kits provided herein, the elution outlet is configured to allow for regulation of the flow of a fluid through the elution outlet.
  • the elution outlet is not configured to allow for regulation of the flow of a fluid through the elution outlet.
  • the elution outlet can be screwed or clicked-on to the pod.
  • the elution outlet can be sealed to the pod using one or more (e.g., two or three) O-rings (e.g., fixed to the pod or the elution outlet).
  • kits that include: (a) any of the pods described herein; and (b) a movable outer sleeve connected to the wall, such that rotation of the movable outer sleeve can cover the at least one area in the wall, such that leakage of a fluid from the interior of the pod to the exterior of the pod through the at least one area in the wall is prevented.
  • the kit further includes a means for stably positioning the pod in the interior of a culture vessel.
  • kits that include: (a) any of the pods described herein; and (b) a means for stably positioning the long axis of the pod in a horizontal position in the interior of a culture vessel.
  • a means for stably positioning the the pod in an interior of a culture vessel are shown in Figures 11-19, 36, and 37.
  • kits that include (a) any of the pods described herein; (b) an inlet system having a top end, an elongated cylindrical body including a fluid conduit, and a bottom end, where the top end of the inlet system includes a fluid inlet and is configured for stable attachment to the top end of the pod and, when stably connected to the pod, allows for flow of a fluid into the pod through the fluid inlet and the fluid conduit, and out of the bottom end of the inlet system, and the elongated cylindrical body of the inlet system is configured to fit inside the wall of the pod; an (c) an outlet system including a fluid outlet that is configured for stable attachment to the bottom end of the pod, and when stably connected to the bottom end of the pod, allows for flow of a fluid out of the pod through the fluid outlet.
  • the top end of the inlet system and the top end of the pod include threading that allows for the stable attachment of the top end of the pod to the top end of the inlet system.
  • the threading in the top end of the inlet system or the threading in the top end of the pod comprises an O- ring.
  • the pod contains a chromatography resin (e.g., any of the exemplary types of chromatography resin described herein); and the inlet system and the pod are configured such that, when the inlet system and the pod are stably attached, the bottom end of the inlet system is proximal to the chromatography resin.
  • the interior surface of the pod and the bottom end of the inlet system are configured to allow for stable attachment of the pod and the bottom end of the inlet system.
  • the interior surface of the pod and the bottom end of the inlet system include threading that allows for stable attachment of the pod and the bottom end of the inlet system.
  • the threading in the interior surface of the pod or the threading in the bottom end of the inlet system includes an O-ring.
  • the outlet system and the bottom end of the pod include threading that allows for stable attachment of the outlet system to the bottom end of the pod.
  • the threading in the outlet system or the threading in the bottom end of the pod includes an O-ring.
  • the fluid inlet is capable of attachment to tubing.
  • the fluid outlet is capable of attachment to tubing.
  • Some embodiments of these kits further include: (d) a means for stably positioning the long axis of the pod in a horizontal position in the interior of a culture vessel (e.g., any of the culture vessels described herein).
  • Some embodiments of these kits further include: (d) a mean for stably positioning the long axis of the pod in a vertical position in the interior of a culture vessel (e.g., any of the culture vessels described herein).
  • Also provided herein are methods of capturing a recombinant protein produced by a liquid cell culture that include contacting any of the pods of described herein, with a liquid cell culture for a period of time, where the contacting step results in at least a portion of the at least one area contacting the liquid cell culture, and the contacting is performed under conditions that result in the capture of the recombinant protein by the chromatography resin.
  • the liquid cell culture is disposed in a culture vessel.
  • Also provided herein are methods of purifying a recombinant protein produced by a liquid cell culture that include: (a) contacting any of the pods of described herein, with a liquid cell culture for a period of time, where the contacting step results in at least a portion of the at least one area contacting the liquid cell culture, and the contacting is performed under conditions that result in the capture of the recombinant protein by the chromatography resin; and (b) eluting the recombinant protein from the chromatography resin after the period of time, thereby purifying the recombinant protein.
  • the method further includes, after (b), (c) performing one or more additional unit operations on the recombinant protein. In some embodiments of any of the methods provided herein, the method further includes, after (c), formulating the recombinant protein into a recombinant protein drug product. In some embodiments of any of the methods provided herein, the method further includes, washing the chromatography resin between step (a) and step (b). In some embodiments of any of the methods described herein, the contacting is performed under sterile conditions.
  • the period of time is about 5 minutes to about 2 weeks. In some embodiments of any of the methods described herein, the period of time is about 5 minutes to about 168 hours. In some embodiments of any of the methods described herein, the period of time is about 5 minutes to about 96 hours. In some embodiments of any of the methods described herein, the period of time is about 5 minutes to about 48 hours. In some embodiments of any of the methods described herein, the period of time is about 5 minutes to about 24 hours. In some embodiments of any of the methods described herein, the period of time is about 5 minutes to about 12 hours. In some embodiments of any of the methods described herein, the period of time occurs when the liquid cell culture is in its exponential growth phase.
  • the period of time occurs when the liquid cell culture is in its stationary growth phase. In some embodiments, the contacting can occur when a liquid cell culture is in its death phase. In some embodiments of any of the methods described herein, at least 30% of the total surface area of the at least one area is in contact with the liquid cell culture over the period of time in step (a). In some embodiments of any of the methods described herein, at least 50% of the total surface area of the at least one area is in contact with the liquid cell culture over the period of time in step (a).
  • At least 75% of the total surface area of the at least one area is in contact with the liquid cell culture over the period of time in step (a). In some embodiments of any of the methods described herein, at least 90% of the total surface area of the at least one area is in contact with the liquid cell culture over the period of time in step (a).
  • the liquid cell culture is disposed in a culture vessel.
  • the culture vessel is a plate (e.g., a multi-well plate), a shake tube, a shake flask, or a bioreactor.
  • the bioreactor is a perfusion bioreactor.
  • the bioreactor is a fed batch bioreactor.
  • the liquid cell culture includes a bacterial cell, a yeast cell, insect cell, a plant cell, or a mammalian cell.
  • the mammalian cell is a human cell.
  • the mammalian cell is a Chinese hamster ovary (CHO) cell or an NS0 cell.
  • the one or more additional unit operations is/are selected from the group of: viral inactivation, viral filtration, polishing, size-exclusion chromatography, cation exchange
  • the recombinant protein is a secreted protein.
  • the recombinant protein is any protein with or without a tag (e.g., a fluorescent tag, a radioisotope, a polyhistidine tag (HIS tag), a c-myc tag, a FLAG tag, a maltose- binding protein (MBP) tag, an albumin-binding protein (ABP) tag, a bacteriophage T7 epitope (T7-tag), a biotin-carboxy carrier protein (BCCP) tag, a bluetongue virus tag (B-tag), a calmodulin binding peptide tag, a cellulose binding domain tag, a chitin binding domain tag, a choline binding domain tag, a galactose-binding protein tag, a Glu-Glu epitope tag, a human influenza hemagglutinin tag,
  • a tag e.g., a fluorescent tag, a radioisotope
  • FIGs.1A to 1E show different exemplary pods and kits provided herein.
  • FIG.2 shows an exemplary embodiment of a pod provided herein, where the longitudinal axis of the pod is approximately horizontal when the pod is disposed in a bioreactor.
  • FIG.3 shows the exemplary pod in FIG.2, after it has been removed from a bioreactor and rotated such that the longitudinal axis is approximately vertical and its bottom end connected to an elution outlet.
  • the top end of the pod can be connected to a buffer reservoir (not shown).
  • FIG.4 is an exemplary depiction of a pod as described herein disposed in a vessel (e.g., a bioreactor) with a rod transiting through holes in the pod and in the side walls of the vessel.
  • a vessel e.g., a bioreactor
  • FIG.5 is a representative image of a sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis comparing the traditional batch capture method (Trad.) and the Capture & Purify method (Cap. & Pure) under reduced and non-reduced conditions.
  • E 1 ⁇ g of elution sample
  • FT flow through sample
  • W wash sample;
  • FIG.6 is a representative diagram comparing the experimental time line of the ⁇ KTA purification method, the traditional batch capture method and the Capture & Purify method, starting at day 4 post-transfection.
  • FIG.7 is a representative graph showing the high-performance liquid chromatography (HPLC) trace under four different conditions as outlined in Table 2.
  • FIG.8 is a representative graph showing the HPLC trace under four different conditions as outlined in Table 3.
  • FIG.9 is a representative graph showing the HPLC trace under two different conditions as outlined in Table 4.
  • FIGs.10-13 are two dimensional drawings of different exemplary means for stably positioning a pod in the interior of a culture vessel.
  • the symmetrical two- pronged claw can hold any of the exemplary pods described herein in a culture vessel.
  • FIGs.14 and 15 are two dimensional drawing of a part of an exemplary means for stably positioning a pod in the interior of a culture vessel; the center of the part is able to attach to a symmetrical two-pronged claw that can hold any of the exemplary pods described herein in a culture vessel (as depicted in Figures 16-19).
  • FIGs.16-19 are two dimensional drawings of different exemplary means for stably positioning a pod in the interior of a culture vessel.
  • the symmetrical two- pronged claw can hold any of the exemplary pods described herein in a culture vessel.
  • FIG.20 shows the overlaid elution profiles of 2 ⁇ g injections of protein samples on an analytical TOSOH Supergel TSK mAb HTP column from concurrent purifications using a device similar to the one shown in Figures 27 and 28 or using traditional batch capture and elution.
  • clarified supernatant containing antibodies was exposed to protein A resin before washing and eluting from the protein A resin after removal of the device or free floating resin from the clarified supernatant.
  • FIG.21 shows the elution profile of a 2 ⁇ g sample injected on an analytical TOSOH Supergel TSK mAb HTP column using the same clarified supernatant as Fig. 20 but using an AKTA system and proA resin contained within a packed column.
  • FIG.22 shows an exemplary pod provided herein.
  • FIG.23 shows an exemplary pod provided herein.
  • FIGs.24-28 show exemplary kits provided herien.
  • FIGs.29-31 show exemplary pods provided herein.
  • FIGs.32 and 33 show elution profiles from a device depicted in Figures 27 and 28 containing a Protein A resin, which was exposed to an unfiltered supernatant and eluted using an ⁇ KTA system.
  • FIG.34 shows an exemplary adapter provided herein.
  • FIG.35 shows an exemplary adapter provided herein attached to an exemplary pod described herein.
  • FIG.36 and 37 show exemplary means for stably positioning the pod in the interior of a culture vessel.
  • filtration and chromatography pods that include a container being formed by a wall and having a top end and a bottom end, where the wall forms a seal with the bottom end, and one or more of the wall, the top end, and the bottom end include at least one area covered by a semi-porous or porous material that allows for a liquid to reversibly flow from the exterior of the pod into the interior of the pod.
  • kits that include any of these pods, methods of using these pods to capture a recombinant protein produced in a liquid cell culture, and methods of using these pods to purify a recombinant protein produced by a liquid cell culture.
  • Non-limiting aspects of these pods, kits, and methods are described below, and can be used in any combination without limitation. Additional aspects of these pods, kits, and methods are known in the art.
  • the pods, kits, and methods provided herein provide for an unexpected decrease in the amount of time necessary to capture or purify a recombinant protein from a protein expression system (e.g., protein produced by a recombinant cell, such as a stable cell, expressing the recombinant protein).
  • a protein expression system e.g., protein produced by a recombinant cell, such as a stable cell, expressing the recombinant protein.
