BUFFERS AND METHODS FOR PURIFYING PROTEINS

Abstract

The invention relates to the field of protein purification processes involving several chromatography steps. The invention pertains to a method for purifying a protein, preferably an antibody or fragment thereof or a protein containing said fragment, from a complex solution, wherein said method comprises at least two chromatography steps which are performed using buffers comprising or consisting of the same chemical compounds. The invention is particularly useful for large scale production and purification of recombinant proteins.

Claims

1-15. (canceled)

16. A method for purifying a protein of interest from a complex solution, said method comprising at least a first liquid chromatography and a second chromatography, wherein for each said liquid chromatography aqueous buffers comprising acetate, phosphate, TRIS base, and optionally sodium hydroxide and/or sodium chloride are used as at least one buffer selected from an equilibration buffer, a loading adjustment buffer, a wash buffer, an elution buffer, a regeneration buffer and a rinse buffer.

17. The method of claim 16, wherein in either the first or the second liquid chromatography, said aqueous buffers are used as at least two buffers selected from an equilibration buffer, a loading adjustment buffer, a wash buffer, an elution buffer, a regeneration buffer and a rinse buffer.

18. The method of claim 16, wherein in the first and in the second liquid chromatography, said aqueous buffers are used as at least two buffers selected from an equilibration buffer, a loading adjustment buffer, a wash buffer, an elution buffer, a regeneration buffer and a rinse buffer.

19. The method of claim 16, wherein in either the first or the second liquid chromatography, said aqueous buffers are used as at least three buffers selected from an equilibration buffer, a loading adjustment buffer, a wash buffer, an elution buffer, a regeneration buffer and a rinse buffer.

20. The method of claim 16, wherein in the first and in the second liquid chromatography, said aqueous buffers are used as at least three buffers selected from an equilibration buffer, a loading adjustment buffer, a wash buffer, an elution buffer, a regeneration buffer and a rinse buffer.

21. The method of claim 16, wherein said aqueous buffers consist of water, acetate, phosphate, TRIS base, and optionally sodium hydroxide and/or sodium chloride.

22. The method of claim 16, wherein said aqueous buffers have the same molar ratio of phosphate over Tris base and the same molar ratio of acetate over phosphate.

23. The method of claim 16, wherein the molar ratio of phosphate over Tris base in said aqueous buffers is between 1.5 and 2.

24. The method of claim 16, wherein the molar ratio of acetate over phosphate in said aqueous buffers is between 1 and 2.5.

25. The method of claim 16, wherein said protein of interest is an antibody, a fragment thereof, or a fusion protein comprising said antibody fragment.

26. The method of claim 16, wherein said protein of interest has an isoelectric point between 6 and 9.5.

27. A method for purifying a protein of interest from a complex solution, wherein said method comprises purifying said complex solution containing said protein of interest through at least a first liquid chromatography and a second chromatography, wherein for each said first and second liquid chromatography, at least one step selected from the group consisting of equilibrating a solid stationary phase, washing a solid stationary phase, eluting the protein of interest, and regenerating a solid stationary phase, is performed using aqueous buffers comprising the compounds acetate, phosphate, TRIS base, and optionally sodium hydroxide and/or sodium chloride.

28. The method of claim 27, wherein said method comprises: a) purifying said complex solution comprising said protein of interest using at least a first liquid chromatography comprising a first solid stationary phase, thereby obtaining a first protein of interest eluent, wherein at least one step selected from the group consisting of equilibrating said first solid stationary phase, washing said first solid stationary phase, eluting the protein of interest, and regenerating said first solid stationary phase, is performed using aqueous buffers comprising the compounds acetate, phosphate, TRIS base, and optionally sodium hydroxide and/or sodium chloride; and b) purifying the first protein of interest eluent of step a) using a second liquid chromatography comprising a second solid stationary phase, thereby obtaining a second protein of interest eluent, wherein at least one step selected from the group consisting of equilibrating said second solid stationary phase, washing said second solid stationary phase, eluting the protein of interest, and regenerating said second solid stationary phase, is performed using aqueous buffers comprising acetate, phosphate, TRIS base, and optionally sodium hydroxide and/or sodium chloride.

