Chromatographic method

Abstract

The present invention relates to a chromatography method. The present invention relates to a method for removing large contaminants such as viruses in a chromatography method for purifying target molecules, preferably monoclonal antibodies (mAbs), by using specially designed chromatography beads having a thin outer layer and a core functionalized with a ligand that absorbs said mAbs or parts thereof.

Description

This application is a divisional application of an invention patent application with an application date of June 4, 2015, application number 201580034231.7 (PCT / EP2015 / 062500), and invention name “Chromatography Method”. Field of the Invention

[0001] The present invention relates to a chromatography method. More specifically, the present invention relates to a method for removing large contaminants, such as viruses, in a chromatography method for purifying target molecules, preferably monoclonal antibodies (mAbs), by using specially designed chromatography beads having a thin outer layer and a core functionalized with a ligand that absorbs said mAbs or a portion thereof. Background of the Invention Immunoglobulins represent the most popular biopharmaceutical products manufactured or in development worldwide. The high commercial demand and resulting value of this particular therapeutic market has led to an emphasis on pharmaceutical companies to maximize the productivity of their respective mAb manufacturing processes while controlling associated costs.

[0003] In most cases, affinity chromatography is used as one of the key steps in purifying these immunoglobulin molecules, such as monoclonal or polyclonal antibodies. Affinity reagents of particular interest are proteins that can specifically bind to the invariant portion of the immunoglobulin molecule, this interaction being independent of the antigen-binding specificity of the antibody. Such reagents can be widely used for affinity chromatography recovery of immunoglobulins from different samples (such as, but not limited to, serum or plasma preparations or cell culture derived materials). An example of such a protein is a staphylococcal protein A that contains domains that can bind to the Fc and Fab portions of IgG immunoglobulins from different species.

[0004] Due to their high affinity and selectivity, reagents based on Staphylococcus protein A (SpA) find wide use in the field of biotechnology, for example in affinity chromatography for capturing and purifying antibodies and for detection. Currently, SpA-based affinity media are probably the most widely used affinity media for the separation of monoclonal antibodies and their fragments from different samples, including industrial raw materials from cell cultures. Therefore, various matrices containing protein A-ligands are commercially available, for example in the form of native protein A (e.g. protein A SEPHAROSE TM , GE Healthcare, Uppsala, Swenden) and also by recombinant protein A (e.g. rProtein A SEPHAROSE TM , GE Healthcare). More specifically, the genetic manipulations performed in commercial recombinant Protein A products are aimed at facilitating their attachment to vectors.

[0005] Virus removal is an essential purification step in the downstream purification of mAbs. Virus removal is routinely performed by passing the mAb feed through a dedicated virus removal filter with a defined cut-off, so that the mAb passes through the filter while the major portion of the virus cannot pass through.

[0006] Despite existing techniques for virus removal during immunoglobulin production, there remains a need for improved products and methods. SUMMARY OF THE INVENTION In a first aspect, the present invention relates to a method for purifying an immunoglobulin-containing protein or a portion thereof from larger contaminants such as viruses, comprising: a) contacting an immunoglobulin-containing sample with chromatography beads comprising a thin lid having a defined pore size and an inner core of defined pore size provided with an affinity ligand; b) adsorbing the immunoglobulin-containing protein to the ligand; c) washing the beads to remove any contaminants; and d) eluting the beads to release the captured immunoglobulin-containing protein or a portion thereof.

[0008] The average diameter size of the chromatography beads is 10-500 μm, such as 10-50 μm, 50-100 μm or 250-450 μm. The chromatography beads can be used, for example, for polishing, capture and midstream use after cell culture.

[0009] The size of the chromatography beads is selected depending on the specific application, eg where high resolution or fast mass transfer is desired, or when feed viscosity is high.The average size of the chromatography medium is for example calculated as the volume weighted median diameter.

[0010] The thickness of the cover is 1-8 μm, preferably 3-5 μm. The cover thickness can be advantageously varied depending on the overall bead size. For example, in some embodiments, it is desirable to have a thinner cover in order to maintain a high binding capacity. In other embodiments, when viral log reduction is more important than binding capacity, it is advantageous to design a chromatography medium with a thicker cover.

[0011] When mAb or Fab is the target, the cap is preferably inactive, ie not provided with any interacting ligand.The cap thickness can be measured by confocal microscopy, by microtome sectioning or conventional microscopy.