  • a recombinant protein can be captured or purifying within about 5 minutes to about 20 hours, about 5 minutes to about 18 hours, about 5 minutes to about 16 hours, about 5 minutes to about 14 hours, about 5 minutes to about 12 hours, about 5 minutes to about 10 hours, about 5 minutes to about 8 hours, about 5 minutes to about 6 hours, about 5 minutes to about 4 hours, about 5 minutes to about 3 hours, about 5 minutes to about 2 hours, about 5 minutes to about 1 hour, about 5 minutes to about 30 minutes, about 5 minutes to about 20 minutes, about 5 minutes to about 10 minutes, about 10 minutes to about 20 hours, about 10 minutes to about 18 hours, about 10 minutes to about 16 hours, about 10 minutes to about 14 hours, about 10 minutes to about 12 hours, about 10 minutes to about 10 hours, about 10 minutes to about 8 hours, about 10 minutes to about 6 hours, about 10 minutes to about 4 hours, about 10 minutes to about 3 hours, about 10 minutes to about 2 hours, about 10 minutes to about 1 hour, about 10 minutes to about 30 minutes, about 10 minutes to about 20 minutes, about 5 minutes to about 10 minutes
  • filtration and chromatography pods that include a container being formed by a wall and having a top end and a bottom end, where the wall forms a seal with the bottom end, and one or more of the wall, the top end, and the bottom end include at least one area covered by a semi-porous or porous material that allows for a liquid to reversibly flow from the exterior of the pod into the interior of the pod.
  • the semi-porous or porous material can be attached to the wall, top end, and/or bottom end of the pod by heating sealing.
  • Other means for attaching the semi- porous or porous material to the wall, top end, and/or bottom end of the pod are known in the art.
  • pods and kits provided herein are shown in Figures 1A to 1E, Figure 2, Figure 3, Figure 4, Figures 22-31, and Figure 35.
  • pod designs such as those set forth in Figure 1A, may optionally include one or more panels adapted to cover some or all of the surface of the sides of the pod which are formed by the semi-porous or porous material.
  • the semi-porous or porous material retains or substantially retains the resin within the pod structure.
  • the semi-porous or porous material is characterized by one or both of: (a) not allowing a cell to flow from the exterior of the pod into the interior of the pod and (b) retaining or substantially retaining the resin within the interior of the pod (c) is coated with a hydrophilic or hydrophobic coating.
  • the semi-porous or porous material does not allow a cell to flow from the exterior of the pod into the interior of the pod.
  • the semi-porous or porous material retaining or substantially retaining the resin within the interior of the pod.
  • the semi-porous or porous material is characterized by not allowing a cell to flow from the exterior of the pod into the interior of the pod and retaining or substantially retaining the resin within the interior of the pod.
  • the semi-porous or porous material is characterized by one or both of: (a) not allowing a mammalian cell to flow from the exterior of the pod into the interior of the pod and (b) retaining or substantially retaining the resin within the interior of the pod. In some examples of any of the pods described herein, the semi-porous or porous material does not allow a mammalian cell to reversibly flow from the exterior of the pod into the interior of the pod.
  • the semi-porous or porous material is characterized by one or both of: (a) not allowing an insect cell to flow from the exterior of the pod into the interior of the pod and (b) retaining or substantially retaining the resin within the interior of the pod. In some examples of any of the pods described herein, the semi-porous or porous material does not allow an insect cell or a yeast cell to reversibly flow from the exterior of the pod into the interior of the pod. In some examples of any of the pods described herein, the semi- porous or porous material does not allow a bacterial cell to reversibly flow from the exterior of the pod into the interior of the pod.
  • the material is a semi-permeable or porous membrane. In some examples, the material is a semi-permeable or porous mesh (e.g., a nylon mesh).
  • the material can be include (at least in part) one or more of the following: polyester, polyester with a hydrophilic coating, cellulose acetate, cellulose nitrate, polyethersulfone (PES), polystyrene, polypropylene, acrylic copolymer, polyvinyl chloride (PVC), polyvinylidene difluoride (PVDF), polytetrafluoroethylene (PTFE), and polycarbonate.
  • the material is a precision woven polymer (e.g., any precision woven polymer).
  • the material is a Sefar film (see, e.g., Sefar’s U.S. website, www.sefar.us).
  • the material is Petex mesh sold by Sefar.
  • the material has a pore size or average pore size of about 50 nm to about 50 ⁇ m, about 50 nm to about 45 ⁇ m, about 50 nm to about 40 ⁇ m, about 50 nm to about 35 ⁇ m, about 50 nm to about 30 ⁇ m, about 50 nm to about 25 ⁇ m, about 50 nm to about 20 ⁇ m, about 50 nm to about 18 ⁇ m, about 50 nm to about 16 ⁇ m, about 50 nm to about 14 ⁇ m, about 50 nm to about 12 ⁇ m, about 50 nm to about 10 ⁇ m, about 50 nm to about 8 ⁇ m, about 50 nm to about 6 ⁇ m, about 50 nm to about 5 ⁇ m, about 50 nm to about 4 ⁇ m, about 50 nm to about 3 ⁇ m, about 50 nm to about 2 ⁇ m, about 50 nm to about 1 ⁇ m
  • the material has a pore size or average pore size of about 30 ⁇ m to about 70 ⁇ m, about 30 ⁇ m to about 68 ⁇ m, about 30 ⁇ m to about 66 ⁇ m, about 30 ⁇ m to about 64 ⁇ m, about 30 ⁇ m to about 62 ⁇ m, about 30 ⁇ m to about 60 ⁇ m, about 30 ⁇ m to about 58 ⁇ m, about 30 ⁇ m to about 56 ⁇ m, about 30 ⁇ m to about 54 ⁇ m, about 30 ⁇ m to about 52 ⁇ m, about 30 ⁇ m to about 50 ⁇ m, about 30 ⁇ m to about 48 ⁇ m, about 30 ⁇ m to about 46 ⁇ m, about 30 ⁇ m to about 44 ⁇ m, about 30 ⁇ m to about 42 ⁇ m, about 30 ⁇ m to about 40 ⁇ m, about 30 ⁇ m to about 38 ⁇ m, about 30 ⁇ m to about 36 ⁇ m, about 30 ⁇ m to about 30 ⁇ m to about
  • one or more of the wall, the top end, and the bottom end include at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) area covered by a semi-porous or porous material that allows for a liquid to reversibly flow from the exterior of the pod into the interior of the pod.
  • the pod includes: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 areas covered by a semi-porous or porous material (that allows for a liquid to reversibly flow from the exterior of the pod into the interior of the pod) in the wall, and/or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 areas covered by a semi-porous or porous material (that allows for a liquid to reversibly flow from the exterior of the pod into the interior of the pod) in the bottom end, and/or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 areas covered by a semi-porous or porous material (that allows for a liquid to reversibly flow from the exterior of the pod into the interior of the pod) in the top end.
  • the wall includes at least one area covered by the material. In some examples of the pods described herein, the bottom end and the wall each include at least one area covered by the material. In some examples, the top end comprises at least one area covered by the material. In some examples, the top end and the bottom end each include at least one area covered by the material.
  • the total number of areas covered by a semi-porous or porous material (that allows for a liquid to reversibly flow from the exterior of the pod into the interior of the pod) in one or more of the wall, the top end, and the bottom end is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30.
  • the total number of areas covered by a semi-porous or porous material (that allows for a liquid to reversibly flow from the exterior of the pod into the interior of the pod) in one or more of the wall, the top end, and the bottom end is 1 to 30, 1 to 28, 1 to 26, 1 to 24, 1 to 22, 1 to 20, 1 to 18, 1 to 16, 1 to 14, 1 to 12, 1 to 10, 1 to 8, 1 to 6, 1 to 4, 1 to 3, 1 or 2, 2 to 30, 2 to 28, 2 to 26, 2 to 24, 2 to 22, 2 to 20, 2 to 18, 2 to 16, 2 to 14, 2 to 12, 2 to 10, 2 to 8, 2 to 6, 2 to 4, 2 or 3, 3 to 30, 3 to 28, 3 to 26, 3 to 24, 3 to 22, 3 to 20, 3 to 18, 3 to 16, 3 to 14, 3 to 12, 3 to 10, 3 to 8, 3 to 6, 3 or 4, 4 to 30, 4 to 28, 4 to 26, 4 to 24, 4 to 22, 4 to 20, 4 to 18, 4 to 16, 4 to 14, 4 to 12, 4 to 10, 4 to 8, 4 to 6, 6 to 30, 6 to 28, 6 to to
  • one or more of the at least one area covered by a semi-porous or porous material is circular, ellipsoidal, square, rectangular, elongated (e.g., an elongated line or rectangle), star-shaped, zig- zag shaped, or serpentine shaped.
  • one or more (e.g., each) of the at least one areas covered by the semi-porous or porous material has a surface area of about 0.5 mm 2 to about 60 cm 2 , about 0.5 mm 2 to about 58 cm 2 , about 0.5 mm 2 to about 56 cm 2 , about 0.5 mm 2 to about 54 cm 2 , about 0.5 mm 2 to about 52 cm 2 , about 0.5 mm 2 to about 50 cm 2 , about 0.5 mm 2 to about 48 cm 2 , about 0.5 mm 2 to about 46 cm 2 , about 0.5 mm 2 to about 44 cm 2 , about 0.5 mm 2 to about 42 cm 2 , about 0.5 mm 2 to about 40 cm 2 , about 0.5 mm 2 to about 38 cm 2 , about 0.5 mm 2 to about 36 cm 2 , about 0.5 mm 2 to about 34 cm 2 , about 0.5 mm 2 to about 32 cm 2 , 0.5 mm 2 to about 30 cm 2 , about 0.5 mm 2 to
  • the total sum of the surface area of the least one areas covered by the semi-porous or porous material is about 0.5 mm 2 to about 150 cm 2 , about 0.5 mm 2 to about 140 cm 2 , about 0.5 mm 2 to about 130 cm 2 , about 0.5 mm 2 to about 120 cm 2 , about 0.5 mm 2 to about 110 cm 2 , about 0.5 mm 2 to about 100 cm 2 , about 0.5 mm 2 to about 90 cm 2 , about 0.5 mm 2 to about 80 cm 2 , about 0.5 mm 2 to about 70 cm 2 , about 0.5 mm 2 to about 60 cm 2 , about 0.5 mm 2 to about 50 cm 2 , about 0.5 mm 2 to about 40 cm 2 , about 0.5 mm 2 to about 30 cm 2 , about 0.5 mm 2 to about 20 cm 2 , about 0.5 mm 2 to about 18 cm 2 , about 0.5 mm 2 to about 16 cm 2 , about 0.5 mm 2 to about
  • the percentage of the total surface area of the wall that is represented by at least one area covered by a semi-porous or porous material that allows for a liquid to reversibly flow from the exterior of the pod into the interior of the pod is about 0.1% to about 95%, about 0.1% to about 90%, about 0.1% to about 85%, about 0.1% to about 80%, about 0.1% to about 75%, about 0.1% to about 70%, about 0.1% to about 65%, about 0.1% to about 60%, about 0.1% to about 55%, about 0.1% to about 50%, about 0.1% to about 45%, about 0.1% to about 40%, about 0.1% to about 35%, about 0.1% to about 30%, about 0.1% to about 25%, about 0.1% to about 20%, about 0.1% to about 18%, about 0.1% to about 16%, about 0.1%
  • the percentage of the total surface area of the bottom end that is represented by at least one area covered by a semi-porous or porous material that allows for a liquid to reversibly flow from the exterior of the pod into the interior of the pod is about 1% to about 100%, about 1% to about 95%, about 1% to about 90%, about 1% to about 85%, about 1% to about 80%, about 1% to about 75%, about 1% to about 70%, about 1% to about 65%, about 1% to about 60%, about 1% to about 55%, about 1% to about 50%, about 1% to about 45%, about 1% to about 40%, about 1% to about 35%, about 1% to about 30%, about 1% to about 25%, about 1% to about 20%, about 1% to about 18%, about
  • the percentage of the total surface area of the top end that is represented by at least one area covered by a semi-porous or porous material that allows for a liquid to reversibly flow from the exterior of the pod into the interior of the pod is about 1% to about 100%, about 1% to about 95%, about 1% to about 90%, about 1% to about 85%, about 1% to about 80%, about 1% to about 75%, about 1% to about 70%, about 1% to about 65%, about 1% to about 60%, about 1% to about 55%, about 1% to about 50%, about 1% to about 45%, about 1% to about 40%, about 1% to about 35%, about 1% to about 30%, about 1% to about 25%, about 1% to about 20%, about 1% to about 18%, about
  • the bottom end of the container is flat, has a hemispherical shape, or has a conical shape.