29. The method of claim 27, wherein in either the first or the second liquid chromatography, at least two steps selected from the group consisting of equilibrating said solid stationary phase, washing said solid stationary phase, eluting the protein of interest, and regenerating said solid stationary phase are performed using aqueous buffers comprising acetate, phosphate, TRIS base, and optionally sodium hydroxide and/or sodium chloride.

30. The method of claim 27, wherein said aqueous buffers consist of water, acetate, phosphate, TRIS base, and optionally sodium hydroxide and/or sodium chloride.

31. The method of claim 27, wherein said aqueous buffers have the same molar ratio of phosphate over Tris base and the same molar ratio of acetate over phosphate.

32. The method of claim 27, wherein the molar ratio of phosphate over Tris base in said aqueous buffers is between 1.5 and 2.

33. The method of claim 27, wherein the molar ratio of acetate over phosphate in said aqueous buffers is between 1 and 2.5.

34. The method of claim 27, wherein said protein of interest is an antibody, a fragment thereof, or a fusion protein comprising said antibody fragment.

35. The method of claim 27, wherein said protein of interest has an isoelectric point between 6 and 9.5.

Description

DESCRIPTION OF THE FIGURES

[0108] FIG. 1: Principle of buffer formulation and uses according to the invention

[0109] FIG. 2: Theorical titration curves with two different formulation using NaOH 1 mol/L

[0110] FIG. 3: Experimental titration curves. Evolution of pH and conductivity of an aqueous buffer solution comprising 55 mM acetate, 20 mM phosphate and 10 mM Tris base upon addition of gradual amount of NaOH

EXAMPLES

[0111] The following examples are meant as an illustration of various embodiments of the invention and shall not be construed as limiting the scope or the definition of the invention in any way.

Example 1. Theoretical and Experimental Titration of Two Buffers According to the Invention Support Versatility of Such Buffer Composition for DSP Processes

[0112] In the following experiments, titration of two aqueous buffers comprising 55 mM acetate, 20 mM phosphate and 10 mM Tris base or 20 mM acetate, 20 mM phosphate and 10 mM Tris base, upon addition of NaOH was performed first theoretically, i.e in silico using mathematical models defined in the art, or experimentally i.e. in laboratory settings.

[0113] 1. Material and Methods

[0114] Solutions

[0115] For the preparation of the buffer, the following solutions were made: [0116] 55 mM acetate, 20 mM phosphate and 10 mM Tris base [0117] NaOH 1M as titration solution [0118] These solutions are made with the corresponding commercial species from Merck KGaA: [0119] Glacial acetic acid (64-19-7) [0120] Orthophosphoric acid 85% (7664-38-2) [0121] Tris base (77-86-1) [0122] NaOH 32% (1310-73-2)

[0123] Buffers are prepared at room temperature.

[0124] Theoretical Titration

[0125] The theoretical titration of two aqueous buffers comprising 55 mM acetate, 20 mM phosphate and 10 mM Tris base or 20 mM acetate, 20 mM phosphate and 10 mM Tris base, upon addition of NaOH is performed based on the Henderson-Hasselbalch equation (thermodynamic model):

[00001]$\mathrm{pH}=\mathrm{pKa}+\log \left(\frac{\left[A-\right]}{\left[\mathrm{HA}\right]}\right)$ [0126] pH: pH of the solution for a given values of species concentration [0127] Ka: acid dissociation constant [0128] [A]: concentration of the conjugate base [0129] [HA]: concentration of the acid

[0130] An in-house software is used to perform the calculation for a multi-equilibrium case. This method is detailed by Baeza-Baeza et al. (Systematic Approach To Calculate the Concentration of Chemical Species in Multi-Equilibrium Problems, Journal of Chemical Education 2011 88 (2), 169-173).

[0131] Experimental Titration

[0132] Titration of the pH was performed using 713 pH Meter device from Metrohm.

[0133] Conductivity measurements was performed using 712 Conductometer from Metrohm.

[0134] Measurements are performed according supplier recommendations. Each point of the titration corresponds to a certain volume addition of NaOH 1M and thus, a total concentration of NaOH in the buffer.

