PURIFICATION OF ANTIBODIES BY MIXED MODE CHROMATOGRAPHY
20240376146 ยท 2024-11-14
Assignee
Inventors
Cpc classification
C07K1/20
CHEMISTRY; METALLURGY
International classification
Abstract
Herein is reported a method for producing or purifying an antibody using a mixed mode chromatography material that comprises ion exchange functional groups and hydrophobic interaction functional groups (MM HIC/TEX) operated in flowthrough mode, wherein the antibody is a hydrophilic antibody, and the antibody is applied in a solution comprising the antibody and an antichaotropic salt to the MM HIC/IEX chromatography material.
Claims
1. A method for producing an antibody using a mixed mode chromatography material that comprises ion exchange functional groups and hydrophobic interaction functional groups (MM HIC/IEX) operated in flowthrough mode, wherein a) the antibody is a hydrophilic antibody, and b) the antibody is applied in a solution comprising the antibody and an antichaotropic salt to the MM HIC/IEX.
2. The method according to claim 1, wherein the method further comprises the following steps: c) optionally a rinsing solution is applied, d) the antibody is recovered in the flowthrough of b) or optionally in the flowthrough of b) and c), and thereby producing the antibody using a MM HIC/IEX operated in flowthrough mode.
3. The method according to claim 1 or claim 2, wherein the method is for producing an antibody composition with reduced antibody-related high molecular weight (HMW) impurity content and/or with reduced viral impurity content, the antibody is applied to the MM HIC/IEX in a solution comprising the antibody, at least one HMW impurity and/or at least one viral impurity and the antichaotropic salt, the antibody composition with reduced HMW impurity content and/or with reduced viral impurity content is recovered from the flowthrough, and thereby an antibody composition with reduced HMW impurity content and/or with reduced viral impurity content is produced.
4. The method according to claim 3, wherein the HMW impurity content and/or the viral impurity content is reduced compared to the solution applied to the MM HIC/IEX in step b).
5. The method according to claim 3, wherein the HMW impurity content and/or the viral impurity content is reduced compared to a solution essentially without an antichaotropic salt; and/or compared to a solution comprising a hydrophobic antibody.
6. The method according to claim 1, wherein the hydrophilic antibody is an antibody that has a retention time on a hydrophobic interaction chromatography (HIC) material that is equal or less than that of rituximab.
7. The method according to claim 6, wherein the HIC material contains polyether groups (ethyl ether groups) as ligand.
8. The method according to claim 1, wherein the antichaotropic salt has a molar surface tension increment in the range of and including 1.285 to 4.183?10E3 dyn*g*cm.sup.?1*mol.sup.?1.
9. The method according to claim 1, wherein the antichaotropic salt is selected from the group consisting of (NH.sub.4).sub.2SO.sub.4, Na.sub.2SO.sub.4, K.sub.2SO.sub.4, NaCl and KCl.
10. The method according to claim 1, wherein the solution comprising the antibody and the antichaotropic salt of step b has a conductivity of from 0.5 to 120 mS/cm.
11. The method according to claim 1, wherein in the solution comprising the antibody and the antichaotropic salt, the antichaotropic salt has a concentration of from 10 mM to 900 mM.
12. The method according to claim 1, wherein the loaded amount to the MM HIC/IEX is from 15 g of protein per Liter of chromatography material (15 g/L) to 350 g of protein per Liter of chromatography material (350 g/L).
13. The method according to claim 1, wherein the solution comprising the antibody and the antichaotropic salt has a pH value of from 4.0 to 9.0.
14. The method according to claim 3, wherein the HMW impurity is an impurity which has a molecular weight of 285 kDa or more.
15. The method according to claim 1, wherein the MM HIC/IEX comprises i) anion exchange functional groups or cation exchange functional groups, or ii) strong anion exchange functional groups, or iii) weak cation exchange functional groups.
Description
DESCRIPTION OF THE FIGURES
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EXPERIMENTAL PART
Material & Methods
1. Proteins
[0314] The molecules used herein were humanized IgG1 monoclonal antibodies (mabs) produced in Chinese hamster ovary cells. Starting material used to load the mixed mode chromatography columns was an affinity chromatography column eluate (denoted as affinity column pool). The molecules encompassed standard IgG-like mabs and complex antibody formats, e.g. bispecific CrossMab format, mabs containing a bound ligand (2+1 C format) and T-cell binding mabs (2+1 N format; TCB). The pI of the molecules was in the range of 8.0-9.4.
[0315] For the RVLP removal studies starting material used to load the mixed mode chromatography columns was a second column chromatography eluate (i.e. an eluate after an affinity chromatography and a subsequent second chromatography run; denoted as second column pool). The second chromatography run could for example be perfomed on a cation exchange chromatography material (as is the case for e.g. mab9), an anion exchange chromatography material or a mixed mode chromatography material such as a mixed mode anion exchange chromatography material (as is the case for e.g. mab1, mab2 or mab7).