  • the container has an interior volume of about 0.05 mL to about 1,000 mL, about 0.05 mL to about 950 mL, about 0.05 mL to about 900 mL, about 0.05 mL to about 850 mL, about 0.05 mL to about 800 mL, about 0.05 mL to about 750 mL, about 0.05 mL to about 700 mL, about 0.05 mL to about 650 mL, about 0.05 mL to about 600 mL, about 0.05 mL to about 550 mL, about 0.05 mL to about 500 mL, about 0.05 mL to about 450 mL, about 0.05 mL to about 400 mL, about 0.05 mL to about 350 mL, about 0.05 mL to about 300 mL, about 0.05 mL to about 250 mL, about 0.05 mL to about 200 mL, about 0.05 mL to about 400 mL, about 0.05 mL
  • the wall can be composed or composed in part of polyethylene (PE), polypropylene (PP), polystyrene (PS), polyvinyl chloride (PVC), an acrylic polymer, a polyester, silicone rubber, silicon, a polyurethane, a halogenated plastic (e.g., chlorinated polyethylene, chlorinated polyvinyl chloride, chlorosulfonated polyethylene, polychloroprene, fluorinated ethylene propylene), biaxially-oriented polypropylene (BOPP), a bioplastic, cellophane, an ethane-derived plastic, nylon, poly-lactic acid (PLA), polyethylene terephthalate (PE or PETE plastic code 1), polypropylene, polystyrene, polycarbonate, polyeurothene, or poly(methyl methacrylate).
  • PE polyethylene
  • PP polypropylene
  • PS polystyrene
  • PVC polyvinyl chloride
  • acrylic polymer e.g., chlorin
  • compositions that the wall can be composed of include: Bakelite (phenol-formaldehyde resin), Kevlar or Twaron (para-aramid), Kynar (polyvinylidene difluoride), Mylar (polyethylene terephthalate film), Neoprene (polychloroprene), nylon (polyamide 6,6), Orlon (polyacrylonitrile), Rilsan
  • the wall can be composed or composed in part of nylon, polypropylene, or polyvinyl alcohol (PVA).
  • the wall has a cylindrical shape, a conical shape, a rectangular shape, a square shape, a spherical shape, a hemispherical shape, an hour glass shape, or an ellipsoidal shape.
  • the pod further includes a chromatography resin disposed in the interior of the container.
  • the chromatography resin is a commercial chromatography resin. In other examples, the chromatography resin is not a commercial chromatography resin.
  • the chromatography resin can be any chromatography resin known in the art.
  • the chromatography resin can be an affinity chromatography resin (e.g., a protein A resin, a protein G resin, a protein L resin, a nickel resin, or a magnetic bead resin), a hydrophobic interaction chromatography resin, a mixed mode chromatography resin, a molecular sieve chromatography resin.
  • the resin is a specific affinity resin, e.g., a resin conjugated to an agent (e.g., an antibody, antibody fragment, peptide, nucleic acid, and the like) that is specific for a recombinant protein of interest.
  • the chromatography resin is an alkali-resistant resin.
  • the chromatography resin is a sterilized chromatography resin.
  • the chromatography resin disposed in the interior of the container can have a volume of about 0.1 mL to about 500 mL, about 0.1 mL to about 490 mL, about 0.1 mL to about 480 mL, about 0.1 mL to about 460 mL, about 0.1 mL to about 440 mL, about 0.1 mL to about 420 mL, about 0.1 mL to about 400 mL, about 0.1 mL to about 380 mL, about 0.1 mL to about 360 mL, about 0.1 mL to about 340 mL, about 0.1 mL to about 320 mL, about 0.1 mL to about 300 mL, about 0.1 mL to about 280 mL, about 0.1 mL to about 260 mL, about 0.1 mL to about 250 mL, about 0.1 mL to about 240 mL, about 0.1 mL to about 220 mL, about
  • the chromatography resin disposed in the pod has a sterility assurance level of about 1 x 10 -6 to about 1 x 10 -10 , about 1 x 10 -6 to about 0.5 x 10 -10 , about 1 x 10 -6 to about 1 x 10 -9 , about 1 x 10 -6 to about 0.5 x 10 -9 , about 1 x 10 -6 to about 1 x 10 -8 , about 1 x 10 -6 to about 0.5 x 10 -8 , about 1 x 10 -6 to about 1 x 10- 7 , about 1 x 10 -6 to about 0.5 x 10 -7 , about 0.5 x 10 -7 to about 1 x 10 -10 , about 0.5 x 10 -7 to about 0.5 x 10 -10 , about 0.5 x 10 -7 to about 0.5 x 10 -10 , about 0.5 x 10 -7 to about 1 x 10 -9 , about 0.5 x 10 -7 to about 0.5 x 10 -9 , about 0.5
  • kits that include any of the pods described herein. Also provided herein are kits that include (a) any of the pods described herein (where the pod includes one or more areas covered by a semi-porous or porous material that allows for a liquid to reversibly flow from the exterior of the pod into the interior of the pod, in the wall of the container), and (b) one or more panels that can be secured over each of the one or more areas in the wall, such that leakage of a fluid from the interior of the pod to the exterior of the pod through the wall is prevented.
  • kits that include (a) any of the pods described herein (where the pod includes one or more areas covered by a semi-porous or porous material that allows for a liquid to reversibly flow from the exterior of the pod into the interior of the pod, in the wall of the container), and (b) an outer sleeve that can be secured over the pod in order to cover the one or more areas in the wall, such that leakage of a fluid from the interior of the pod to the exterior of the pod through the at least one area in the wall is prevented.
  • kits that include (a) any of the pods described herein and (b) one or both of an elution outlet that can be stably fixed onto the bottom end, and a buffer reservoir that can be stably fixed onto the top end. Also provided herein are kits that include (a) any of the pods described herein (where the pod includes one or more areas covered by a semi-porous or porous material that allows for a liquid to reversibly flow from the exterior of the pod into the interior of the pod, in the wall of the container), and (b) a movable outer sleeve connected to the wall, such that rotation of the movable sleeve can cover the one or more areas in the wall, such that leakage of a fluid from the interior of the pod to the exterior of the pod through the at least one area in the wall is prevented. Also provided herein are kits that include (a) any of the pods described herein and (b) a means for stably positioning the long axis of the pod in a horizontal position in the
  • kits described herein can include a means for stably positioning the pod in the interior of a culture vessel.
  • Exemplary means for stably positioning the pod in the interior of a culture vessel can include a bracket, a brace, and an adapter that fits over an opening in a culture vessel and allows for the insertion of the pod into the culture vessel (without dropping the pod into the culture vessel.
  • the means for stably positioning the pod in the interior of a culture vessel is flexible and allows the pod to move with movement of the liquid culture without dropping into the culture vessel.
  • the means for stably positioning the pod in the interior of a culture vessel is rigid and allows the pod to withstand movement of the liquid culture without dropping into the culture vessel.
  • Non-limiting examples of means for stably positioning the pod in the interior of a culture vessel are shown in Figures 10-19, 36, and 37.
  • the pod is stably fixed by taping the pod into the interior of a culture vessel.
  • the pod is stably fixed by snapping the pod in the interior of a culture vessel.
  • the kit includes an adapter manifold to allow the compilation of multiple pods into a plate array, e.g., to further facilitate automated, high-throughput purification approaches.
  • Non-limiting examples of culture vessels include a multi-well plate (e.g., a 6- well plate, a 12-well plate, an 18-well plate, a 24-well plate, a 48-well plate, a 96-well plate, or a 384-well plate), shake flask (e.g., an Erlenmeyer flask or a T-flask), a shake tube (e.g., an Eppendorf tube), a wave bag, a bioreactor (e.g., a benchtop bioreactor, a perfusion bioreactor, a fed-batch bioreactor), or a rolling tube bioreactor. Additional examples of bioreactors are known in the art.
  • a multi-well plate e.g., a 6- well plate, a 12-well plate, an 18-well plate, a 24-well plate, a 48-well plate, a 96-well plate, or a 384-well plate
  • shake flask e.g., an Erlen
  • kits described herein can further include one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) panels that can be secured over each of the one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) areas in the wall, such that leakage of the fluid from the interior of the pod to the exterior of the pod through the wall is prevented.
  • the one or more panels can be made of one or more of any of the exemplary materials described herein that can be used to make the wall.
  • the one or more panels are designed to cover any of the areas described herein.
  • the one or more panels can be designed to click, snap, or screw into the wall to cover the one or more areas in the wall described herein.
  • kits described herein can further include an outer sleeve that can be secured over the pod in order to cover the at least one area in the wall, such that leakage of a fluid from the interior of the pod to the exterior of the pod through the at least one area in the wall is prevented.
  • the sleeve can be composed, at least in part, of flexible rubber or silicon, such that it is capable of being slid over the container to cover the at least one area in the wall.
  • the sleeve can be an empty chromatography column that fits over the container.
  • kits described herein can further include a movable outer sleeve connected to the wall, such that rotation of the movable outer sleeve can cover the at least one area in the wall, such that leakage of a fluid from the interior of the pod to the exterior of the pod through the at least one area in the wall is prevented.
  • the necessary rotation of the movable outer sleeve to cover the at least one area in the wall is clockwise. In some embodiments, the necessary rotation of the movable outer sleeve to cover the at least one area in the wall is counter-clockwise.
  • a movable outer sleeve is connected to the wall, such that sliding the movable outer sleeve up or down can cover the at least one area in the wall, such that leakage of a fluid from the interior of the pod to the exterior of the pod through the at least one area in the wall is prevented.
  • kits further include an elution outlet that can be stably fixed onto the bottom end.
  • the elution outlet can be a funnel or a flat surface having a port.
  • the elution outlet can include a means for controlling the flow of the fluid out of the interior of the container.
  • means for controlling the flow of a fluid out of the interior of the container include a screw valve or a stopcock. Additional exemplary means for controlling the flow of fluid out of the interior of the container are known in the art.
  • the elution outlet does not include a means for controlling the flow of a fluid out of the interior of the container.
  • the elution outlet can be designed such that it can be snapped, clicked, or screwed onto the bottom end of the container.
  • the buffer reservoir can be sealed to the pod using one or more (e.g., two or three) O-rings (e.g., fixed to the pod or the buffer reservoir).