[0012] The pore size of the cap and core corresponds to a K of 0.1-1 D, preferably 0.3-0.7, which is measured using a dextran with a Mw of 110 kDa as a probe molecule. The present invention is applicable to target molecules larger than 20,000 D, such as Mabs of about 150,000 D. The pore size is important for capacity but also for selectivity. In swelling materials such as agarose and other polysaccharide gels, the pore size is best assessed by reverse exclusion chromatography, in which the volume fraction K through which the probe molecule (e.g., a dextran with a Mw of 110 kDa) can pass is determined. d This method is described, for example, in L Hagel et al.: J Chromatogr A 743, 33-42 (1996).

[0013] The pore size of the lid and the core can be the same or different. For purification of Mabs and Fabs, they are preferably the same.

[0014] In a preferred embodiment, the porosity of the cap and the core is 0.30-0.95, preferably about 0.60-0.85. The porosity of the cap and the core may be the same or different.

[0015] The porosity or pore density differences in the cap and core can be obtained by variations in the conditions under which the chromatographic medium is produced, such as using different amounts and percentages of bead material, crosslinking agents, salts, and shrinking agents. The porosity of the chromatographic medium can also be modified by selectively adding different charged or uncharged chemical entities that will physically and chemically reduce the porosity in different layers of the bead particle.

[0016] According to the invention, the cap and the core are made of agarose, but may also be made of other natural or synthetic polymers.

[0017] Preferably, the affinity ligand is a proteinaceous affinity ligand, and the ligand is most preferably protein A or an affinity ligand derived from protein A. The ligand can be a monomer, a dimer or any polymer. For Mabs, the ligand is preferably a pentamer (Z4).

[0018] In another embodiment, the affinity ligand is protein L or an affinity ligand derived from protein L. In this case, the immunoglobulin is a monoclonal antibody having a structure other than a naturally occurring antibody, such as a monospecific antibody (antigen-binding fragment, F(ab')2 fragment, Fab' fragment, single-chain variable fragment, bi-scFv, single domain antibody).

[0019] In yet another embodiment, the affinity ligand is protein G or an affinity ligand derived from protein G.

[0020] The chromatography beads may be provided in a column and the contaminants are obtained in the flow through. Alternatively, the chromatography beads are provided in an intermittent mode. The chromatography beads may be magnetic. In yet another alternative, the chromatography beads are provided in a fluidized bed mode.

[0021] The method according to the present invention is particularly suitable for removing contaminating viruses from Mab feed. In the downstream processing of antibodies, it is required to investigate and ensure that viruses that may be present in the feed are removed. Obtaining official approval, such as approval from the FDA, becomes a key issue. The effective steps of virus removal significantly simplify the verification of the method. The method of the present invention can be preferably used to obtain a verification process for therapeutic drugs / proteins (especially Mabs) that do not contain contaminating viruses.

[0022] As will be shown in the Examples section, the methods of the present invention provide significantly improved viral reduction compared to prior art methods.

[0023] It should be understood that the term "immunoglobulin-containing protein" includes antibodies and fusion proteins or conjugates and variant antibodies comprising antibody parts or Fc chains and antibody fragments such as FAbs, as long as they substantially maintain the binding properties of antibodies. The immunoglobulin-containing protein can be monospecific or bispecific (trifunctional antibodies, chemically linked F(ab')2, bispecific T-cell engagers). The antibody can be a monoclonal antibody or a polyclonal antibody. The antibody is preferably IgG, IgA and / or IgM from mammalian species such as humans.

[0024] The terms chromatography medium, chromatography beads, gel or resin cover the same concept and are used interchangeably. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 The chromatogram of Experiment 1 described below is shown, in which 1 mg of pure mAb and 1.2 x 10 7 pfu virus was injected into the prototype LS-02213C.

[0026] Figure 2 The chromatogram of experiment 2 is shown, in which 1.5 mg of semi-purified mAb mixed with phage was injected over the prototype LS-02213C.

[0027] Figure 3 A chromatogram is shown where 1.5 mg of semi-purified mAb mixed with phage was injected onto the commercial chromatography resin MabSelect Sure LX. DETAILED DESCRIPTION OF THE INVENTION According to a preferred embodiment of the present invention, viruses are removed in the mAb capture step by using a thin, inactive cover with a defined pore size and chromatography beads with adsorptive ligands inside the core of the beads. This will make it impossible for the virus to penetrate the cover, and the virus in the final feed will come out with the flow-through fraction, while the mAb will diffuse through the cover and absorb into the ligand inside the core of the beads. After column washing, the mAb is then conventionally eluted with acetic acid.

[0029] The invention will be described more closely in connection with the drawings and the experimental part.