[0135] Results

[0136] As shown in FIGS. 1 and 2, upon addition of NaOH, the pH of the aqueous buffer solution roughly evolves according to a linear curve. No striking pH rise is observed, confirming the buffer properties of the solution over the entire range comprised between pH 2 and 10.

Example 2. Example of an Implementation of a Purification Cascade Using the Method of the Invention

[0137] A monoclonal IgG1 antibody (hereafter A1) having an isoelectric point of about 8.5 was recombinantly produced in CHO cells using usual cell culture methods, and the respective harvested cell culture fluids were purified according to two different embodiments of the method of the invention.

[0138] A. Purification Method 1

[0139] For purification method 1, the following downstream purification cascade was performed, in that order: [0140] 1. Protein A affinity chromatography using Amsphere A3 column (from JSR Life Science) [0141] a) The column was equilibrated using a buffer consisting of water, 0.05M Acetate, 0.02M Phosphate, 0.01M Tris Base, adjusted at pH 8 with NaOH 1M [0142] b) The cell culture harvest was filtrated on Stericup 0.22 m from Merck Millipore then loaded on the column; [0143] c) A first wash buffer was flown through the column, which consisted of water, 0.05M Acetate, 0.02M Phosphate, 0.01 M Tris Base, adjusted at pH 8 with NaOH 1M [0144] d) A second wash buffer was flown through the column, which consisted of water, 0.05M Acetate, 0.02M Phosphate, 0.01 M Tris Base, 1.5M NaCl, adjusted at pH 5.5 with NaOH 1M [0145] e) A third wash buffer was flown through the column, which consisted of water, 0.05M Acetate, 0.02M Phosphate, 0.01 M Tris Base, adjusted at pH 8 with NaOH 1M [0146] f) The protein of interest was eluted from the column using a buffer which consisted of water, 0.05M Acetate, 0.02M Phosphate, 0.01 M Tris Base, adjusted at pH 3.2 with NaOH 1M. The eluate was conserved for the next chromatography; [0147] g) Regeneration was performed using NaOH 0.1M [0148] h) Sanitation was performed using NaOH 0.5M [0149] i) Rinse was performed using a buffer which consisted of water, 0.05M Acetate, 0.02M Phosphate, 0.01 M Tris Base, adjusted at pH 8 with NaOH 1M [0150] 2. CEX chromatography using Fractogel EMD COO.sup. (M) column (from Merck Millipore); [0151] a) The column was equilibrated using a buffer consisting of water, 0.05M Acetate, 0.02M Phosphate, 0.01 M Tris Base, adjusted at pH 5.5 with NaOH 1M [0152] b) The eluate of step 1. f) was adjusted at pH 5.5 with NaOH 1M prior to the load on the column. [0153] c) A wash buffer was flown through the column, which consisted of water, 0.05M Acetate, 0.02M Phosphate, 0.01 M Tris Base, adjusted at pH 5.5 with NaOH 1M. The resulting flowthrough (2.b and 2.c) was conserved for the next chromatography; [0154] d) Regeneration was performed using a buffer which consisted of water, 0.05M Acetate, 0.02M Phosphate, 0.01 M Tris Base, 1M NaCl, adjusted at pH 8 with NaOH 1M [0155] e) Sanitation was performed using NaOH 0.5M [0156] f) Rinse was performed using a buffer which consisted of water, 0.05M Acetate, 0.02M Phosphate, 0.01 M Tris Base, adjusted at pH 5.5 with NaOH 1M [0157] 3. AEX chromatography using Capto Adhere column (from GE Healthcare); [0158] a) The column was equilibrated using a buffer consisting of water, 0.025M Acetate, 0.01M Phosphate, 0.005 M Tris Base, adjusted at pH 7.5 with NaOH 1M [0159] b) The CEX flowthrough was adjusted at pH 7.5 with NaOH 1M prior to the load on the column. [0160] c) A wash buffer was flown through the column, which consisted of water, 0.025M Acetate, 0.01M Phosphate, 0.005 M Tris Base, adjusted at pH 7.5 with NaOH 1M. The resulting flowthrough (3.b and 3.c) was conserved for analyses; [0161] d) Regeneration was performed using a buffer which consisted of water, 0.05M Acetate, 0.02M Phosphate, 0.01 M Tris Base, 1.5M NaCl, adjusted at pH 5.5 with NaOH 1M [0162] e) Sanitation was performed using NaOH 0.5M

[0163] Quality and impurities analysis for the flowthrough of step 3b)c) was performed. The results are presented in table 1.