  • kits described herein further include a buffer reservoir that can be stably fixed onto the top end.
  • the buffer reservoir can be snapped, clicked, or screwed onto the top end of the container.
  • the buffer reservoir can have any shape such that it can hold a volume of fluid that can be flowed into the container.
  • a buffer reservoir can be cylindrical, rectangular, hemispherical, spherical, or ellipsoidal in shape.
  • the buffer reservoir can include a means for controlling the flow of the fluid into of the interior of the container from the buffer reservoir.
  • means for controlling the flow of a fluid out of the buffer reservoir into the container include a screw valve or a stopcock.
  • the buffer reservoir does not include a means for controlling the flow of a fluid out of the buffer reservoir into the interior of the container.
  • the buffer reservoir can be designed to hold a volume of fluid between 0.1 mL to about 500 mL, about 0.1 mL to about 450 mL, about 0.1 mL to about 400 mL, about 0.1 mL to about 350 mL, about 0.1 mL to about 300 mL, about 0.1 mL to about 250 mL, about 0.1 mL to about 200 mL, about 0.1 mL to about 150 mL, about 0.1 mL to about 100 mL, about 0.1 mL to about 80 mL, about 0.1 mL to about 60 mL, about 0.1 mL to about 50 mL, about 0.1 mL to about 40 mL, about 0.1 mL to about 30 mL, about
  • kits that include: (a) any of the pods described herein (e.g., a pod having any of the characteristics or aspects described herein in any combination); (b) an inlet system having a top end, an elongated cylindrical body comprising a fluid conduit, and a bottom end, where the top end of the inlet system includes a fluid inlet and is configured for stable attachment to the top end of the pod and, when stably connected to the pod, allows for flow of a fluid into the pod through the fluid inlet and the fluid conduit, and out of the bottom end of the inlet system, and the elongated cylindrical body of the inlet system is configured to fit inside the wall of the pod; and (c) an outlet system including a fluid outlet that is configured for stable attachment to the bottom end of the pod, and when stably connected to the bottom end of the pod, allows for flow of a fluid out of the pod through the fluid outlet.
  • any of the pods described herein e.g., a pod having any of the characteristics or aspects described herein in any combination
  • the top end of the inlet system and the top end of the pod include threading that allows for the stable attachment of the top end of the pod to the top end of the inlet system.
  • the top end of the inlet system and the top end of the pod form a seal e.g., a fluid-tight seal).
  • the threading in the top end of the inlet system or the threading in the top end of the pod includes an O-ring. Other means for providing a stable connection between the top end of the inlet system and the top end of the pod are known in the art.
  • the pod contains a chromatography resin; and the inlet system and the pod are configured such that when the inlet system and the pod are stably attached, the bottom end of the inlet system is proximal to the chromatography resin.
  • an interior surface of the pod and the bottom end of the inlet system are configured to allow for stable attachment of the pod and the bottom end of the inlet system.
  • the interior surface of the pod and the bottom end of the inlet system form a seal (e.g., a fluid-tight seal).
  • the interior surface of the pod and the bottom end of the inlet system include threading that allows for stable attachment of the pod and the bottom end of the inlet system.
  • the threading in the interior surface of the pod or the threading in the bottom end of the inlet system includes an O-ring.
  • Other means for providing a stable connection between the interior surface of the pod and the bottom end of the inlet system are known in the art.
  • the outlet system and the bottom end of the pod include threading that allows for stable attachment of the outlet system to the bottom end of the pod.
  • the threading in the outlet system or the threading in the bottom end of the pod includes an O-ring.
  • the fluid inlet is capable of attachment to tubing (e.g., tubing which is connected to a chromatography system).
  • the fluid outlet is capable of attachment to tubing (e.g., tubing which is connected to a chromatography system).
  • Some embodiments of these kits further include: (d) a means for stably positioning the long axis of the pod in a horizontal position in the interior of a culture vessel.
  • Some embodiments of these kits further include: (d) a means for stably positioning the long axis of the pod in a vertical position in the interior of a culture vessel.
  • the elongated cylindrical body of the inlet system has a length of about 0.1 cm to about 50 cm, about 0.1 cm to about 45 cm, about 0.1 cm to about 40 cm, about 0.1 cm to about 35 cm, about 0.1 cm to about 30 cm, about 0.1 cm to about 25 cm, about 0.1 cm to about 20 cm, about 0.1 cm to about 18 cm, about 0.1 cm to about 16 cm, about 0.1 cm to about 14 cm, about 0.1 cm to about 12 cm, about 0.1 cm to about 10 cm, about 0.1 cm to about 8 cm, about 0.1 cm to about 6 cm, about 0.1 cm to about 5 cm, about 0.1 cm to about 4.5 cm, about 0.1 cm to about 4.0 cm, about 0.1 cm to about 3.5 cm, about 0.1 cm to about 3.0 cm, about 0.1 cm to about 2.5 cm, about 0.1 cm to about 2.0 cm, about 0.1 cm to about 1.5 cm, about 0.1 cm to about 1.0 cm, about 0.1 cm to about 0.5 cm, about 0.5 cm to about 50 cm, about 0.1 cm to about
  • the fluid conduit in the inlet system has an internal diameter of about 0.1 mm to about 10 mm, about 0.1 mm to about 9.5 mm, about 0.1 mm to about 9.0 mm, about 0.1 mm to about 9.0 mm, about 0.1 mm to about 8.5 mm, about 0.1 mm to about 8.0 mm, about 0.1 mm to about 7.5 mm, about 0.1 mm to about 7.0 mm, about 0.1 mm to about 6.5 mm, about 0.1 mm to about 6.0 mm, about 0.1 mm to about 5.5 mm, about 0.1 mm to about 5.0 mm, about 0.1 mm to about 4.5 mm, about 0.1 mm to about 4.0 mm, about 0.1 mm to about 3.5 mm, about 0.1 mm to about 3.0 mm, about 0.1 mm to about 2.5 mm, about 0.1 mm to about 2.0 mm, about 0.1 mm to about 1.5 mm, about 0.1 mm to about 1.0 mm, about
  • the pod contains a chromatography resin (e.g., any of the types of chromatography resin described herein), and the inlet system and the pod are configured such that when the inlet system and the pod are stably attached, the bottom end of the inlet system is approximately about 1 mm to about 30 mm (e.g., about 1 mm to about 25 mm, about 1 mm to about 20 mm, about 1 mm to about 15 mm, about 1 mm to about 10 mm, about 1 mm to about 5 mm) from the chromatography resin.
  • the pod can contain any of the volumes of chromatography resin described herein.
  • the inlet system has an approximately conical shape.
  • the outlet system has an approximately conical shape.
  • a non-limiting example of a kit including a pod, an inlet system, and an outlet system is shown in Figures 27 (non-assembled) and Figure 28 (assembled).
  • Non-limiting examples of the other kits described herein are shown in Figures 1A-1E, Figure 3, Figures 23-26, and Figure 35.
  • kits can further include: (d) an adapter having a top end and a bottom end, wherein the top end of the adapter is configured for stable attachment to the bottom end of the pod, and the bottom end of the adapter has one or more blades, where when the top end of the adapter is stably attached to the bottom end of the pod, the positioning of the one or more blades promotes the flow of a fluid into the pod (e.g., through the material that allows for a liquid to reversibly flow from the exterior of the pod into the interior of the pod).
  • a fluid into the pod e.g., through the material that allows for a liquid to reversibly flow from the exterior of the pod into the interior of the pod.
  • an interior surface of the pod and the top end of the adapter are configured to allow for stable attachment of the pod and the top end of the adapter.
  • the interior surface of the pod and the top end of the adapter form a seal (e.g., a fluid-tight seal).
  • the interior surface of the pod and the top end of the adapter include threading that allows for stable attachment of the pod and the top end of the adapter.
  • the threading in the interior surface of the pod or the threading in the top end of the adapter includes an O-ring. Other means for providing a stable connection between the interior surface of the pod and the top end of the adapter are known in the art.
  • kits described herein can further include instructions for performing any of the methods described herein.
  • methods of capturing a recombinant protein produced by a liquid cell culture that include: contacting any of the pods provided herein, with a liquid cell culture for a period of time, where the contacting step results in at least a portion of the at least one area contacting the liquid cell culture, and the contacting is performed under conditions that result in the capture of the recombinant protein by the chromatography resin.
  • Also provided herein are methods of purifying a recombinant protein produced by a liquid cell culture that include: (a) contacting any of the pods provided herein, with a liquid cell culture for a period of time, where the contacting step results in at least a portion of the at least one area contacting the liquid cell culture, and the contacting is performed under conditions that result in the capture of the recombinant protein by the chromatography resin; and (b) eluting the recombinant protein from the chromatography resin after the period of time, thereby purifying the recombinant protein.
  • the eluting (and optional washing step) can be performed using a chromatography system (e.g., an ⁇ KTA system).
  • the pod is any of the pods described herein, where the pod includes a chromatography resin (e.g., any of the chromatography resins described herein or known in the art) disposed in the interior of the container.
  • the pod is disposed such that the longitudinal axis of the pod is placed about horizontally (e.g., horizontally) in a liquid cell culture.
  • the pod is disposed such that the longitudinal axis of the pod is placed about vertically (e.g., vertically) in a liquid cell culture.
  • the pod includes a chromatography resin that is a commercial chromatography resin.
  • the chromatography resin is an affinity chromatography resin (e.g., a protein A resin, a protein G resin, a protein L resin, a nickel resin, or a magnetic bead resin).
  • the pod or the chromatography resin can be frozen or stored for a period of time before the recombinant protein is eluted from the chromatography resin in the pod.
  • the pod can or chromatography resin can be frozen or stored for a period of time after the contacting step and after the pod or chromatography resin has been contacted with a wash buffer.
  • the contacting results in at least a portion of the at least one area (e.g., at least 10% of the at least one area, at least 15% of the at least one area, at least 20% of the at least one area, at least 25% of the at least one area, at least 30% of the at least one area, at least 35% of the at least one area, at least 40% of the at least one area, at least 45% of the at least one area, at least 50% of the at least one area, at least 55% of the at least one area, at least 60% of the at least one area, at least 65% of the at least one area, at least 70% of the at least one area, at least 75% of the at least one area, at least 80% of the at least one area, at least 85% of the at least one area, at least 90% of the at least one area, at least 95% of the at least one area, at least 97% of the at least one area, at least 98% of the at least one area, at least 99% of the at least one area) contacting the liquid cell culture.
  • At least 20% e.g., at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) of the total surface area of the at least one area is in contact with the liquid culture.
  • the contacting is performed under conditions that result in the capture of the recombinant protein by the chromatography resin.