[0030] In the present invention, two chromatography bead prototypes were synthesized: Protein A core beads with an inactive outer layer and the Protein A (Z4) ligand in the interior of the bead, referred to as prototypes LS-02213A, B, and C.

[0031] Protein L core beads having an inactive outer layer and a Protein L ligand in the interior of the bead, referred to as Prototype LS-002480.

[0032] Bacteriophage Φx174 (Escherichia coli phage 13706-1 TM ) was used as the test virus. Due to its small diameter of 25 nm, it is challenging to remove this virus from mAb by size exclusion. This virus particle has an icosahedral structure.

[0033] Bacteriophage Φx174 infects and proliferates in E. coli. Φx174 can be detected by a plaque agar overlay test using E. coli as a host. This test is extremely sensitive because it can detect single virus particles.

[0034] In the present invention, Φx174 13706 TM Escherichia coli strain C (also purchased from ATCC) is propagated. The phage is purified and the mAb sample is invaded with phage. This sample is loaded onto a chromatography column comprising a core bead with an inactive layer and an affinity ligand inside the bead. The column is cleaned after sample application and the mAb is finally eluted. During chromatography, fractions are collected to measure the viral titer in the selected fraction. The viral titer in the flow-through fraction, the first cleaning fraction and the mAb elution fraction is measured. The viral logarithm of the mAb elution fraction is calculated to reduce.

[0035] Experimental Section Example 1: Creation of cap / core beads Partially brominated and lid-deactivated allylated high flow agarose (HFA) was prepared according to US 6602990, 30 μmol allyl / mL gel, Kd: 0.675, d50v: 87.6, Dw: 70.3.

[0036] HFA beads were deactivated in the outer layer to form three different cap thickness sizes of 2, 4 and 6 μm.

[0037] Partial bromination The amount of bromine added was calculated to give a cap thickness greater than the target cap thickness of 0.5 μm, as experience has shown that the cap often becomes thinner than the target thickness due to bromine loss.

[0038] 100mL bromine solution was prepared in 100mL E-flask under magnetic stirring. The bromine solution was then added during ~60 seconds under vigorous stirring (200rpm). The flask was left at room temperature for 15 minutes. The gel was then washed with 12xGV distilled water.

[0039] 160mL allylated HFA 35Z from LS-001647A was washed with 5xGV distilled water, and then transferred to a 2L round-bottom flask. 1200mL distilled water was added and mechanical propeller was used for stirring. A solution of 68 μL bromine in 100mL distilled water was prepared in a 100mL E-flask under magnetic stirring. Bromine solution was then added during ~60 seconds under vigorous stirring (300rpm). The flask was left at room temperature for 15 minutes. The gel was then washed with 12xGV distilled water.

[0040] 100mL bromine solution was prepared in 100mL E-flask under magnetic stirring. The bromine solution was then added during ~60 seconds under vigorous stirring (300rpm). The flask was left at room temperature for 15 minutes. The gel was then washed with 12xGV distilled water.

[0041] Deactivate the activation cap A (2 μm cap): The partially activated gel from A above was transferred to a 500 mL round bottom flask. 53.7 mL of distilled water and 6.34 mL of 50% NaOH (1 M) were added and stirred with a mechanical propeller. The flask was immersed in a 50° C. water bath for 14.5 h. The gel was then washed with 10×GV distilled water.

[0042] Cap): The partially activated gel from B above was transferred to a 500 mL round bottom flask. 143.1 mL of distilled water and 16.9 mL of 50% NaOH (1 M) were added and stirred with a mechanical propeller. The flask was immersed in a 50°C water bath for 14.5 h. The gel was then washed with 10 x GV of distilled water.

[0043] Cap): The partially activated gel from C above was transferred to a 500 mL round bottom flask. 53.7 mL of distilled water and 6.34 mL of 50% NaOH (1 M) were added and stirred with a mechanical propeller. The flask was immersed in a 50°C water bath for 15 h. The gel was then washed with 10 x GV of distilled water.

[0044] Titration Wash the gel with plenty of water. Measure 1.0 mL of gel with a Teflon cube and transfer to a suction flask with 9 ml of distilled water. Add a saturated solution of bromine in water until the yellow color is due to Br. 2 The sample was left under magnetic stirring for 5 min. The sample was placed under vacuum (water suction) under magnetic stirring to remove excess bromine. The sample was then transferred to the titration beaker by rinsing the flask with 10 mL of distilled water. 2-3 drops of concentrated HNO 3 And start with 0.1MAgNO 3 Titration to indirectly measure the allyl content. Results are given in μmol / mL gel.