[0164] B. Purification Method 2

[0165] For purification method 2, the following downstream purification cascade was performed, in that order: [0166] 1. Protein A affinity chromatography using Amsphere A3 column (from JSR Life Science) [0167] a) The column was equilibrated using a buffer consisting of water, 0.02M Acetate, 0.02M Phosphate, 0.01 M Tris Base, adjusted at pH 8 with NaOH 1M [0168] b) The cell culture harvest was filtrated on Stericup 0.22 m from Merck Millipore then loaded on the column; [0169] c) A first wash buffer was flown through the column, which consisted of water, 0.02M Acetate, 0.02M Phosphate, 0.01 M Tris Base, adjusted at pH 8 with NaOH 1M [0170] d) A second wash buffer was flown through the column, which consisted of water, 0.02M Acetate, 0.02M Phosphate, 0.01 M Tris Base, 1.5M NaCl, adjusted at pH 5.5 with NaOH 1M [0171] e) A third wash buffer was flown through the column, which consisted of water, 0.02M Acetate, 0.02M Phosphate, 0.01 M Tris Base, adjusted at pH 8 with NaOH 1M [0172] f) The protein of interest was eluted from the column using a buffer which consisted of water, 0.02M Acetate, 0.02M Phosphate, 0.01 M Tris Base, adjusted at pH 3.2 with NaOH 1M. The eluate was conserved for the next chromatography; [0173] g) Regeneration was performed using NaOH 0.1M [0174] h) Sanitation was performed using NaOH 0.5M [0175] i) Rinse was performed using a buffer which consisted of water, 0.02M Acetate, 0.02M Phosphate, 0.01 M Tris Base, adjusted at pH 8 with NaOH 1M [0176] 2. CEX chromatography using Fractogel EMD COO.sup. (M) column (from Merck Millipore); [0177] a) The column was equilibrated using a buffer consisting of water, 0.02M Acetate, 0.02M Phosphate, 0.01 M Tris Base, adjusted at pH 5.5 with NaOH 1M [0178] b) The eluate of step 1. f) was adjusted at pH 5.5 with NaOH 1M prior to the load on the column. [0179] c) A wash buffer was flown through the column, which consisted of water, 0.02M Acetate, 0.02M Phosphate, 0.01 M Tris Base, adjusted at pH 5.5 with NaOH 1M. The resulting flowthrough (2.b and 2.c) was conserved for the next chromatography; [0180] d) Regeneration was performed using a buffer which consisted of water, 0.02M Acetate, 0.02M Phosphate, 0.01 M Tris Base, 1M NaCl, adjusted at pH 8 with NaOH 1M [0181] e) Sanitation was performed using NaOH 0.5M [0182] f) Rinse was performed using a buffer which consisted of water, 0.02M Acetate, 0.02M Phosphate, 0.01 M Tris Base, adjusted at pH 5.5 with NaOH 1M [0183] 3. AEX chromatography using Capto Adhere column (from GE Healthcare); [0184] a) The column was equilibrated using a buffer consisting of water, 0.02M Acetate, 0.02M Phosphate, 0.01 M Tris Base, adjusted at pH 7.5 with NaOH 1M [0185] b) The CEX flowthrough was adjusted at pH 7.5 with NaOH 1M prior to the load on the column. c) A wash buffer was flown through the column, which consisted of water, 0.02M Acetate, [0186] 0.02M Phosphate, 0.01 M Tris Base, adjusted at pH 7.5 with NaOH 1M. The resulting flowthrough (3.b and 3.c) was conserved for analyses; [0187] d) Regeneration was performed using a buffer which consisted of water, 0.1M Acetate, 0.1M Phosphate, 0.05 M Tris Base [0188] e) Sanitation was performed using NaOH 0.5M [0189] f) Rinse was performed using a buffer which consisted of water, 0.05M Acetate, 0.02M Phosphate, 0.01 M Tris Base, adjusted at pH 7.5 with NaOH 1M

[0190] Quality and impurities analysis for the flowthrough of step 3b)c) was performed. The results are presented in table 2.