[0316] The method for determination of the retention times is described below in Materials and Methods item 10. The retention times of the mabs determined with this method were in the range from 19 min. to 41 min. An overview of the retention times of the mabs is given in Table. The retention time of Rituximab was found to be the cut-point for defining hydrophilic and hydrophobic mabs. Mabs with a retention time?retention time.sub.rituximab, i.e. that have the same or a shorter retention time as rituximab, are defined to be hydrophilic, mabs with retention time>retention time.sub.rituximab, i.e. have a longer retention time, are defined to be hydrophobic.
TABLE-US-00001 TABLE MM-1 Mabs and retention times retention denoted time mab as [min] bivalent, monospecific full-length IgG1 antibody mab1 19.4 specifically binding to antigen 1 bivalent, monospecific full-length IgG1 antibody mab2 21.7 specifically binding to antigen 2 antibody in 2 + 1 N format specifically binding to mab4 29.5 antigens 3 and 4 bivalent, monospecific full-length IgG1 antibody mab5 29.6 specifically binding to antigen 5 Rituximab Cut-point 32.0 bivalent, bispecific full-length IgG1 antibody in mab6 35.8 CrossMab format specifically binding to antigens 6 and 7, variant 1 bivalent, bispecific full-length IgG1 antibody in mab7 35.8 CrossMab format specifically binding to antigens 6 and 7, variant 2 antibody in 2 + 1 C format specifically binding to mab8 41.2 antigens 8 and 9 antibody in 2 + 1 N format specifically binding to mab9 41.2 antigens 4 and 8
2. Chemicals
[0317] K.sub.2HPO.sub.4, KH.sub.2PO.sub.4, KCl, Na.sub.2HPO.sub.4?.sub.2H.sub.2O, NaH.sub.2PO.sub.4?H.sub.2O, ethanol, Tris(hydroxymethyl)-aminomethan, acetic acid, citric acid, sodium sulfate, ammonium sulfate, sodium chloride, potassium chloride, potassium sulfate, guanidinium/hydrochloride, urea, NaOH were purchased from the manufacturers as listed below and used as provided by the manufacturer.
[0318] Merck KGaA: K.sub.2HPO.sub.4, KH.sub.2PO.sub.4, KCl, Na.sub.2HPO.sub.4?2H.sub.2O, NaH.sub.2PO.sub.4?H.sub.2O, ethanol, acetic acid, citric acid, sodium sulfate, ammonium sulfate, sodium chloride, potassium chloride, guanidinium chloride, urea, NaOH [0319] ANGUS Chemie GmbH: Tris(hydroxymethyl)-aminomethan [0320] Sigma Aldrich and Merck KGaA: ammonium sulfate [0321] Thermo Fisher Scientific GmbH: potassium sulfate
3. Robocolumns (RCs)
[0322] Robocolumns? (RC) were purchased from Repligen GmbH (Ravensburg, Germany): [0323] Capto? adhere ImpRes; PN 01100408R; 200 ?L
4. Chromatography Resins
[0324] The following chromatography resins were used herein: [0325] Capto? adhere ImpRes (Cytiva (Formerly GE Healthcare), Uppsala, Sweden) [0326] Capto? adhere (Cytiva (Formerly GE Healthcare)) [0327] Nuvia aPrime4A (Bio-Rad Laboratories, Inc., USA) [0328] Q Sepharose FF (Cytiva (Formerly GE Healthcare)) [0329] Phenyl Sepharose 6 FF (high sub) (Cytiva (Formerly GE Healthcare)) [0330] Capto? MMC ImpRes (Cytiva (Formerly GE Healthcare))
5. Robotic Labware
[0331] Filter plates: PALL; AcroPrep Advance 96 Well, 1 mL, 0.45 ?m, REF 8184 [0332] MTP UV Plates: UV Microtiter Plates, Thermo Scientific
6. Materials for Load Preparation
[0333] Centrifuge: Heraeus Multifige 3 S-R; Rotor: 75006445 [0334] Amicon Ultra Centrifugal Filters (Merck Millipore, Ultracel-30K) [0335] Slide-A-Lyzer Dialysis Cassettes (Thermo Scientific, 20 000-30 000 MWCO) [0336] Minisart Syringe Filter (Sartorius Minisart High Flow, 0.22 ?m)
7. Robotic Systems
7.1. Tecan Freedom EVO 150
[0337] A Tecan Freedom EVO 150 (Tecan Deutschland GmbH, Crailsheim, Germany) liquid handling system (LHS) was used to perform the RC runs. The EVO 150 was equipped with one liquid handling arm (LiHa) and one excentric gripper, an atoll bridge for the RCs and an Infinite M200 NanoQuant plate reader (Tecan Deutschland GmbH, Crailsheim, Germany). The LHS was controlled by the software Freedom EVOware (Tecan Deutschland GmbH, Crailsheim, Germany). The software used to control the plate reader was Magellan (Tecan Deutschland GmbH, Crailsheim, Germany). The platform was additionally equipped with a Te-Stack? for storage of 96 well collection plates (microtiter plates) and a Te-Slide? for plate transport and fraction collection. The LiHa was capable of processing volumes of 10 ?L to 1000 ?L and was equipped with 1000 ?L dilutor syringes. The LiHa consisted of eight separately controllable channels equipped with eight fixed stainless steel needles. All RC runs were performed with Capto? adhere ImpRes resin. The column dimensions were 1 cm length?0.5 cm diameter and a bed volume of 200 ?L.