[0045] The allylated gel LS-001647A was titrated again (#2) and at the same time the remaining allyl content was determined. This time a lower allyl content was determined, 25.1 instead of 29.8 μmol allyl / mL gel. The total amount of partially activated bromine added was calculated based on the originally determined allyl content of LS-001647A, i.e. 29.8 μmol allyl / mL gel. The actual cap thickness was evaluated by using the remaining allyl content and the initial allyl content of 25.1 μmol / mL gel. The cap thickness could not be completely the target thickness, but three different levels were still obtained, which is satisfactory. The cap thickness can be detected using a confocal microscope.

[0046] Example 2: Construction of Protein A Core Beads In this example, protein A was immobilized into the core of allylated HFA with different cap inactivations.

[0047] Tetramer Z (N3A, N6D, N23T) using protein Z 4 –Cys (hereinafter referred to as Z4) serves as a ligand for protein A.

[0048] Gel activation The same procedure is used for LS-001840A, B and C.

[0049] 55 mL (g) of the dry gel was placed in an E-flask with 55 ml of distilled water and 2.2 g of NaOAc. The flask was swirled and then a saturated aqueous solution of bromine was added until a yellow color persisted. Sodium formate was then added to quench the excess bromine.

[0050] Reduced protein To 55 mL Z4 (51.7 mg / mL, based on AAA), add 570 mg NaHCO 3 (should be 465mg), 58mg Na 2 CO 3 , 481 mg NaCl and 20 mg EDTA. The E-flask was shaken and 212 mg DTE was added and then the flask was placed on a shaking table. The reduction was carried out for 90 minutes before filling the super loop.

[0051] Desalination Use Connect to A Sephadex G-25 column (~400 mL) of the system was used to desalt the tetramer. Before starting the run, the column was equilibrated with 0.15 M NaCl / 1 mM EDTA until conductivity and pH were stable. 52 mL of reducing solution produced 90.18 g of desalted tetramer. Fractions 9-17 (~90 mL) were collected.

[0052] Determination of protein concentration by UV The desalted solution was diluted 20-fold and had an absorbance of 0.286 at 276 nm. This equates to a protein concentration of 26.4 mg / mL (87% yield).

[0053] Coupling The activated gel was washed with 3xGV 0.1 M phosphate / 1 mM EDTA pH 8.6.

[0054] A: 51mL gel (LS-001840A) + 14mg Z4 / mL gel (27.1mL) + 10.5mL desalting buffer (should be 8.615mL) + 1.45M Na 2 SO 4 (17.86 g) were mixed in a 250 mL flask.

[0055] B: 51 mL gel (LS-001840B) + 15 mg Z4 / mL gel (29.0 mL) + 6.68 mL desalting buffer + 1.45 M Na 2SO 4 (17.86 g) were mixed in a 250 mL flask.

[0056] C: 51 mL gel (LS-001840C) + 16 mg Z4 / mL gel (31.0 mL) + 4.74 mL desalting buffer + 1.45 M Na 2 SO 4 (17.86 g) were mixed in a 250 mL flask.

[0057] Mechanical propeller stirring was applied and the flask was immersed in a 33°C water bath for 3 h.

[0058] Inactivation The gel was washed with 3xGV distilled water. The gel + 1GV 0.1M phosphate / 1mM EDTA / 7.5% thioglycerol pH8.5 was mixed and the flask was left at room temperature for 17h. The gel was then washed three times alternately with 1xGV 0.5M HAc and 2xGV 0.1M TRIS / 0.15M NaCl pH8.5 and then with 10xGV mL distilled water.

[0059] Drying method The dry weight of the prototype was determined by a single measurement. 1 mL of gel was measured using a Teflon cube and transferred to a pre-dried and pre-weighed glass filter. The gel was drained and washed twice with acetone. Drying was carried out overnight in an oven at 50° C. under vacuum, and the dry weight of the gel was determined by subtracting the mass of the pre-weighed glass filter.

[0060] Amino acid analysis 1 mL of each gel was dried and analyzed.

[0061] Dry weight: 81.9 mg / mL Ligand density: 3.8 mg / mL LS-002213B dry weight: 83.5 mg / mL ligand density: 5.0 mg / mL LS-002213C dry weight: 82.4 mg / mL ligand density: 5.0 mg / mL Example 3: Construction of protein L core beads Protein L was immobilized into the core with 3.5 μm cap-inactivated allylated HFA.