  • the capturing can result in the recombinant protein being bound to a level of up to 60% to 100% (e.g., up to 60% to 70%, up to 60% to 75%, up to 60% to 80%, up to 60% to 85%, up to 60% to 90%, up to 60% to 95%, up to 65% to 70%, up to 65% to 75%, up to 65% to 80%, up to 65% to 85%, up to 65% to 90%, up to 65% to 95%, up to 65% to 100%, up to 70% to 75%, up to 70% to 80%, up to 70% to 85%, up to 70% to 90%, up to 70% to 95%, up to 70% to 100%, up to 75% to 80%, up to 75% to 85%, up to 75% to 90%, up to 75% to 95%, up to 75% to 100%, up to 80% to 85%, up to 80% to 90%, up to up to 75% to 9
  • the capturing can result in the capture of about 0.05 mg to about 80 mg (e.g., about 0.05 mg to about 0.1 mg, about 0.05 mg to about 0.2 mg, about 0.05 mg to about 0.5 mg, about 0.05 mg to about 0.6 mg, about 0.05 mg to about 0.8 mg, about 0.05 mg to about 1 mg, about 0.05 mg to about 2 mg, about 0.05 mg to about 5 mg, about 0.05 mg to about 10 mg, about 0.05 mg to about 15 mg, about 0.05 mg to about 20 mg, about 0.05 mg to about 25 mg, about 0.05 mg to about 30 mg, about 0.05 mg to about 35 mg, about 0.05 mg to about 40 mg, about 0.05 mg to about 45 mg, about 0.05 mg to about 50 mg, about 0.05 mg to about 55 mg, about 0.05 mg to about 60 mg, about 0.05 mg to about 65 mg, about 0.05 mg to about 70 mg, about 0.05 mg to about 75 mg, about 0.05 mg to about 80 mg, about 0.1 mg to about 0.2 mg, about 0.1 mg to about 80 mg,
  • the purity of capture is at least 45% (e.g., at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%
  • the contacting of any of the pods provided herein with a liquid cell culture is for a period of time (e.g., about 30 seconds to about 2 weeks).
  • the eluting of the recombinant protein from the chromatography resin is after a period of time (e.g., about 30 seconds to about 2 weeks).
  • the period of time for contacting and/or for eluting the recombinant protein from the chromatography resin is about 30 seconds to about 1 minute, about 30 seconds to 45 seconds, about 30 seconds to about 90 seconds, about 30 seconds to about 2 minutes, about 30 seconds to about 150 seconds, about 30 seconds to about 3 minutes, about 30 seconds to about 210 seconds, about 30 seconds to about 4 minutes, about 30 seconds to about 270 seconds, about 30 seconds to about 5 minutes, about 1 minute to about 2 minutes, about 1 minute to about 3 minutes, about 1 minute to about 4 minutes, about 1 minute to about 5 minutes, about 2 minutes to about 3 minutes, about 2 minutes to about 4 minutes, about 2 to about 5 minutes, about 3 minutes to about 4 minutes, about 3 minutes to about 5 minutes, about 4 minutes to about 5 minutes, about 5 minutes to about 2 weeks, about 5 minutes to about 168 hours, about 5 minutes to about 162 hours, about 5 minutes to about 156 hours, about 5 minutes to about 150 hours, about 5 minutes to about 144 hours, about 5 minutes to about 138 hours,
  • the period of time occurs when the liquid cell culture is in its exponential growth phase. In some embodiments of the contacting step of any of the methods provided herein, the period of time occurs when the liquid cell culture is in its stationary growth phase. In some embodiments, the contacting can occur when a liquid cell culture is in its death phase. In some embodiments of the contacting step of any of the methods provided herein, the period of time occurs on day 4 of cell culture until day 5.
  • the contacting step can include contacting one or more pods (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) sequentially or at the same time with a liquid cell culture for a period of time (e.g., any of the period of times described herein).
  • Recombinant proteins described herein include, without limitation, receptors, antibodies, antigen-binding antibody fragments, antibody fragments, fusion proteins, ligands, secreted proteins, enzymes, recombinant protein drug products, and any recombinant protein with or without a tag.
  • Non-limiting examples of tags include: a fluorescent protein (e.g., green fluorescent protein, red fluorescent protein, cyan fluorescent protein, yellow fluorescent protein), a radioisotope, a His-tag, a FLAG- tag, a c-myc tag, a maltose-binding protein (MBP) tag, an albumin-binding protein (ABP) tag, a bacteriophage T7 epitope (T7-tag), a biotin-carboxy carrier protein (BCCP) tag, a bluetongue virus tag (B-tag), a calmodulin binding peptide tag, a cellulose binding domain tag, a chitin binding domain tag, a choline binding domain tag, a galactose-binding protein tag, a Glu-Glu epitope tag, a human influenza hemagglutinin tag, a Halo Tag®, a histidine affinity tag (HAT), a polyarginine tag (Asp-tag), a polyphen
  • Exemplary recombinant antibodies and recombinant antibody fragments that can be expressed in any of the cells described herein can include recombinant IgG1 (e.g., recombinant human IgG1), recombinant IgG2 (e.g., recombinant human IgG2), recombinant IgG3 (e.g., recombinant human IgG3), recombinant IgG4 (e.g., recombinant human IgG4), recombinant IgM (e.g., recombinant human IgM), recombinant IgE (e.g., recombinant human IgE), recombinant IgD (e.g., recombinant human IgD), recombinant IgA (e.g., recombinant human IgA), a recombinant Fab, a recombinant scFv,
  • the recombinant protein is an antibody.
  • the term“antibody” refers to a whole or intact antibody (e.g., IgM, IgG, IgA, IgD, or IgE) molecule.
  • An antibody is tetrameric in nature, containing two identical heavy chain protein portions and two identical light chain protein portions, which associate together to adopt a“Y”-shaped conformation held together by disulfide bridges at the hinge region.
  • the term“antibody” includes a monoclonal antibody, a chimerized or chimeric antibody, a humanized antibody, a deimmunized human antibody, and a fully human antibody.
  • the antibody can be made in or derived from any of a variety of species, e.g., mammals such as humans, non-human primates (e.g., monkeys, baboons, or chimpanzees), horses, cattle, pigs, sheep, goats, dogs, cats, rabbits, guinea pigs, gerbils, hamsters, rats, and mice.
  • the antibodies can be generated synthetically, e.g., via synthetically produced oligonucleotides, as described in, e.g, Fuh et al., J. Mol. Biol.340:1073-1093, 2004, and Knappik et al., J. Mol. Biol.296(11):57-86, 2000, the disclosures of each of which are incorporated herein by reference in their entirety.
  • the antibody can be generated by antibody phage display in yeast and/or human cells.
  • the term“antibody fragment,”“antigen-binding fragment,” or similar terms refer to a fragment of an antibody that retains the ability to bind to an antigen, e.g., a single chain antibody (scFv), an Fd fragment, an Fab fragment, an Fab′ fragment, or an F(ab′)2 fragment.
  • An scFv is a single polypeptide chain that includes both the heavy and light chain variable regions of the antibody from which the scFv is derived.
  • diabodies Polyabodies (Poljak, Structure 2(12):1121-1123, 1994; Hudson et al., J. Immunol. Methods 23(1-2):177-189, 1999, the disclosures of both of which are incorporated herein by reference in their entirety), minibodies, triabodies
  • domain antibodies also known as “heavy chain immunoglobulins” or camelids; Holt et al., Trends Biotechnol.
  • the recombinant protein is a bispecific antibody.
  • Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens.
  • bispecific antibodies is based on the co-expression of two immunoglobulin heavy-chain/light-chain pairs, where the two heavy chain/light-chain pairs have different specificities (Milstein and Cuello, Nature 305:537-539, 1983).
  • Antibody variable domains with the desired binding specificities can be fused to immunoglobulin constant domain sequences.
  • the fusion of the heavy chain variable region is preferably with an immunoglobulin heavy-chain constant domain, including at least part of the hinge, CH2, and CH3 regions.
  • bispecific antibodies have been produced using leucine zippers. See, e.g., Kostelny et al., J. Immunol.148(5):1547-1553, 1992.
  • the leucine zipper peptides from the Fos and Jun proteins may be linked to the Fab′ portions of two different antibodies by gene fusion.
  • the antibody homodimers may be reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers. This method can also be utilized for the production of antibody homodimers.
  • The“diabody” technology described by Hollinger et al., Proc. Natl. Acad.
  • the fragments include a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) by a linker which is too short to allow pairing between the two domains on the same chain. Accordingly, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen-binding sites.
  • VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen-binding sites.
  • scFv single-chain Fv
  • the antibodies can be“linear antibodies” as described in, e.g., Zapata et al., Protein Eng.8(10):1057-1062, 1995. Briefly, these antibodies include a pair of tandem Fd segments (VH-CH1-VH-CH1) which form a pair of antigen binding regions. Linear antibodies can be bispecific or monospecific.
  • Antibodies with more than two valencies are contemplated and described in, e.g., Tutt et al., J. Immunol.147:60, 1991.
  • the disclosure also embraces variant forms of multi-specific antibodies such as the dual variable domain immunoglobulin (DVD-Ig) molecules described in Wu et al., Nat Biotechnol.25(11): 1290-1297, 2007.
  • DVD-Ig molecules are designed such that two different light chain variable domains (VL) from two different parent antibodies are linked in tandem directly or via a short linker by recombinant DNA techniques, followed by the light chain constant domain.
  • the heavy chain includes two different heavy chain variable domains (VH) linked in tandem, followed by the constant domain CH1 and Fc region.
  • Methods for making DVD-Ig molecules from two parent antibodies are further described in, e.g., WO 08/024188 and WO 07/024715.
  • the bispecific antibody is a Fabs-in-Tandem immunoglobulin, in which the light chain variable region with a second specificity is fused to the heavy chain variable region of a whole antibody.
  • Fabs-in-Tandem immunoglobulin in which the light chain variable region with a second specificity is fused to the heavy chain variable region of a whole antibody.
  • the recombinant protein is a nanobody, such as a camelid or dromedary antibodies (e.g., antibodies derived from Camelus bactrianus, Calelus dromaderius, or Lama paccos).
  • a nanobody such as a camelid or dromedary antibodies (e.g., antibodies derived from Camelus bactrianus, Calelus dromaderius, or Lama paccos).
  • Such antibodies unlike the typical two-chain (fragment) or four-chain (whole antibody) antibodies from most mammals, generally lack light chains. See U.S. Patent No.5,759,808; Stijlemans et al., J. Biol. Chem. 279:1256-1261, 2004; Dumoulin et al., Nature 424:783-788, 2003; and Pleschberger et al., Bioconjugate Chem.14:440-448, 2003.
  • an amino acid sequence of a camelid antibody can be altered recombinantly to obtain a sequence that more closely resembles a human sequence, i.e., the nanobody can be“humanized” to thereby further reduce the potential immunogenicity of the antibody.
  • the recombinant protein is a non-antibody, scaffold protein.
  • These proteins are, generally, obtained through combinatorial chemistry- based adaptation of preexisting antigen-binding proteins.
  • the binding site of human transferrin for human transferrin receptor can be diversified using the system described herein to create a diverse library of transferrin variants, some of which have acquired affinity for different antigens.
  • the scaffold portion of the non- antibody scaffold protein can include, e.g., all or part of: the Z domain of S. aureus protein A, human transferrin, human tenth fibronectin type III domain, kunitz domain of a human trypsin inhibitor, human CTLA-4, an ankyrin repeat protein, a human lipocalin (e.g., anticalins), human crystallin, human ubiquitin, or a trypsin inhibitor from E. elaterium.
  • the Z domain of S. aureus protein A human transferrin, human tenth fibronectin type III domain, kunitz domain of a human trypsin inhibitor, human CTLA-4, an ankyrin repeat protein, a human lipocalin (e.g., anticalins), human crystallin, human ubiquitin, or a trypsin inhibitor from E. elaterium.
  • the recombinant protein is a fusion protein.