7.2. Hamilton Microlab STARlet
[0338] A Hamilton Microlab STARlet roboter was used for the preparation of the filter plate used herein. The roboter was equipped with eight 1000 ?L pipetting channels and a shaker. A 50% slurry of resin in water (v/v) was produced and placed in a glass vial on the shaker. The LiHa was equipped with wide bore 1000 ?L tips (cut) to transfer the resin from the shaker to the filter plate. A filter plate containing 50 ?L resin per well was produced. The storage solution was water.
7.3. Tecan Freedom EVO 200
[0339] A Tecan Freedom EVO 200 (Tecan Deutschland GmbH, Crailsheim, Germany) liquid handling system was used to prepare the load plate and buffer plate as used herein and to perform the runs. The Tecan Freedom EVO 200 was equipped with one liquid handling arm (LiHa), one excentric gripper, a Te-Shake?, a Te-Stack? for storage of microplates, a Te-Slide? for plate transport and an Infinite M200 plate reader (Tecan Deutschalnd GmbH, Crailsheim, Germany). Additionally, a centrifuge (Rotanta 46RSC, Hettich, Germany) was integrated into the worktable to remove the supernatant after incubation. The LiHa was capable of processing volumes of 10 ?L to 1000 ?L and was equipped with 1000 ?L dilutor syringes and consisted of eight separately controllable channels equipped with eight fixed stainless steel pipette tips. The Tecan robot was controlled by the software Evoware. The software used to control the plate reader was Magellan.
8. Determination of Protein Purity
[0340] Protein purity in terms of monomer content and high molecular weights species was determined by size-exclusion High Performance Liquid Chromatography (SE-HPLC) using a HPLC system (Thermo Fisher). Protein separation was performed on a TSK-Gel G3000SWXL (7.8?300 mm; 5 ?m) column (TOSOH Bioscience, P/N 08541) with a flow rate of 0.5-1.0 ml/min using 0.2 M K.sub.2HPO.sub.4/KH.sub.2PO.sub.4, 0.25 M KCl, pH 7.0 as eluent.
[0341] The following conditions were used: [0342] wavelength: 280 nm [0343] isocratic; 30 min [0344] column temperature: 25? C. [0345] sample temperature: 10? C. [0346] applied quantity: 150 g protein
9. Determination of Protein Concentration
9.1. Determination Using Cuvette
[0347] Protein concentrations were determined by UV spectroscopy using a Spectramax Plus (Molecular Devices, Munich, Germany). The measurement was executed in a cuvette. Protein samples were diluted in water. Concentrations were determined according to the following Equation 1 deriving from Lambert-Beer law:
9.2. Determination Using Microplate
[0349] Protein concentrations were determined by UV spectroscopy using an Infinite plate reader (Tecan Deutschland GmbH, Crailsheim, Germany). The measurement was executed in a microplate. Protein samples were diluted in water. Concentrations were determined according to Equation (1).
[0350] The path length d was calculated with the following Equation 2:
10. Determination of Retention Time and Hydrophobicity
10.1. Equipment
[0352] HPLC device with integrated data collection system; Dionex (now Thermo Fisher Scientific) [0353] 0.2 ?m membrane filters; e.g. Pall Life Sciences Supor?-200, Catalog no. 60301 [0354] Tosoh Bioscience, TSKgel? Ether-5 PW HPLC Column, 10 ?m, 7.5 mm?75 mm, Catalog no. 0008641, i.e. a hydrophobic interaction chromatography column with an inner diameter of 2 mm, a column length of 75 mm and a particle size of 10 ?m. The column has a polymethacrylate base material (matrix) with polyether groups as ligand (ethyl ether groups).