[0062] Reference: Structure of Peptostreptococcal Protein L and Identification of a Repeated Immunoglobulin Light Chain-binding Domain, The Journal of Biological Chemsitry, Volume 267, Issue 18, June 25, Pages 12820-12825, 1992, William Kastern, Ulf Sjobring and Lars

[0063] Gel activation 55 mL (g) of the xerogel from LS-001840B was placed in an E-flask with 55 ml of distilled water and 2.2 g of NaOAc. The flask was rotated and then a saturated aqueous solution of bromine was added until the yellow color persisted. Sodium formate was then added to remove excess bromine.

[0064] coupling The activated gel was washed with 3xGV 0.2M phosphate / 1 mM EDTA pH 11.5.

[0065] 52mL gel (LS-001840B) + 15mg PrL / mL gel (15.2mL) + 21.2mL coupling buffer + 1.30M Na 2 SO 4 (16.32 g) were mixed in a 250 mL flask.

[0066] Mechanical propeller stirring was applied and the flask was immersed in a 30 °C water bath for 17.5 h.

[0067] Inactivation The gel was washed with 3xGV distilled water and then 4xGV 0.1 phosphate / 1 mM EDTA pH 8.5. The gel + 1GV 0.1M phosphate / 1 mM EDTA / 7.5% thioglycerol pH 8.5 was mixed and the flask was immersed in a 45°C water bath for 2h under stirring. The gel was then washed three times alternately with 3xGV 0.1M HAc and 3xGV 0.1M TRIS / 0.15M NaCl pH 8.5 and then 10xGVmL distilled water.

[0068] Drying method The dry weight of the prototype was determined by a single measurement. 1 mL of gel was measured using a Teflon cube and transferred to a pre-dried and pre-weighed glass filter. The gel was drained and washed twice with acetone. Drying was carried out overnight in an oven at 50° C. under vacuum, and the dry weight of the gel was determined by subtracting the mass of the pre-weighed glass filter.

[0069] Amino acid analysis, AAA 1 mL of the gel was dried and analyzed.

[0070] LS-002480 Dry weight: 91.4 mg / mL gel Ligand density: 11.9 mg PrL / mL gel Multi-point attachment: 10.6 lysine / PrL of attachment gel When calculating ligand density and the extent of multipoint attachment, AAA data for the amino acids alanine, valine, isoleucine, and leucine were used.

[0071] Experiment 4: Phage and bacterial proliferation Nutrient Agar 23 g of nutrient agar and 5.0 g of NaCl were weighed into a 1000 mL glass beaker. The glass beaker was filled with 1000 milli-Q water, and the solution was mixed. The solution was autoclaved at 121° C. for 15 minutes. After autoclaving, the solution was stored in a 45° C. water bath.

[0072] Nutrient Broth 16 g of nutrient broth and 5.0 g of NaCl were weighed into a 1000 mL glass beaker. The glass beaker was filled with 1000 milli-Q water and the solution was mixed. The solution was autoclaved at 121° C. for 15 minutes. After autoclaving, the solution was stored in a 45° C. water bath.

[0073] Top agar 7.7 g of nutrient agar and 5.0 g of NaCl were weighed into a 1000 mL glass beaker. The glass beaker was filled with 1000 milli-Q water, and the solution was mixed. The solution was autoclaved at 121° C. for 15 minutes. After autoclaving, the solution was stored in a 45° C. water bath.

[0074] Preparation of agar plates 10 cm plates were prepared by adding 10 mL of 45°C nutrient agar to each plate. The plates were stored at 5°C.

[0075] 1 mL of nutrient broth was added to a freeze-dried vial from ATCC containing E. coli strain C. 50 μL of glycerol was also added and the vial was mixed thoroughly. The vials were stored at -70°C.

[0076] To propagate bacteria, a serological loop was placed in frozen bacteria and some bacteria were captured in the loop. The loop containing bacteria was pulled onto an agar plate. The agar plate was placed in a 37°C incubator overnight. Bacterial growth was seen in the agar plate the next day and bacteria were scraped from the serological loop and placed in a tube containing 3 mL of nutrient broth. The tube was incubated in an environmental shaker for 18 h and turbidity development of bacterial growth occurred after 18 hours.