TABLE-US-00001 TABLE 1 A1 purification specifications further to purification according to method 1 Buffer Mix 1 Yield Cumulative HCP DNA HMW LMW Steps % yield % ppm ppb % % Harvest 212690 148234 4.8 2.7 Capture 97.1 97.1 7354 1893 9.2 2.6 CEX 88.2 85.6 3214 2.5 2.3 MM 85.3 73.1 11 0.3 1.8

TABLE-US-00002 TABLE 2 A1 purification specifications further to purification according to method 2 Buffer Mix 2 Yield Cumulative HCP DNA HMW LMW Steps % yield % ppm ppb % % Harvest 209462 2136078 3.5 6.6 Capture 100 100 4614 4996 7.5 7.0 CEX 87.7 87.7 1808 2.0 6.3 MM 78.3 68.7 20 0.3 4.7

[0191] Conclusion: The yield of both purification cascades are comparable (around 70%). Despite the use of different harvest lot, the aggregate percentage and HOP concentration are very low and comparable for both cascades (less than 20 ppm of HOP and 0.3% of HMW at the end of the purification). The high value of clipped form in the second example is due to the initial value of the corresponding harvest and a limited impact of the purification on the LMW. To conclude, the different buffer ratio used show great and comparable result in term of performance and purification.

Example 3. Example of an Implementation of a Purification Cascade Using the Method of the Invention

[0192] A fusion protein comprising an antibody fragment (hereafter A2) having an isoelectric point of 6.09, was recombinantly produced in CHO cells using usual cell culture methods, and the respective harvested cell culture fluids were purified according to two embodiments of method of the invention.