10.2. Working Solutions:
[0355] Eluent A: 25 mM Na-phosphate buffer comprising 1.5 M (NH.sub.4).sub.2SO.sub.4 adjusted to pH 7.0 [0356] Eluent B: 25 mM Na-phosphate buffer, adjusted to pH 7.0
10.3. Hydrophobic Interaction Chromatography (HIC) Standard
[0357] For standard preparation Rituximab is diluted to 1 mg/ml with a suitable dilution buffer. 20 ?l (=20 ?g) of the HIC standard are injected. The peak of Rituximab will appear close to 32 min run time, i.e. has a retention time close to 32 min.
10.4. Samples
[0358] Samples are diluted to a concentration of 1 mg/ml. If samples are already at a concentration of 1 mg/ml, no dilution is necessary. 20 ?l (=20 ?g) of the 1 mg/ml solution are injected.
10.5. Blank
[0359] 20 ?l of the dilution buffer were injected.
10.6. Conditioning of New Columns
[0360] Columns were provided in ultrapure water. Before the first run a new column was prepared as follows:
[0361] 1) the column was transferred at ambient temperature to Eluent B: the flow was slowly increased from 0.0 ml/min to 0.8 ml/min within a minimum of 40 min; the column was washed thereafter with Eluent B at 0.8 ml/min until a stable baseline was reached (usually after 60 min).
[0362] 2) Gradient run: a linear gradient from 0% Eluent A to 100% Eluent A within 20 min. was run; the column was washed thereafter with Eluent A until a stable baseline was reached (usually after 60 min).
[0363] 3) Saturation: the available standard was injected and processed according to 10.8. multiple times until three successive chromatograms were identical with regard to peak form, height and area; the column was thereafter used.
10.7. Conditioning of Used Columns
[0364] After mounting, column was washed at ambient temperature with ultrapure water. The flow was slowly increased from 0.0 ml/min to 0.8 ml/min within a minimum of 40 min. Thereafter the column was washed with Eluent A at 0.8 ml/min until a stable baseline was reached. The column was thereafter used.
10.8. Working Conditions and Sequence Layout
[0365] Before any sequence 1? Eluent A was injected, followed by an injection of 20 ?l of 0.1 M NaOH and another injection of Eluent A.
[0366] The following working conditions were used: [0367] Flow rate: 0.8 ml/min [0368] Gradient:
TABLE-US-00002 Time [min] Eluent A [%] Eluent B [%] 0.0 100 0 2.0 100 0 3.0 87 13 5.0 87 13 57.0 0 100 62.0 0 100 63.0 100 0 65.0 100 0 [0369] Maximum pressure: 22 bar [0370] Wavelength: 214 nm (in addition record 220 nm and 280 nm) [0371] Injected protein: 20 ?g [0372] Column temperature: 40? C.?2? C. [0373] Temperature in autosampler: 10? C.?4? C.
[0374] HIC standard and samples were measured in the following sequence: [0375] 1. Eluent A [0376] 2. 20 ?l 0.1 M NaOH [0377] 3. Eluent A [0378] 4. HIC/reference [0379] 5. Blank (dilution buffer) [0380] 6. Samples 1 to n [0381] 7+n Blank (dilution buffer)
[0382] The retention times were determined at peak maximum.
[0383] Mabs eluting simultaneously with or prior to Rituximab (with shorter retention time) have found to be hydrophilic (retention time.sub.mab?retention time.sub.rituximab) whereas mabs eluting after Rituximab (with longer retention time) have found to be hydrophobic (retention time.sub.mab>retention time.sub.rituximab).
11. Determination of RNA Concentration
[0384] In order to determine the removal of RVLPs, the RNA concentrations in the samples are determined as representative measurements.
[0385] Automated RNA-analytics are performed via the FLOW PCR setup system (Roche Diagnostics Gmbh). The system consists of 3 modules: FLOW PCR SETUP instrument (Roche Diagnostics GmbH, order no. 07101996001), MagNA Pure 96 instrument (Roche Diagnostics GmbH, order no. 06541089001) and LightCycler?480 instrument (Roche Diagnostics GmbH, order no. 05015278001). The determination of the RNA content is performed according to the manufacture's operating manuals. In brief, the RNA is isolated using the MagNA Pure 96 instrument. After a treatment with DNAse, the probes are measured in the LightCycler?480 instrument to quantify the RNA by means of the PCR technology (quantitative RT PCR). The RNA is therefore first converted to cDNA by reverse transcription (RT). Then, the cDNA is amplified in a PCR reaction and the concentration is quantified by way of comparison to a standard curve. Subsequently the result is converted to RNA content.