[0077] Pipette 1 mL of nutrient broth onto the plate containing the E. coli phage 13706-01TM (Φx174) freeze-dried vials. 10cm agar plates were preheated for 1 hour at 37°C. 2 drops of 3mL bacterial culture were mixed with 2.5mL top agar. The aliquots were poured onto the preheated plates and the plates were solidified. 0.5mL of the phage solution rehydrated was serologically pipetted onto the surface of the top agar in the agar plates. The agar plates were incubated overnight and E. coli cells were seen to dissolve. The soft agar surface was scraped into a sterile 10mL centrifuge tube. The suspension was centrifuged at 4000rpm for 10 minutes, and the supernatant was aseptically filtered through a 0.2μm sterile filter into a sterile 1.5mL collection tube. The proliferation and filtered phage solution in the tube was stored at 5°C.

[0078] To determine the viral titer of the propagated phage solution, a plaque coverage assay was used.

[0079] Bacteria were propagated as above, but when cells were grown in broth tubes, growth ceased after 3 hours. 8 agar plates were pre-warmed at 37°C. Phage were serially diluted in sterile 1.5 mL tubes by pipetting 10 μL of phage stock and mixing with 990 μL of nutrient broth. This was 10 2 dilution.

[0080] The phage was further serially diluted up to 10 by taking 100 μL of each dilution and mixing with 900 μL of nutrient broth. 9 .

[0081] 100 μL of each dilution of phage was mixed with 300 μL of bacteria in a 10 mL sterile centrifuge tube, incubated for 15 minutes and then 3 mL of warm (45°C) top agar was added to each tube. The top agar mixed with bacteria and phage was poured onto an agar plate. The top agar was solidified at room temperature and placed in a 37°C incubator for 3 hours. After 3 hours, visible 3-5 mm plaques were seen and these plaques were countable in a colony counter at a certain dilution. At 10 717 plaques were found in the dilution, which means that the virus concentration in the stock solution is 17x10 7 / 0.1ml=17x10 8 =1.7x10 9 pfu (plaque forming units) / mL.

[0082] Example 5: Removal of viruses from mAb samples by chromatography Chromatography system avant 25sn 1536118, GE Healthcare Column Tricorn 5 / 100, GE Healthcare Resin LS-02213C Core beads 4.5-5μm OH-cap Ligand: 5.0 mg Z4 / mL gel Basement matrix: HFA Reference resin MabSelect SuRe LX, GE Healthcare sample: mAb, pure 3.0 mg / mL, purified with Mabselect SuRe, Capto S to remove acid and base variants and Capto bind to remove HCP and DNA mAb 5, semi-purified 15.0 mg / mL, purified only with MabSelect SuRe Test 1 15 μL of phage stock solution (1.7 x 10 9 pfu / mL), 1.0 mL of 3.0 mg / mL pure mAb in PBS and 0.48 mL PBS solution were mixed in a 1.5 mL tube. The solution was sterile filtered using a 0.2 μm sterile filter. This was the starting material. Note that sterile filtration may reduce the amount of phage. Trial 1 was performed on a prototype of the present invention.

[0083] Tests 2 and 3 50 μL of phage stock solution (1.7 x 10 9 pfu / mL), 1.0 mL 15 mg / mL PrA purified Mab (impure) and 3.95 mL PBS solution were mixed in a 10 mL centrifuge tube. The solution was filtered through a 0.45 μm filter. This was the starting material. Note that 0.45 μm filtration may reduce the amount of phage. Test 2 was performed on a prototype of the present invention. Test 3 is a comparative example performed on a commercial resin.

[0084] Chromatography Two tricorn columns were packed with prototype LS-002213C in 10 mM NaCl at a flow rate of 8 mL / min. The bed height was adjusted to 5.2 cm and the top adapter was adjusted to ~1 mm below the mark. 1.0 mL of resin was obtained in the column. The same packing procedure was performed for MaBSelectSuRe LX resin.

[0085] Check system flow at 1.0 mL / min using a 10 mL volumetric flask. Equilibrate the column for 5 cv at 0.25 mL / min (4 min residence time).

[0086] 500 μL of Mab mixture with phage was injected to each column using a 500 μL capillary loop. Fraction collection with 0.5 mL fractions was started.

[0087] After injection, the column was cleaned with 5cv PBS at 0.25mL / min followed by 5cv 0.1M NaOAc pH 6.0 at 0.25mL / min. mAbs were eluted with 5cv 0.1M HAc at 0.25mL / min, and the fraction collector fractionated only the eluted peaks using watchcommandos (peak start>50mAU, peak end<100mAU). The column was CIPed with 2cv 0.1M NaOH at 0.25mL / min (including a 20min hold time after 1cv), followed by equilibration with PBS 5cv at 0.25mL / min.