[0193] A. Purification Method 1

[0194] For purification method 1, the following downstream purification cascade was performed, in that order: [0195] 1. Protein A affinity chromatography using Amsphere A3 column (from JSR Life Science) [0196] a) The column was equilibrated using a buffer consisting of water, 0.05M Acetate, 0.02M Phosphate, 0.01 M Tris Base, adjusted at pH 8 with NaOH 1M [0197] b) The cell culture harvest was filtrated on Stericup 0.22 m from Merck Millipore then loaded on the column; [0198] c) A first wash buffer was flown through the column, which consisted of water, 0.05M Acetate, 0.02M Phosphate, 0.01 M Tris Base, adjusted at pH 8 with NaOH 1M [0199] d) A second wash buffer was flown through the column, which consisted of water, 0.05M Acetate, 0.02M Phosphate, 0.01 M Tris Base, 1.5M NaCl, adjusted at pH 5.5 with NaOH 1M [0200] e) A third wash buffer was flown through the column, which consisted of water, 0.05M Acetate, 0.02M Phosphate, 0.01 M Tris Base, adjusted at pH 8 with NaOH 1M [0201] f) The protein of interest was eluted from the column using a buffer which consisted of water, 0.05M Acetate, 0.02M Phosphate, 0.01 M Tris Base, adjusted at pH 3.2 with NaOH 1M. The eluate was conserved for the next chromatography; [0202] g) Regeneration was performed using NaOH 0.1M [0203] h) Sanitation was performed using NaOH 0.5M [0204] i) Rinse was performed using a buffer which consisted of water, 0.05M Acetate, 0.02M Phosphate, 0.01 M Tris Base, adjusted at pH 8 with NaOH 1M [0205] 2. CEX chromatography using Fractogel EMD COO.sup. (M) column (from Merck Millipore); [0206] a) The column was equilibrated using a buffer consisting of water, 0.05M Acetate, 0.02M Phosphate, 0.01 M Tris Base, adjusted at pH 5.5 with NaOH 1M [0207] b) The eluate of step 1. f) was adjusted at pH 5.5 with NaOH 1M prior to the load on the column. [0208] c) A wash buffer was flown through the column, which consisted of water, 0.05M Acetate, 0.02M Phosphate, 0.01 M Tris Base, adjusted at pH 5.5 with NaOH 1M. The resulting flowthrough (2.b and 2.c) was conserved for the next chromatography; [0209] d) Regeneration was performed using a buffer which consisted of water, 0.05M Acetate, 0.02M Phosphate, 0.01 M Tris Base, 1M NaCl, adjusted at pH 8 with NaOH 1M [0210] e) Sanitation was performed using NaOH 0.5M [0211] f) Rinse was performed using a buffer which consisted of water, 0.05M Acetate, 0.02M Phosphate, 0.01 M Tris Base, adjusted at pH 5.5 with NaOH 1M [0212] 3. AEX chromatography using Eshmuno Q column (from Merck Millipore); [0213] a) The column was equilibrated using a buffer consisting of water, 0.025M Acetate, 0.01M Phosphate, 0.005 M Tris Base, adjusted at pH 5 with NaOH 1M [0214] b) The CEX flowthrough was adjusted at pH 5 with Acetic acid 1M prior to the load on the column. [0215] c) A wash buffer was flown through the column, which consisted of water, 0.025M Acetate, 0.01M Phosphate, 0.005 M Tris Base, adjusted at pH 5 with NaOH 1M. The resulting flowthrough (3.b and 3.c) was conserved for analyses; [0216] d) Regeneration was performed using a buffer which consisted of water, 0.05M Acetate, 0.02M Phosphate, 0.01 M Tris Base, 1.5M NaCl, adjusted at pH 5.5 with NaOH 1M [0217] e) Sanitation was performed using NaOH 0.5M [0218] f) Rinse was performed using a buffer which consisted of water, 0.05M Acetate, 0.02M Phosphate, 0.01 M Tris Base, adjusted at pH 5 with NaOH 1M Quality and impurities analysis for the flowthrough of step 3b)c) was performed. The results are presented in table 1.