Overview of the Examples
[0386] The examples section can be subdivided into four parts:
Part I: Impact of Mab Hydrophobicity on High Molecular Weight (HMW) Impurity Reduction at Constant Conductivity
[0387] Flowthrough (FT) runs were performed with Capto? adhere ImpRes RCs (Repligen) on a robotic system (Tecan Freedom 150). Up to 5 antichaotropic (ac) salts were chosen (Na.sub.2SO.sub.4, NaCl, (NH.sub.4).sub.2SO.sub.4, KCl, K.sub.2SO.sub.4). The FT of 7 mabs was collected and purity was analyzed by SE-HPLC. The HMW reduction achieved by adding an antichaotropic salt to the load were compared with the HMW reduction determined with a Tris/Acetate buffer, pH 8 and accordingly a Tris/Citrate buffer, pH 6 at same conductivity without containing an antichaotropic salt. It was shown that only for hydrophilic mabs (retention time.sub.mab?retention time.sub.rituximab) an improved HMW reduction was achieved by addition of an antichaotropic salt to the load material compared to a load material having same conductivity under absence of an antichaotropic salt. In contrast to that, for hydrophobic mabs (retention time.sub.mab>retention time.sub.rituximab) no positive impact was observed with addition of an antichaotropic salt.
Table MM-2
[0388] Table MM-2: summarizes the examples of part I.
TABLE-US-00003 TABLE MM-2 Part I-examples example conductivity number mabs pH [mS/cm] salts used Example 1 mab 1, 2, 8.0 20 Na.sub.2SO.sub.4, NaCl, (NH.sub.4).sub.2SO.sub.4, 4-8 KCl, K.sub.2SO.sub.4 Example 2 mab 1, 2, 6.0 20 Na.sub.2SO.sub.4, KCl 4-8 Example 3 mab2 & 8.0 & 10 Na.sub.2SO.sub.4, NaCl, (NH.sub.4).sub.2SO.sub.4, mab7 6.0 KCl, K.sub.2SO.sub.4
Part II: Impact of Antichaotropic Salt Molarity on HMW Removal
[0389] In the 2.sup.nd experimental part RC experiments and Kp (partition coefficient) screens were performed to investigate the impact of different molarities of antichaotropic salts on HMW reduction. In Example 4 a hydrophilic mab was loaded on Capto? adhere ImpRes RCs with a molarity range up to 500 mM. Different salts, Na.sub.2SO.sub.4, NaCl, (NH.sub.4).sub.2SO.sub.4, KCl and K.sub.2SO.sub.4 were investigated at pH 8.0. It was shown that an increase in salt molarity could improve reduction of HMWs in the FT fractions.
[0390] In addition to these RC runs, Kp screens were run to show the impact of salt molarity on HMW reduction for a broader range of pH and molarity using Capto? adhere ImpRes resin/chromatography material (Examples 5 and 6). For Example 5 three mabs were chosen, two hydrophilic mabs and one hydrophobic mab. For the Kp screens the investigated pH range was pH 5.5-8.0 and the molarity range 10-800 mM. The Kp screen HMW reduction confirmed the RC data. It was shown that for hydrophilic mabs an increasing salt molarity resulted in an improved HMW reduction whereas HMW reduction for the hydrophobic mab was not improved by increasing the antichaotropic salt molarity. To emphasize the need of an antichaotropic salt, chaotropic salts were investigated additionally. The Contour plots using the chaotropic salts did not show an improved HMW reduction with increasing salt molarity.
[0391] In Example 6 Kp screens were performed with mab2 and a screening range of pH 4-9 and molarities up to ?900 mM. Within these Kp screens two sets of buffers were compared: one buffer containing the antichaotropic salt Na.sub.2SO.sub.4 and one buffer without an antichaotropic salt (only Tris/Acetate buffer).
[0392] Table MM-3 summarizes the examples of part II.
TABLE-US-00004 TABLE MM-3 Part II - examples salt example molarity number procedure mab pH [mM] salts used Example 4 robocolumn mab2 8.0 0-500 Na.sub.2SO.sub.4, NaCl, (NH.sub.4).sub.2SO.sub.4, KCl, K.sub.2SO.sub.4 Example 5 Kp screen mab2; 5.5-8.0 10-800 antichaotropic: mab4; (NH.sub.4).sub.2SO.sub.4, KCl mab7 chaotropic: Urea, Gua/HCl Example 6 Kp screen mab2 4.0-9.0 10-~900 Na.sub.2SO.sub.4, Tris/Acetate
Part III: Comparison of HMW Removal with Other Resins
[0393] The 3.sup.rd part consists of two examples: In example 7 HMW removals of a hydrophilic mab in a pH range of 5.5-8.0 and salt molarities of 10-800 mM were investigated. In this example the following mixed mode anion exchange (MMAEX) resins were compared: Capto? adhere ImpRes, Capto? adhere and Nuvia aPrime. All three resins showed an improved HMW reduction when an antichaotropic salt was added to the load solution comprising a hydrophilic mab.