[0088] The mAb elution peaks in fractions b10 and b11 were combined (if necessary).

[0089] Virus count Bacteria were propagated as above, but on a 9 mL scale. Growth ceased after 3 hours. 90 agar plates were pre-warmed at 37°C. The various fractions selected and the initial sample were serially diluted in sterile 1.5 mL tubes by pipetting 100 μL of the fraction and mixing with 900 μL of nutrient broth. The samples were further serially diluted up to 10 by taking 100 μL of each dilution and mixing with 900 μL of nutrient broth. 7 .

[0090] 100 μL of each diluted sample was mixed with 300 μL of bacteria in a 10 mL sterile centrifuge tube, incubated for 15 minutes and then 3 mL of warm (45° C.) top agar was added to each tube. The top agar mixed with bacteria and phage was poured onto an agar plate. The top agar solidified at room temperature and placed in a 37° C. incubator for 3 hours. Visible 3-5 mm plaques were seen after 3 hours and these plaques were countable in a colony counter at a certain dilution.

[0091] result Figure 1 Shown is the chromatogram from experiment 1, where 1 mg of pure mAb mixed with phage was injected into prototype LS-02213C. Select fractions used for plaque coverage assay: initial sample, A1, A2, A3, A4 and mAb elution peaks B10 and B11 (combined volume = 0.56 mL).

[0092] The scheme for counting plaques (pfu) can be seen in Table 1. The numbers in bold are the numbers used for the calculations.

[0093] Table 1. Pfu counting scheme for selected fractions from the chromatogram at different dilutions, Experiment 1.

[0094] Mass balance calculation, test 1: Initial sample: 2.4x10 6 pfu / 0.1x0.5=1.2x10 7 pfu loaded A1: 1.1x10 6 / 0.1x0.5=5.5x10 6 pfu A2:5.0x10 5 / 0.1x0.5=2.5x10 6 pfu A3: 2.9x10 5 / 0.1x0.5=1.45x10 6 pfu A4: 8x10 4 / 0.1x0.5=0.4x10 6 pfu All A1-A4=9.85x10 6 pfu, yield = 0.985 / 1.20*100 = 82% Elution fraction: 1.8x10 2 / 0.1x0.56mL=1.0x10 3 pfu Reduce: log 10 (1.2x10 7 / 1.0x10 3 )=4.1 Mab yield: Mab loaded 1.0 mg, mAb eluted in pool = 0.83 mg Yield = 83% (measured by UV at 280 nm using an extinction coefficient (ext. coeff.) of 1.4) Figure 2The chromatogram of Experiment 2 is shown, in which 1.5 mg of semi-purified mAb and 2.8 x 10 7 The pfu is loaded onto the prototype LS-02213C.

[0095] The protocol for counting plaques (pfu) for Experiment 2 can be seen in Table 2. The numbers in bold are the numbers used for the calculations.

[0096] Table 2. Pfu counting scheme for selected fractions from the chromatogram at different dilutions, Experiment 2.

[0097] Mass balance calculation, test 2: Initial sample: 5.6x10 6 pfu / 0.1x0.5=2.8x10 7 pfu loaded A1: 1.1x10 5 / 0.1x0.5=0.55x10 6 pfu A2:5.0x10 5 / 0.1x0.5=2.5x10 6 pfu A3: 3.2x10 5 / 0.1x0.5=1.6x10 6 pfu A4: 2.5x10 5 / 0.1x0.5=1.25x10 6 pfu A5: 1.6x10 5 / 0.1x0.5=0.8x10 6 pfu Total A1-A5=0.67x10 7 pfu, yield = 0.67 / 2.8*100 = 24% Elution fraction: 5.3x10 3 / 0.1x0.58mL=3.1x10 4 pfu Reduce: log 10 (2.8x10 7 / 3.1x10 4 )=3.0 Mab yield: Mab loaded 1.5 mg, mAb eluted in pool = 1.34 mg Yield = 89% (measured by UV at 280 nm using an extinction coefficient (ext. coeff.) of 1.4) Figure 3A chromatogram is shown where 1.5 mg of semi-purified mAb mixed with phage was injected onto MabSelectSure LX. Select fractions used for plaque coverage assay: initial sample, A1, A2, A3, A4, A5 and mAb elution peak.

[0098] In the chromatogram, 1.5 mg of semi-purified mAb and 1.4 x 107 pfu were loaded onto MaBSelect SuRe.

[0099] Table 3. Pfu counting scheme for selected fractions from the chromatogram at different dilutions, Experiment 3.