  • fusion proteins that can be produced by any of the cells described herein include chimeric antibodies (e.g., humanized antibodies), labelled antibodies (e.g., antibodies labelled with an enzyme or fluorescent protein tag), proteins ligands that bind specifically to a cancer cell surface receptor, where the protein ligand is labelled with a proteinaceous cytotoxin, an antibody with a fusion partner that extends the half life of the antibody in vivo (e.g., an antibody fused to human albumin), a bispecific antibody, where one light chain variable domain and one heavy chain variable domain bind to an antigen on an immune cell (e.g., a T cell, a macrophage, a neutrophil, or a monocyte) and the other light chain variable domain and the other heavy chain variable domain bind to an antigen on a target cell (e.g., a cancer cell or a virus infected cell) or a pathogen (
  • a target cell e
  • recombinant proteins include, without limitation, ligands, enzymes, peptides, scFvs, non-antibody scaffold proteins, chimeric antigen receptor molecules, and the like, in which the diversity element is fused in frame with one or more constant elements.
  • the recombinant protein is a multimeric protein, such as, but not limited to, Fabs, whole antibodies, MHC class I molecules, MHC class I molecules, T cell receptor molecules, bispecific antibodies, trispecific antibodies, integrins, tumor growth factor receptor dimers, G-protein coupled receptors, and the like.
  • the liquid cell culture can include any cell, e.g., a eukaryotic cell.
  • a eukaryotic cell refers to a cell having a distinct, membrane-bound nucleus.
  • Such cells may include, for example, mammalian (e.g., rodent, non-human primate, or human), insect, fungal, or plant cells.
  • the eukaryotic cell is a yeast cell, such as Saccharomyces cerevisiae.
  • the eukaryotic cell is a higher eukaryote, such as mammalian, avian, plant, or insect cells.
  • Non-limiting examples of mammalian cells that can produce a recombinant protein include, e.g., rodent (e.g., mouse, rat, or hamster) or human cells.
  • the mammalian cell is a Chinese hamster ovarian (CHO) cell, a human retina PER.C6 cell, a human aminocyte CAP cell, a mouse melanoma NS0 or Sp2/0 cell, a duck EB66 cell, a hamster BHK cell, a human embryonic kidney (HEK 293) cell, a COS cell, or a chicken-derived B cell line DT40.
  • the HEK 293 cell is a freestyle HEK293-F cell, a HEK 2936F, a HEK293FT, a HEK 293T cell, or an Expi293F cell.
  • CHO cells are epithelial cells which grow as an adherent monolayer or in suspension.
  • CHO cells lines that were derived from the original cells isolated by Tjio and Puck may include point mutations, deletions, or integrations of nucleic acid sequences relative to the original CHO cell lines.
  • the subgroups of CHO cells may include, but is not limited to, CHO DP-12 cells, CHO-K1 cells, CHO/dhfr- cells, CHO-S cells, CHO-GS cells, CHO-K1 DUX B11 cells, CHO pro3- cells, CHO- DG44 cells, CHO GAT- cells, or freestyle CHO-S cells.
  • the eukaryotic cell is a primary cell or a cell line.
  • the mammalian cell is a cancer cell.
  • the mammalian cell is a human cancer cell line, including those listed in the“Cancer Cell Line Encyclopedia” (Barretina et al., Nature 483(7391):603-607, 2012, or Forbes et al., Curr. Protoc. Hum. Genet.2008).
  • the eukaryotic cell is a T lymphocyte lineage cell (e.g., a primary T cell or a T-cell line), a T-cell derived cell line which lacks TCR expression (see, e.g., Torres et al., J.
  • B lymphocyte lineage cell such as pro-B cells (see, e.g., Urashima et al., Am. J. Hematol.1994), pre-B cells (see, e.g., Adachi et al., DNA Cell Biol.2006), and B-cells, and cell lines derived from these.
  • the B lineage cell line is a murine pre-B cell line, such as 1624-5 (Holms et al., J. Exp. Med.1986; Principato et al., Mol. Cell Biol.1990), or a pro-B cell line, such as Ba/F3 (commercially available from Thermo Scientific).
  • the mammalian cells can express a secreted version of the recombinant protein.
  • the secreted version of the recombinant protein is transiently expressed.
  • the secreted version of the recombinant protein is stably expressed.
  • the liquid cell culture can include an insect cell (e.g., a baculovirus-insect cell, a Drosophila cell (e.g., a Drosophila S2 cell), a Trichoplusia ni cell, a Lepidopteran cell), a yeast cell (e.g., a Saccharoymyces cerevisiae cell, a Pichia pastoris cell), a plant cell or a bacterial cell (e.g., an E.coli cell, a Baccillus brevis cell, a Bacillus megaterium cell, a Bacillus subtilis cell, a Caulobacter crescentis cell).
  • insect cell e.g., a baculovirus-insect cell, a Drosophila cell (e.g., a Drosophila S2 cell), a Trichoplusia ni cell, a Lepidopteran cell
  • yeast cell e.g., a Saccharoymyces cerevisiae
  • proteins and vectors described herein can be introduced into the cells (e.g., mammalian cells, insect cells, yeast cells, bacterial cells, or plant cells).
  • cells e.g., mammalian cells, insect cells, yeast cells, bacterial cells, or plant cells.
  • Non-limiting examples of vectors and methods for introducing vectors into cells are described herein.
  • Cells can be maintained in vitro under conditions that favor growth, proliferation, continued viability, and differentiation.
  • cells can be cultured by contacting a cell with a liquid culture medium that includes the necessary growth factors and supplements to support cell viability and growth.
  • liquid culture medium means a fluid that contains sufficient nutrients to allow a cell (e.g., any of the cells described herein) to grow or proliferate in vitro.
  • a liquid culture medium can contain one or more of: amino acids (e.g., 20 amino acids), a purine (e.g., hypoxanthine), a pyrimidine (e.g., thymidine), choline, inositol, thiamine, folic acid, biotin, calcium, niacinamide, pyridoxine, riboflavin, thymidine, cyanocobalamin, pyruvate, lipoic acid, magnesium, glucose, sodium, potassium, iron, copper, zinc, and sodium bicarbonate.
  • amino acids e.g., 20 amino acids
  • a purine e.g., hypoxanthine
  • a pyrimidine e.g., thymidine
  • choline inositol
  • thiamine e.g., thymidine
  • choline e.g., inositol
  • thiamine e.g., thy
  • a liquid culture medium can contain serum from a mammal. In some embodiments, a liquid culture medium does not contain serum or another extract from a mammal (a defined liquid culture medium). In some embodiments, a liquid culture medium can contain trace metals, a mammalian growth hormone, and/or a mammalian growth factor. Another example of liquid culture medium is minimal medium (e.g., a medium containing only inorganic salts, a carbon source, and water). Non-limiting examples of liquid culture medium are described herein. Additional examples of liquid culture medium are known in the art and are commercially available. A liquid cell culture medium can contain any density of any cell described herein (e.g., any density of mammalian cells).
  • the term“animal-derived component free liquid culture medium” means a liquid culture medium that does not contain any components (e.g., proteins or serum) derived from a mammal.
  • the term“serum-free liquid culture medium” means a liquid culture medium that does not contain a mammalian serum.
  • the term“serum-containing liquid culture medium” means a liquid culture medium that contains a mammalian serum.
  • the term“chemically-defined liquid culture medium” is a term of art and means a liquid culture medium in which all of the chemical components are known.
  • a chemically-defined liquid culture medium does not contain fetal bovine serum, bovine serum albumin, or human serum albumin, as these preparations typically contain a complex mix of albumins and lipids.
  • protein-free liquid culture medium means a liquid culture medium that does not contain any protein (e.g., any detectable protein).
  • a cell may be cultured for an extended period of time, e.g., for about 1 day to about 90 days, about 85 days, about 80 days, about 75 days, about 70 days, about 65 days, about 60 days, about 55 days, about 50 days, about 45 days, about 40 days, about 35 days, about 30 days, about 20 days, about 25 days, about 20 days, about 15 days, about 10 days, about 8 days, about 6 days, about 4 days, or about 2 days (inclusive); about 2 days to about 90 days, about 85 days, about 80 days, about 75 days, about 70 days, about 65 days, about 60 days, about 55 days, about 50 days, about 45 days, about 40 days, about 35 days, about 30 days, about 20 days, about 25 days, about 20 days, about 15 days, about 10 days, about 8 days, about 6 days, or about 4 days (inclusive); about 4 days to about 90 days, about 85 days, about 80 days, about 75 days, about 70 days, about 65 days, about 60 days, about 55 days, about 50 days, about 45 days, about
  • the liquid cell culture is agitated for a period of time (e.g., continuously, periodically) during culturing.
  • agitation means stirring or otherwise moving a portion of liquid culture medium in a bioreactor. This is performed in order to, e.g., increase the dissolved oxygen concentration in the liquid culture medium in a bioreactor.
  • Agitation can be performed using any art known method, e.g., an instrument (e.g., a shaking, rocking, or tilting platform) or propellor.
  • an instrument e.g., a shaking, rocking, or tilting platform
  • propellor e.g., a propellor
  • Exemplary devices and methods that can be used to perform agitation of a portion of the liquid culture medium in a bioreactor are known in the art.
  • the liquid cell culture is a perfusion culture.
  • perfusion culture is a term of art and refers to the culturing of cells present in a culture vessel (e.g., any of the culture vessels described herein) by periodically or continuously removing of a first liquid culture medium and at the same time or shortly thereafter adding substantially the same volume of a second liquid culture medium to the culture vessel.
  • an incremental change e.g., increase or decrease
  • the volume of the first liquid culture medium removed and added over incremental periods (e.g., an about 24-hour period, a period of between about 1 minute and about 24-hours, or a period of greater than 24 hours) during the culturing period (e.g., the culture medium refeed rate on a daily basis).
  • the fraction of media removed and replaced each day can vary depending on the particular cells being cultured, the initial seeding density, and the cell density at a particular period of time.
  • the liquid cell culture is a fed-batch culture.
  • fed-batch culture is a term of art and refers to culturing of cells present in a culture vessel by periodically or continuously adding of a second liquid culture medium to a first liquid culture medium without substantial or significant removing the first liquid culture medium or second liquid culture medium from the liquid cell culture.
  • the second liquid culture medium can be the same as the first liquid culture medium.
  • the second liquid culture medium is a concentrated form of the first liquid culture medium.
  • the second liquid culture medium is added as a dry powder.
  • the liquid cell culture can be disposed in a culture vessel.
  • culture vessels include: a shake flask, a shake tube, a wave bag, a T- flask, a triple flask, a spinner flask, a roller bottle, a bioreactor (e.g., a perfusion bioreactor, a fed-batch bioreactor), or a multi-well plate (e.g., a 4-well cell culture plate, a 6-well cell culture plate, a 8-well cell culture plate, a 12-well cell culture plate, a 24-well cell culture plate, a 24-well cell culture plate).
  • a bioreactor e.g., a perfusion bioreactor, a fed-batch bioreactor
  • a multi-well plate e.g., a 4-well cell culture plate, a 6-well cell culture plate, a 8-well cell culture plate, a 12-well cell culture plate, a 24-well cell culture plate, a 24-well cell culture plate.
  • the culture vessel has a total interior volume of about 0.2 mL to 10,000 L, (e.g., about 0.2 mL to about 9,000 L, about 0.2 mL to about 8,000 L, about 0.2 mL to about 7,000 L, about 0.2 mL to about 6,000 L, about 0.2 mL to about 5,000 L, about 0.2 mL to about 4,000 L, about 0.2 mL to about 3,000 L, about 0.2 mL to about 2,000 L, about 0.2 mL to about 1,000 L, about 0.2 mL to about 950 L, about 0.2 mL to about 900 L, about 0.2 mL to about 850 L, about 0.2 mL to about 800 L, about 0.2 mL to about 750 mL, about 0.2 mL to about 700 L, about 0.2 mL to about 650 mL, about 0.2 mL to about 600 L, about 0.2 mL to about 550 L, about 0.2 mL to about
  • cells in a liquid cell culture can include a nucleic acid or a vector encoding a recombinant protein (e.g., any of the recombinant proteins described herein).