[0394] Example 8 summarizes Kp screens for a MMAEX, an Anion exchange (AEX) resin, a HIC resin and a mixed mode cation exchange (MMCEX) resin with one hydrophilic and one hydrophobic mab. The investigated pH range was pH 4.0-9.0 and 5-850 mM salt. The flowthrough samples of the hydrophilic mab indicated an improved HMW reduction with increasing salt molarity for both ionic mixed mode resins (MMAEX and MMCEX). In contrast to that for the hydrophobic mab HMW reduction on the MMAEX resin was constant over the investigated range of salt molarity. For the MMCEX resin HMW reduction for the hydrophobic mab was not improved by increasing salt molarity below 500 mM Na.sub.2SO.sub.4. With the single mode resins Q Sepharose FF (AEX) no positive effect on HMW reduction was observed neither for the hydrophilic nor for the hydrophobic mab. For the Phenyl Sepharose 6 FF (high sub) (HIC) a positive impact of increasing salt molarity on HMW reduction was measured for the hydrophilic and hydrophobic mab.
[0395] Table MM-4 illustrates the examples for part III.
TABLE-US-00005 TABLE MM-4 Part III - examples salt example molarity number resins mab pH [mM] Salts used Example 7 Capto adhere mab2 5.5-8.0 10-800 antichaotropic: ImpRes, (NH.sub.4).sub.2SO.sub.4, Capto adhere, KCl chaotropic: Nuvia aPrime Urea, Gua/HCl Example 8 Q Sepharose mab2 4.0-9.0 5-850 (NH.sub.4).sub.2SO.sub.4, FF, Phenyl & Na.sub.2SO4 Sepharose mab6 6FF (high sub), Capto MMC ImpRes
Part IV: Upscaling ?KTA Column Runs
[0396] For the 4th part scale-up runs on an ?KTA system were performed with Na.sub.2SO.sub.4 and mab2. Here an upscaling from Kp screen resin volumes of 50 ?L and RC volumes of 200 ?L to a column volume of 6.8 mL was done with Capto? adhere ImpRes resin. Example 9 shows the Mainpeak value of the FT fractions for two runs with the same conductivity but different Na.sub.2SO.sub.4 molarities. Although the conductivity of both loads was equal, the HMW reduction for the load containing the higher molarity of Na.sub.2SO.sub.4 was significantly better. In Example 10 runs at pH 7 and pH 8 with different Na.sub.2SO.sub.4 molarities (and different conductivities) were described. An improved HMW reduction was determined with increasing salt molarity. These lab scale results confirmed the results achieved with the robotic systems in part II and III.
[0397] Table MM-5 illustrates the examples for part IV.
TABLE-US-00006 TABLE MM-5 Part IV - examples example conductivity number resins mab pH [mS/cm] salts used Example 9 Capto mab2 8 9 (different Na.sub.2SO.sub.4 adhere molarities: ImpRes 20 & 40 mM) Example 10 Capto mab2 7 5-12 Na.sub.2SO.sub.4 adhere 8 (0-60 mM) ImpRes
Part V: Removal of RVLPs (Retrovirus Like Particles)
[0398] In addition to the removal of product-related impurities (like HMWs) it was also investigated whether the use of an antichaotropic salt in the purification of antibodies on a MM chromatography resin does also have an effect of the removal of retrovirus like particles (RVLPs).
[0399] In this respect two KpScreens were performed (Example 11 and Example 12).
KpScreen: Impact of Antichaotropic Salt and Mab Hydrophobicity on RVLP Removal
[0400] Kp (partition coefficient) screens were performed to investigate the impact of an antichaotropic salt and mab hydrophobicity on RVLP reduction. The concentration of (RNA-containing) RVLPs was measured via the quantification of the concentration of RNA by means of quantitative RT PCR. Within these Kp screens two sets of buffers were compared: one buffer containing the antichaotropic salt Na.sub.2SO.sub.4 (Example 11) and one buffer without an antichaotropic salt (only Tris/Acetate buffer) (Example 12). Additionally four mabs were chosen, two hydrophilic mabs (mab1 and mab2) and two hydrophobic mabs (mab7 and mab9). The chromatography resin was Capto? adhere ImpRes.