[0100] Mass balance calculation, test 3: Initial sample: 2.8x10 6 pfu / 0.1x0.5=1.4x10 7 pfu loaded A1: 0.3x10 5 / 0.1x0.5=0.15x10 6 pfu A2: 2.2x10 5 / 0.1x0.5=1.1x10 6 pfu A3: 2.4x10 5 / 0.1x0.5=1.2x10 6 pfu A4: 1.0x10 5 / 0.1x0.5=0.5x10 6 pfu A5: 0.35x10 5 / 0.1x0.5=0.17x10 6 pfu Total A1-A5=3.1x10 6 pfu, yield = 0.31 / 1.4*100 = 22% Elution fraction: 2.4x10 4 / 0.1x0.66mL=1.6x10 5 pfu Reduce: log 10 (1.4x10 7 / 1.6x10 5 )=1.9 Mab yield: Mab loaded 1.5 mg, mAb eluted in pool = 1.36 mg Yield = 91% (measured by UV at 280 nm using an extinction coefficient (ext. coeff.) of 1.4) result For the prototype LS-02213C, the virus reduction with the pure mAb was better (1 log) than the assay using the semi-purified mAb. The virus recovery in the assay using the pure mAb was also better (82%) compared to the assay using the semi-purified mAb (only 24%).

[0101] In experiments 2 and 3, where the same starting material was used, it was clearly shown that prototype LS-02213C had better viral reduction performance than MabSelect SuRe using semi-purified mAb feed.Prototype LS-02213C showed better viral reduction (1 log) than MabSelectSuRe.

[0102] The results showed that using pure mAb, the prototype LS-02213C was able to reduce the amount of phage with a 4.1 log reduction and 83% mAb yield.

[0103] Using semi-purified mAbs, LS-02213C achieved a viral log reduction of 3.0, while MabSelect Sure LX achieved a viral log reduction of 1.9.

[0104] It should be noted that the log reduction depends on the residence / absorption time (flow rate) and the wash volume prior to eluting the protein. In the above examples, a standard column residence time of 4 min and a standard wash (prior to elution) of 10 column volumes were used.

Claims

1. A method for purifying an immunoglobulin-containing protein or a portion thereof from larger contaminants, comprising: a) contacting an immunoglobulin-containing sample with chromatography beads comprising a thin cover having a defined pore size and an inner core of defined pore size provided with an affinity ligand; b) adsorbing the immunoglobulin-containing protein to the ligand; c) washing the beads to remove any contaminants; and d) eluting the beads to release the captured immunoglobulin-containing proteins or portions thereof.

2. The method according to claim 1, wherein the contaminant is a virus.

3. The method according to claim 1 or 2, wherein the average diameter size of the chromatography beads is 10-500 μm, such as 10-50 μm, 50-100 μm or 250-450 μm.

4. A method according to claim 1, 2 or 3, wherein the cover has a thickness of 1-8 μm, preferably 3-5 μm.

5. A method according to any one of the preceding claims, wherein the pore size of the cap and the core corresponds to K D 0.1-1, preferably about 0.3-0.

7.

6. A method according to any one of the preceding claims, wherein the porosity of the cap and core is between 0.30 and 0.95, preferably about 0.60 and 0.

85.

7. A method according to any one of the preceding claims, wherein the cap and core are made of agarose.

8. A method according to any one of the preceding claims, wherein the pore size and / or porosity of the cap and core are the same or different.

9. The method according to any one of the preceding claims, wherein the affinity ligand is a proteinaceous affinity ligand.

10. The method according to claim 9, wherein the ligand is protein A or an affinity ligand derived from protein A, such as protein Z4.

11. The method according to claim 10, wherein the immunoglobulin-containing protein is a mAb.

12. The method according to claim 9, wherein the affinity ligand is protein L or an affinity ligand derived from protein L.

13. The method according to claim 12, wherein the immunoglobulin is Fab.

14. The method according to claim 9, wherein the affinity ligand is protein G or an affinity ligand derived from protein G.

15. The method according to any one of the preceding claims, wherein the chromatography beads are provided in a column and the contaminants are obtained in the flow-through.

16. The method according to any one of the preceding claims, wherein the chromatography beads are provided in batch mode.

17. A method according to claim 15 or 16, wherein the chromatography beads are magnetic.

18. The method according to claim 15 or 17, wherein the chromatography beads are provided in fluidized bed mode.

19. A method according to any one of the preceding claims for use in validating a method to obtain a therapeutic immunoglobulin substantially free of contaminating viruses.

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