[0219] C. Purification Method 2

[0220] For purification method 2, the following downstream purification cascade was performed, in that order: [0221] 1. Protein A affinity chromatography using Amsphere A3 column (from JSR Life Science) [0222] a) The column was equilibrated using a buffer consisting of water, 0.02M Acetate, 0.02M Phosphate, 0.01 M Tris Base, adjusted at pH 8 with NaOH 1M [0223] b) The cell culture harvest was filtrated on Stericup 0.22 m from Merck Millipore then loaded on the column; [0224] c) A first wash buffer was flown through the column, which consisted of water, 0.02M Acetate, 0.02M Phosphate, 0.01 M Tris Base, adjusted at pH 8 with NaOH 1M [0225] d) A second wash buffer was flown through the column, which consisted of water, 0.02M Acetate, 0.02M Phosphate, 0.01 M Tris Base, 1.5M NaCl, adjusted at pH 5.5 with NaOH 1M [0226] e) A third wash buffer was flown through the column, which consisted of water, 0.02M Acetate, 0.02M Phosphate, 0.01 M Tris Base, adjusted at pH 8 with NaOH 1M [0227] f) The protein of interest was eluted from the column using a buffer which consisted of water, 0.02M Acetate, 0.02M Phosphate, 0.01 M Tris Base, adjusted at pH 3.2 with NaOH 1M. The eluate was conserved for the next chromatography; [0228] g) Regeneration was performed using NaOH 0.1M [0229] h) Sanitation was performed using NaOH 0.5M [0230] i) Rinse was performed using a buffer which consisted of water, 0.02M Acetate, 0.02M Phosphate, 0.01 M Tris Base, adjusted at pH 8 with NaOH 1M [0231] 2. CEX chromatography using Fractogel EMD COO.sup. (M) column (from Merck Millipore); [0232] a) The column was equilibrated using a buffer consisting of water, 0.02M Acetate, 0.02M Phosphate, 0.01 M Tris Base, adjusted at pH 5.5 with NaOH 1M [0233] b) The eluate of step 1. f) was adjusted at pH 5.5 with NaOH 1M prior to the load on the column. [0234] c) A wash buffer was flown through the column, which consisted of water, 0.02M Acetate, 0.02M Phosphate, 0.01 M Tris Base, adjusted at pH 5.5 with NaOH 1M. The resulting flowthrough (2.b and 2.c) was conserved for the next chromatography; [0235] d) Regeneration was performed using a buffer which consisted of water, 0.02M Acetate, 0.02M Phosphate, 0.01 M Tris Base, 1M NaCl, adjusted at pH 8 with NaOH 1M [0236] e) Sanitation was performed using NaOH 0.5M [0237] f) Rinse was performed using a buffer which consisted of water, 0.02M Acetate, 0.02M Phosphate, 0.01 M Tris Base, adjusted at pH 5.5 with NaOH 1M [0238] 3. AEX chromatography using Eshmuno Q column (from Merck Millipore); [0239] a) The column was equilibrated using a buffer consisting of water, 0.02M Acetate, 0.02M Phosphate, 0.01 M Tris Base, adjusted at pH 5 with NaOH 1M [0240] b) The CEX flowthrough was adjusted at pH 5 with acetic acid 1M prior to the load on the column. [0241] c) A wash buffer was flown through the column, which consisted of water, 0.02M Acetate, 0.02M Phosphate, 0.01 M Tris Base, adjusted at pH 5 with NaOH 1M. The resulting flowthrough (3.b and 3.c) was conserved for analyses; [0242] d) Regeneration was performed using a buffer which consisted of water, 0.1 M Acetate, 0.1 M Phosphate, 0.05 M Tris Base [0243] e) Sanitation was performed using NaOH 0.5M [0244] f) Regeneration was performed using a buffer which consisted of water, 0.02M Acetate, 0.02M Phosphate, 0.01 M Tris Base, 1M NaCl, adjusted at pH 5.5 with NaOH 1M [0245] g) Rinse was performed using a buffer which consisted of water, 0.02M Acetate, 0.02M Phosphate, 0.01 M Tris Base, adjusted at pH 5 with NaOH 1M

[0246] Quality and impurities analysis for the flowthrough of step 3b)c) was performed. The results are presented in table 2.

TABLE-US-00003 TABLE 3 A2 purification specifications further to purification according to method 1 Buffer Mix 1 Clipped Yield Cumulative HCP DNA HMW forms Steps % yield % ppm ppb % % Harvest 296993 1591777 1.9 9.6 Capture 98.0 95.6 100 64 2.7 8.2 CEX 83.5 79.8 42 0.3 4.2 AEX 85.3 68.1 26 0.3 4.6

TABLE-US-00004 TABLE 4 A2 purification specifications further to purification according to method 2 Buffer Mix 2 Clipped Yield Cumulative HCP DNA HMW form Steps % yield % ppm ppb % % Harvest 217938 Not yet N/A 21.0 Capture 90.3 90.3 255 Not yet N/A 19.3 CEX 87.4 78.9 150 N/A 22.3 AEX 78.4 61.9 42 0.3 21.0

[0247] Conclusion: The yield of both purification cascades are comparable (around 65%). Despite the use of different harvest lots, the aggregate percentage and HCP concentration are very low and comparable for both cascades (around 30 ppm of HCP and 0.3% of HMW at the end of the purification). The high value of clipped form in the second example is due to the initial value of the corresponding harvest and a limited impact of the purification on the LMW. To conclude, the different buffer ratio used show great and comparable result in terms of performance and purification.

REFERENCES

[0248] Goyon et al.: Determination of isoelectric points and relative charge variants of 23 therapeutic monoclonal antibodies. Journal of Chromatography B, Volumes 1065-1066, 15 Oct. 2017, Pages 119-128 [0249] Baeza-Baeza, J. J., & Garcia-Alvarez-Coque, M. C. (2011). Systematic Approach To Calculate the Concentration of Chemical Species in Multi-Equilibrium Problems. Journal of Chemical Education, 88(2), 169-173 [0250] Carta G. and Jungbauer A. (2010) Protein Chromatography, Wiley-VCH Verlag GMBH.