[0401] For Example 11 the investigated pH range was pH 5.0-8.0 and the molarity range 25-400 mM Na.sub.2SO.sub.4 (corresponding to a conductivity range of 3-34 mS/cm). For the hydrophilic mabs an improved RVLP reduction was shown compared to the hydrophobic mabs (
[0404] LF: load fraction of RC [0405] total load volume: volume required to apply 350 g.sub.protein/L.sub.resin [0406] total loaded amount: sum of the amount of antibody applied in all applied respective load fractions together [0407] loaded amount: used in calculation of HMW removal with best fit line [0408] HMW value: analytically determined HMW content in a flowthrough fraction [0409] HMW removal value: HMW removal calculated for a single load fraction [0410] HMW removal: calculated HMW removal based on best fit line [0411] pool HMW removal value: calculated HMW removal obtained for a total loaded amount applied in a single fraction [0412] single, pool loaded amount: theoretical amount of antibody loaded in a single fraction of the respective calculated pool HMW removal value [0413] FT: flowthrough fraction [0414] RC: Robocolumn? [0415] RC-run: Robocolumn? run [0416] CV: column volume
EXAMPLES
Part I: Impact of Mab Hydrophobicity on High Molecular Weight (HMW) Impurity Reduction at Constant Conductivity
Example 1
Robocolumn? Runs with Loads at pH 8 and 20 mS/Cm
[0417] Robotic runs were performed with 4 hydrophilic and 3 hydrophobic mabs at pH 8 and a conductivity of 20 mS/cm. From run to run the buffer condition were varied by adding the following antichaotropic salts: sodium chloride, sodium sulfate, ammonium sulfate, potassium chloride and potassium sulfate. For each run, all 7 mabs were investigated in parallel.
Buffers
[0418] The pH values and conductivities of the respective equilibration buffers are summarized in the following Table X-1.1a. The pH value was each adjusted by adding the respective acid of the buffer (acetic acid). Conductivity was determined after combining all components of the solutions. The buffer 1.5 M Tris/Acetate, pH 8 is the reference condition, i.e. not including any antichaotropic salt but having the same conductivity (buffer only).
TABLE-US-00007 TABLE X-1.1a Buffer conditions for the RC-runs at pH 8 and 20 mS/cm. conductivity buffer pH [mS/cm] 1.5M Tris/Acetate, pH 8 (reference) 8.09 18.71 70 mM Tris/Acetate, 125 mM Na.sub.2SO.sub.4, pH 8 8.03 20.70 70 mM Tris/Acetate, 200 mM NaCl, pH 8 7.97 21.80 70 mM Tris/Acetate, 100 mM (NH.sub.4).sub.2SO.sub.4, pH 8 7.90 20.20 70 mM Tris/Acetate, 150 mM KCl, pH 8 7.90 20.70 70 mM Tris/Acetate, 100 mM K.sub.2SO.sub.4, pH 8 7.98 21.50
Concentrating of Starting Material and Buffer Exchange of Antibody Solution
[0419] The antibody solutions (mabs) were adjusted to pH and conductivities comparable to the respective buffer. To achieve this the respective affinity column elution pools were buffer exchanged to the respective buffer (e.g. to 70 mM Tris/Acetate, 125 mM Na.sub.2SO.sub.4, pH 8; see Table X-1.1a) and concentrated by using Amicon Ultra Centrifugal Filters. After centrifugation, the mabs were diluted to a concentration of approximately 20 g/L and pH and conductivity were determined. The solutions to be loaded (loads) were filtered through a 0.2 ?m sterile filter and protein concentration was determined. This procedure was performed for all Robocolumn? runs (RC-runs).
Loads
[0420] The pH values, conductivities and antibody concentrations of the loads are summarized in the following Table X-1.1b.
TABLE-US-00008 TABLE X-1.1b Load pH, conductivity and concentration for the RC-runs at pH 8 and 20 mS/cm. Ranges are based on the loads comprising the different antibodies. conductivity concentration pH range range of loads range of loads load in of loads [mS/cm] [g/L] 1.5M Tris/Acetate, pH 8 8.03-8.12 17.35-17.58 19.98-25.17 (reference) 70 mM Tris/Acetate, 8.04-8.07 19.24-19.60 18.15-23.69 125 mM Na.sub.2SO.sub.4, pH 8 70 mM Tris/Acetate, 7.91-8.00 20.1-20.60 19.63-22.94 200 mM NaCl, pH 8 70 mM Tris/Acetate, 7.86-7.89 19.38-19.81 14.69-20.76 100 mM (NH.sub.4).sub.2SO.sub.4, pH 8 70 mM Tris/Acetate, 7.89-7.93 19.22-19.59 18.20-25.02 150 mM KCl, pH 8 70 mM Tris/Acetate, 7.98-7.99 19.85-20.20 14.93-22.50 100 mM K.sub.2SO.sub.4, pH 8
[0421] For all experiments of Example 1, the pH range of the loads was within a range of pH 8.0?0.2. The conductivities of the loads varied ?2.0 mS/cm from the conductivity of the equilibration buffer. The protein concentration of the loads was between 14.5-25.5 g/L.