  • the nucleic acid that encodes any of the recombinant proteins described herein can be included in an expression vector. Skilled practitioners will be capable of selecting suitable expression vectors for making the recombinant proteins described herein, and using the expression vectors to express a recombinant protein in a cell. For example, in selecting an expression vector, the cell must be considered because the vector may need to be able to replicate in it and/or be able to integrate into a chromosome of the cell. Exemplary expression vectors that can be used to produce a recombinant protein are also described below.
  • A“vector” is a polynucleotide capable of inducing the expression of a recombinant protein (e.g., any of the recombinant proteins as described herein) in a cell.
  • a vector functions like a“molecular carrier,” delivering fragments of nucleic acids into a host cell (e.g., any of the cell described herein). It may include an expression cassette including regulatory sequences.
  • Foreign polynucleotides may be inserted into the expression cassette(s) of the vector nucleic acid in order to be expressed therefrom.
  • the vectors according to the present invention may be present in circular or linearized form.
  • the term“vector” also includes artificial chromosomes or similar respective polynucleotides allowing the transfer of foreign nucleic acid fragments.
  • a respective vector can be used as expression vector in order to produce a recombinant protein as described herein in a cell.
  • a variety of different methods known in the art can be used to introduce any of the nucleic acids and expression vectors disclosed herein into a cell (e.g., a eukaryotic cell, a plant cell).
  • a cell e.g., a eukaryotic cell, a plant cell.
  • methods that can be used to introduce a nucleic acid into a cell include lipofection, transfection, electroporation, microinjection, calcium phosphate transfection, dendrimer-based transfection, cationic polymer transfection, cell squeezing, sonoporation, transformation, optical transfection, impalection, hydrodynamic delivery, magnetofection, viral transduction (e.g., adenoviral and lentiviral transduction), and nanoparticle transfection.
  • the introduced nucleic acid into a cell is transiently expressed. In some embodiments, the introduced nucleic acid into a cell is integrated into the cell genome.
  • the contacting step results in at least a portion (e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%) of the at least one area contacting the liquid cell culture.
  • the contacting is performed under conditions that result in the capture of the recombinant protein by the chromatography resin.
  • the contacting is performed under sterile conditions.
  • the contacting can be performed during agitation of the liquid cell culture (e.g., by rocking, by tilting, by rotary agitation (e.g., using a propeller), by vortexing, and/or aeration).
  • the contacting can occur when a liquid cell culture is in its exponential growth phase or stationary growth phase. In some embodiments, the contacting can occur when a liquid cell culture is in its death phase. In some embodiments of any of the methods described herein, the contacting can occur in a clarified supernatant of a liquid cell culture.
  • the contacting is performed at a temperature of about 20 °C to about 40 °C (e.g., about 20 °C to about 40 °C, about 20 °C to about 25 °C, about 20 °C to about 30 °C, about 20 °C to about 35 °C, about 20 °C to about 37 °C, about 20 °C to about 38 °C, about 20 °C to about 39 °C, about 27 °C to about 30 °C, about 25 °C to about 30 °C, about 25 °C to about 37 °C, about 25 °C to about 38 °C, about 25 °C to about 39 °C, about 25 °C to about 40 °C, about 27 °C to about 30 °C, about 27 °C to about 35 °C, about 27 °C to about 37 °C, about 27 °C to about 38 °C, about 27 °C to about 39 °C, about 30 °C to about
  • the contacting is performed at a temperature of about 4 °C to about 18 °C (e.g., about 4 °C to about 16 °C, about 4 °C to about 14 °C, about 4 °C to about 12 °C about 4 °C to about 10 °C about 4 °C to about 8 °C about 4 °C to about 6 °C, about 6 °C to about 18 °C, about 6 °C to about 16 °C, about 6 °C to about 14 °C, about 6 °C to about 12 °C about 6 °C to about 10 °C, about 6 °C to about 8 °C, about 8 °C to about 18 °C, about 8 °C to about 16 °C, about 8 °C to about 14 °C, about 8 °C to about 12 °C, about 8 °C to about 10 °C, about 10 °C to about 18 °C, about 10 °C to about 18 °C, about 10
  • contacting can occur when the pod is at a 90° angle ⁇ 50 o (e.g., at a 50 o to 140° angle, at a 50 o to 135° angle, at a 50 o to 130° angle, at a 50 o to 125° angle, at a 50 o to 120° angle, at a 50 o to 115° angle, at a 50 o to 110° angle, at a 50 o to 105° angle, at a 50 o to 100° angle, at a 50 o to 95° angle, at a 50 o to 90° angle, at a 50 o to 85° angle, at a 50 o to 80° angle, at a 50 o to 75° angle, at a 50 o to 70° angle, at a 50 o to 65° angle, at a 50 o to 60° angle, at a 55 o to 140° angle, at a 55 o to 135° angle, at a 55 o to 130° angle,
  • contacting can occur when the pod is at a 180 o angle ⁇ 35 o (e.g., at a 145 o to 180 o angle, at a 145 o to 175 o angle, at a 145 o to 170 o angle, at a 145 o to 165 o angle, at a 145 o to 160 o angle, at a 145 o to 155 o angle, at a 145 o to 150 o angle, at a 150 o to 180 o angle, at a 150 o to 175 o angle, at a 150 o to 170 o angle, at a 150 o to 165 o angle, at a 150 o to 160 o angle, at a 150 o to 155 o angle, at a 155 o to 180 o angle, at a 155 o to 175 o angle, at a 155 o to 170 o angle, at a 155 o to 180 o angle, at a
  • a pod including a chromatography resin can be sterilized.
  • methods of sterilization include: filtration, gamma-irradiation, electron-beam radiation, steam sterilization, autoclaving, and chemical sterilization (e.g., hydrogen chloride, sodium-N-lauroyl sarcosinate, sodium hydroxide, or ethanol).
  • the chromatography resin is sterile.
  • sterile assurance level refers to the probability of a single unit (e.g., a chromatography resin) being non-sterile after it has been subjected to a sterilization process.
  • the chromatography resin has a sterility assurance level of about 1 x 10 -5 to about 1 x 10 -15 (e.g., about 1 x 10 -5 to about 1 x 10 -10 , about 1 x 10 -5 to about 1 x 10 -15 , about 1 x 10 -6 to about 1 x 10 -10 , about 1 x 10 -6 to about 1 x 10 -12 , about 1 x 10 -6 to about 1 x 10 -15 , about 1 x 10 -5 , about 1 x 10 -6 , about 1 x 10 -7 , about 1 x 10 -7 , about 1 x 10 -8 , about 1 x 10 -9 , about 1 x 10 -10 , about 1 x 10 -11 , about 1 x 10 -12 , about 1 x 10 -13 , about 1 x 10 -14 , about 1 x 10 -15 ).
  • the liquid cell culture medium present in the liquid cell culture can be sterile and/or sterilized. Washing Chromatography Resin
  • the method can further include a washing step.
  • the washing step is performed in between the contacting step and the eluting step.
  • the washing step includes one or more washes (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) using a wash buffer.
  • the washing step removes or substantially removes contaminants and/or non-specific proteins.
  • the washing step equilibrates the chromatography resin of the pod before it is contacted with a liquid cell culture.
  • the washing step is performed before removing the pod from the liquid cell culture and/or the culture vessel. In some embodiments, the washing step is performed after removing the pod from the liquid cell culture and/or the culture vessel. In some embodiments, the washing step is performed after removing the pod from the liquid cell culture and/or the culture vessel, and can involve pipetting a wash buffer directly into the interior of the pod.
  • the washing step is performed at about 2 °C to about 25 °C (e.g., 2 °C to about 4 °C, about 2 °C to about 6 °C, about 2 °C to about 8 °C, about 2 °C to about 10 °C, about 2 °C to about 12 °C, about 4 °C to about 6 °C, about 4 °C to about 8 °C, about 4 °C to about 10 °C, about 4 °C to about 12 °C, about 6 °C to about 8 °C, about 6 °C to about 10 °C, about 6 °C to about 12 °C, about 10 °C to about 12 °C, about 10 °C to about 25 °C, about 10 °C to about 22 °C, about 10 °C to about 20 °C, about 10 °C to about 18 °C, about 10 °C to about 14 °C, about 12 °C to about 14 °C, about 12 °
  • the washing step is performed using a wash buffer that contains about 2 mM to about 6 mM (e.g., about 2 mM to about 5 mM, about 2 mM to about 4 mM, about 2 mM to about 3 mM, about 3 mM to about 6 mM, about 3 mM to about 5 mM, about 3 mM to about 4 mM, about 4 mM to about 6 mM, about 4 mM to about 5 mM, about 5 mM to about 6 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM) of ammonium acetate.
  • the wash buffer can further include salts and/or detergents. As is known in the art, the washing buffer is not meant to dissociate the recombinant protein from the chromatography resin.
  • the washing step is performed using about 0.05 mL to about 1,000 mL, about 0.05 mL to about 950 mL, about 0.05 mL to about 900 mL, about 0.05 mL to about 850 mL, about 0.05 mL to about 800 mL, about 0.05 mL to about 750 mL, about 0.05 mL to about 700 mL, about 0.05 mL to about 650 mL, about 0.05 mL to about 600 mL, about 0.05 mL to about 550 mL, about 0.05 mL to about 500 mL, about 0.05 mL to about 450 mL, about 0.05 mL to about 400 mL, about 0.05 mL to about 350 mL, about 0.05 mL to about 300 mL, about 0.05 mL to about 250 mL, about 0.05 mL to about 200 mL, about 0.05 mL to about 150 mL, about 0.05 mL to
  • the washing step can include one or more wash buffers (e.g., 2, 3, 4, 5, 6, 7, or 8) of any of the wash buffers described herein). Eluting a Recombinant Protein
  • any of the pods described herein can be removed from the liquid culture medium before performing the eluting step.
  • the affinity chromatography resin of the pod is a protein A resin.
  • the eluting is performed using an elution buffer with an acidic pH.
  • the elution buffer includes arginine or citrate.
  • the elution buffer includes about 0.01 M to about 1 M (e.g., about 0.01 M to about 1 M, about 0.01 M to about 0.8 M, about 0.01 M to about 0.6 M, about 0.01 M to about 0.4 M, about 0.01 M to about 0.2 M, about 0.01 M to about 0.1 M, about 0.01 M to about 0.08 M, about 0.01 M to about 0.06 M, about 0.01 M to about 0.05 M, about 0.01 M to about 0.04 M, about 0.01 M to about 0.02 M; 0.02 M to about 1 M, about 0.02 M to about 0.8 M, about 0.02 M to about 0.6 M, about 0.02 M to about 0.4 M, about 0.02 M to about 0.2 M, about 0.02 M to about 0.1 M, about 0.02 M to about 0.08 M, about 0.02 M to about 0.06 M, about 0.02 M to about 0.05 M, about 0.02 M to about 0.04 M, 0.04 M to about 1 M, about 0.04 M to about 0.8 M, about 0.02 M to about 0.04
  • the elution buffer includes about 0.1 to about 1 M (e.g., about 0.1 to about 0.9 M, about 0.1 M to about 0.8 M, about 0.1 M to about 0.7 M, about 0.1 M to about 0.6 M, about 0.1 M to about 0.5 M, about 0.1 M to about 0.4 M, about 0.1 M to about 0.3 M, about 0.1 M to about 0.2 M, about 0.2 M to about 1 M, about 0.2 M to about 0.9 M, about 0.2 M to about 0.8 M, about 0.2 M to about 0.7 M, about 0.2 M to about 0.6 M, about 0.2 M to about 0.5 M, about 0.2 M to about 0.4 M, about 0.2 M to about 0.3 M, about 0.3 M to about 1 M, about 0.3 M to about 0.9 M, about 0.3 M to about 0.8 M, about 0.3 M to about 0.7 M, about 0.3 M to about 0.6 M, about 0.3 M to about 0.5, about 0.3 M to about 0.8 M, about 0.3 M to about 0.7
  • the elution buffer includes about 0.1 M of glycine (pH 3.0) or about 0.1 M of citric acid (pH 3.0).
  • the eluted recombinant proteins are neutralized using a buffer that contains about 1 M Tris (pH 9.0).
  • the eluted recombinant proteins are neutralized using a buffer that contains about 1 M HEPES (pH 7.0) (e.g., between about 0.01 M to about 1 M, about 0.01 M to about 0.8 M, about 0.01 M to about 0.6 M, about 0.01 M to about 0.4 M, about 0.01 M to about 0.2 M, about 0.01 to about 0.1 M, about 0.01 M to about 0.05 M; about 0.02 M to about 1 M, about 0.02 M to about 0.8 M, about 0.02 M to about 0.6 M, about 0.02 M to about 0.4 M, about 0.02 M to about 0.2 M, about 0.02 to about 0.1 M, about 0.05 M to about 1 M, about 0.05 M to about 0.8 M, about 0.05 M to about 0.6 M, about 0.05 M to about 0.4 M, about 0.05 M to about 0.2 M, about 0.05 M to about 0.1 M, about 0.05 M to about 1 M, about 0.05 M to about 0.8 M, about 0.05 M to about 0.6 M, about
  • the eluting is performed using about 0.05 mL to about 1,000 mL, about 0.05 mL to about 950 mL, about 0.05 mL to about 900 mL, about 0.05 mL to about 850 mL, about 0.05 mL to about 800 mL, about 0.05 mL to about 750 mL, about 0.05 mL to about 700 mL, about 0.05 mL to about 650 mL, about 0.05 mL to about 600 mL, about 0.05 mL to about 550 mL, about 0.05 mL to about 500 mL, about 0.05 mL to about 450 mL, about 0.05 mL to about 400 mL, about 0.05 mL to about 350 mL, about 0.05 mL to about 300 mL, about 0.05 mL to about 250 mL, about 0.05 mL to about 200 mL, about 0.05 mL to about 150 mL, about
  • the eluting is performed at about 2 °C to about 25 °C (e.g., 2 °C to about 4 °C, about 2 °C to about 6 °C, about 2 °C to about 8 °C, about 2 °C to about 10 °C, about 2 °C to about 12 °C, about 4 °C to about 6 °C, about 4 °C to about 8 °C, about 4 °C to about 10 °C, about 4 °C to about 12 °C, about 6 °C to about 8 °C, about 6 °C to about 10 °C, about 6 °C to about 12 °C, about 10 °C to about 12 °C, about 10 °C to about 25 °C, about 10 °C to about 22 °C, about 10 °C to about 20 °C, about 10 °C to about 18 °C, about 10 °C to about 14 °C, about 12 °C to about 14 °C, about 12
  • the total amount of recombinant protein eluted is about 0.05 mg to about 80 mg (e.g., about 0.05 mg to about 0.1 mg, about 0.05 mg to about 0.2 mg, about 0.05 mg to about 0.5 mg, about 0.05 mg to about 0.6 mg, about 0.05 mg to about 0.8 mg, about 0.05 mg to about 1 mg, about 0.05 mg to about 2 mg, about 0.05 mg to about 5 mg, about 0.05 mg to about 10 mg, about 0.05 mg to about 15 mg, about 0.05 mg to about 20 mg, about 0.05 mg to about 25 mg, about 0.05 mg to about 30 mg, about 0.05 mg to about 35 mg, about 0.05 mg to about 40 mg, about 0.05 mg to about 45 mg, about 0.05 mg to about 50 mg, about 0.05 mg to about 55 mg, about 0.05 mg to about 60 mg, about 0.05 mg to about 65 mg, about 0.05 mg to about 70 mg, about 0.05 mg to about 75 mg, about 0.05 mg to about 80 mg, about 0.1 mg to about 0.2 mg (e.g
  • one or more additional unit operations can be performed on the recombinant protein.
  • the one or more additional unit operations is selected from the group of: viral inactivation, viral filtration, polishing, size-exclusion chromatography, cation exchange chromatography, anion exchange chromatography, hydrophobic interaction chromatography, mixed mode chromatography, reverse phase chromatography and ultrafiltration/diafiltration.
  • the one or more additional unit operations includes cleaning in place (CIP), where the precipitated and/or denatured substances are removed.
  • CIP cleaning in place
  • the method can further include a step of formulating the recombinant protein.
  • the recombinant protein is formulated into a recombinant protein drug product.
  • Example 1 Comparative Study of Traditional Purification Methods and “Capture & Purify” Method with Clarified Supernatant
  • Construct C26 a traditional IgG antibody, is expressed in HEK 293 suspension cells for 5 days at 1 L cell culture scale.
  • the supernatant is clarified by centrifugation and by filtration through a filter with a pore size of 0.22 ⁇ m.
  • the clarified supernatant is divided into four x 250 mL aliquots and stored at 4 o C until needed for testing.
  • One hundred-fifty mL of the clarified supernatant (or 185 mL for ⁇ KTA purification) is used to test three different methods of purification. All three methods of purification are performed using the same elution buffer and the same neutralization buffer.
  • the ⁇ KTA method uses a PBS wash and at least one citrate wash.
  • the first purification method ⁇ KTA is a programmed purification method using an immobilized resin within a prepacked column (i.e. commercial column).
  • the ⁇ KTA purification method is directional flow-based.
  • the second purification method is a traditional batch capture method. This traditional batch capture method includes over-night shaking with the resin floating freely in the clarified supernatant.
  • the third purification method called “Capture & Purify”, includes the use of a chromatography filtration pod as described herein.
  • the Capture & Purify method also includes over-night shaking identical to the traditional batch capture method. Results
  • the ⁇ KTA purification method used a 5 mL resin, while both the traditional batch capture method and the Capture & Purify method used a 3 mL. Nevertheless, 3-mL of resin was sufficient to capture a comparable amount of IgG antibody in the supernatant based on the observed binding capacity of the resin.
  • SDS-PAGE was performed to analyze 20 ⁇ L of flow through (FT) samples, 20 ⁇ L of wash samples (W), 1 ⁇ g of elution sample (E) taken during the traditional batch purification method and the Capture & Purify method (Figure 5). Reduced and non-reduced samples of both purification methods were run on a Life Tech 4-12% gel. SDS analysis suggests that protein capture was complete in both methods, as the protein of interest was not lost in the flow through or during the wash steps of either method.
  • the experimental time line of the three methods is outlined in Figure 6.
  • the ⁇ KTA purification method can take 7 days post-transfection to complete, with over 5-6 hours being dedicated to clarifying and setting up the experimental equipment.
  • the ⁇ KTA purification method is restricted by flow rate, and thus for larger volumes (greater than 1 L), the time to completion can increase dramatically, and sometimes can take longer than indicated in Figure 6.
  • only one protein can be purified at a time using the ⁇ KTA purification method.
  • the traditional batch purification method and the Capture & Purify method can capture and purify several proteins simultaneously in the timeframe as diagrammed in Figure 6.
  • the traditional batch purification method can take 7 days post-transfection to complete, is the most labor-intensive method of the three tested methods.
  • Both set ups were disposed in filtered (as described in Example 1 and 2) or unfiltered liquid cell culture, approximately 175 mL of culture media in a 150 mL bottle, by positioning the pod (in the filtered or unfiltered liquid cell culture) such that its longitudinal axis is approximately vertical. After the pod was removed from the liquid cell culture, the column was washed and then eluted using a PBS and citrate buffer, respectively. Table 3. Capture & Purify experimental conditions for IgG purification
  • Example 5 Study of Purification Methods Using a Pod having 20 ⁇ m or 40 ⁇ m Porous Mesh and the addition of a stop cock to control the flow rate after absorption
  • a transient expression 0.9L of a IgG and subsequent purification was used to compare the traditional batch capture method to a method using a pod and unfiltered culture media.
  • the pods in this instance, had openings, which were covered by either 20 or 40 micron porous mesh, on their vertical sides and bases.
  • the 0.9L culture was subdivided into 5 one hundred eighty mL aliquots. One of these aliquots was clarified by centrifugation and filtered through a filter with 0.22 micron pores, in the other four aliquots the cell culture media was not processed and termed “unfiltered”. All aliquots were added to 250 ml bottles.
  • the first set-up used 2 mL of free floating protein A chromatography resin exposed to filtered supernatant in free-floating batch capture environment with 150 RPM agitation. Subsequent purification employed a gravity flow column to capture the resin from the supernatant and then perform washes and elute the antibody from the resin. This gravity column was washed with 25 mL PBS, and eluted with 20 mL of elution buffer. This chromatography column was loaded with the same filtered cell culture medium containing an antibody as was used to contact the device/pod used in the third set-up.
  • the second set-up used 2.5 mL of free floating protein A chromatography resin exposed to filtered supernatant in a free-floating batch capture environment with 150 RPM agitation.
  • Subsequent purification employed a gravity flow column to capture the resin from the supernatant and then perform washes and elute the antibody from the resin.
  • This gravity column was washed with 37.5 mL of PBS and eluted with 30 mL of elution buffer.
  • This chromatography column was loaded with the same filtered cell culture medium as was used to contact the device/pod used in the fourth set-up.
  • the third set-up used the device shown in Figures 27 and 28 loaded with 2-mL protein A chromatography resin.
  • This resin in the pod/device was washed with 75 mL PBS, and eluted with 30 mL of elution buffer. After the elution buffer was first contacted with the resin in the pod/device, a 5 minute pause was implemented before additional elution buffer was flowed through the resin in the pod/device.
  • the chromatography performed using an ⁇ KTA system.
  • the resin in the pod/device was loaded with the same filtered cell culture medium as was used to contact the device/pod used in the first set-up. More specifically, the pod was contacted with 350 mL of the filtered cell culture medium in a 500 mL bottle.
  • the fourth set-up used the device shown in Figures 27 and 28 loaded with 3- mL protein A chromatography resin.
  • This resin in the pod/device was washed with 65 mL PBS, and eluted with 25 mL of elution buffer (at a flow rate of 3 mL/min).
  • the chromatography performed using an ⁇ KTA system.
  • the resin in the pod/device was loaded with the same filtered cell culture medium as was used to contact the device/pod used in the second set-up. More specifically, the pod was contacted with 350 mL of the filtered cell culture medium in a 500 mL bottle. Results

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Abstract

L'invention concerne des procédés de capture et de purification de protéines recombinantes à partir d'une culture cellulaire liquide comprenant une capsule de filtration et de chromatographie à double usage, des capsules de filtration et de chromatographie, et l'utilisation de ces capsules.
PCT/US2018/044863 2017-08-01 2018-08-01 Capsules de filtration et de chromatographie et leurs procédés d'utilisation WO2019028172A1 (fr)

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