[0422] In the following a description of an exemplary RC-run is provided. All RC-runs were performed alike, except that the buffer for preparing the loads was different (see Tables X-1.1a and b above). For example, for loads using the buffer (70 mM Tris/Acetate, 125 mM Na.sub.2SO.sub.4, pH 8) the pH ranged from pH 8.04-8.07, which means that the load for the 7 different mabs in the equilibration buffer was in this pH range after buffer exchange and concentrating.
[0423] The following Table X-1.2 shows the properties of the individual loads for the RC-runs for the different antibodies in 70 mM Tris/Acetate, 125 mM Na.sub.2SO.sub.4, pH 8, flowthrough-mode:
TABLE-US-00009 TABLE X-1.2 Example 1 - loads in 70 mM Tris/Acetate, 125 mM Na.sub.2SO.sub.4, pH 8. protein conductivity concentration mab pH [mS/cm] [mg/mL] mab1 8.07 19.38 19.86 mab2 8.06 19.25 22.89 mab4 8.04 19.25 20.77 mab5 8.05 19.34 23.69 mab6 8.05 19.46 19.96 mab7 8.05 19.24 19.15 mab8 8.04 19.60 18.15
[0424] These loads were individually applied to different Capto adhere ImpRes RCs.
Chromatography
[0425] RC-runs were performed on a Tecan Freedom Evo 150. The RCs were first equilibrated (pH and conductivity adjustment) with 10 CV of buffer.sub.without antibody (e.g. 70 mM Tris/Acetate, 125 mM Na.sub.2SO.sub.4, pH 8). Thereafter each RC was loaded stepwise in 200 ?L load fractions (LFs) up to 350 g.sub.protein/L.sub.resin and the flowthrough was collected in 200 ?L flowthrough fractions (FT fractions). After the 350 g.sub.protein/L.sub.resin had been applied, 8 column volumes (CV) of buffer without antibody were applied to the columns to wash residual unbound material from the column before regeneration. The wash following the load was not collected.
[0426] The flow rate for all RC-runs was 18 CV/hr which corresponds to a residence time of 3.3 min. The wash was followed by regeneration and storage of the RCs.
TABLE-US-00010 TABLE X-1.3 Example 1 - exemplary chromatography steps. step buffer CV equilibration 70 mM Tris/Acetate, 125 mM Na.sub.2SO.sub.4, pH 8 10 load see Table X-1.2 14-20 wash 70 mM Tris/Acetate, 125 mM Na.sub.2SO.sub.4, pH 8 8
[0427] The following Table X-1.4 summarizes the load concentrations and volumes.
TABLE-US-00011 TABLE X-1.4 Example 1-load concentration and volumes used in RC-runs using the scheme of Table X-1.3. load total load concentration volume mab [mg/mL] [?L] mab1 19.86 3530 mab2 22.89 3060 mab4 20.77 3370 mab5 23.69 2950 mab6 19.95 3510 mab7 19.15 3660 mab8 18.15 3860
[0428] Pipetting of the load fractions by the Tecan Freedom Evo 150 was executed in a mode that all loads for one joint RC-run were completed at the same time. Therefore the starting point for applying the load varied depending on the antibody concentration in the load. Loads with a higher concentration, e.g. mab5, resulted in lower required total load volumes (and thereby loading steps) and resulted in a later start of the loading steps. For each load step a respective flowthrough fraction (FT) was collected. Thus, for each run a different number of 200 ?L fractions were applied, collected and analyzed, respectively (at most 20 fractions for mab8).
[0429] The following Table X-1.5 shows the increasing total loaded amount [g/L] after each load step. After at most 20 consecutive load steps a total loaded amount of 350 g.sub.protein/L.sub.resin had been applied to each RC.
TABLE-US-00012 TABLE X-1.5a Example 1load step depending total loaded amounts [g/L] for the runs using the schemes of Tables X-1.3 and X-1.4. load step 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 mab1 total loaded 0 0 13 33 53 72 92 112 132 152 172 192 211 231 251 271 291 311 331 350 amount [g/L] mab2 total loaded 0 0 0 0 7 30 53 76 98 121 144 167 190 213 236 259 282 304 327 350 amount [g/L] mab4 total loaded 0 0 0 18 38 59 80 101 121 142 163 184 205 225 246 267 288 308 329 350 amount [g/L] mab5 total loaded 0 0 0 0 0 18 41 65 89 113 136 160 184 207 231 255 278 302 326 350 amount [g/L] mab6 total loaded 0 0 11 31 51 71 91 111 131 151 171 191 211 230 250 270 290 310 330 350 amount [g/L] mab7 total loaded 0 6 25 44 63 82 101 121 140 159 178 197 216 236 255 274 293 312 331 350 amount [g/L] mab8 total loaded 5 24 42 60 78 96 114 132 151 169 187 205 223 241 260 278 296 314 332 350 amount [g/L]
Analytics
[0430]
TABLE-US-00013 TABLE X-1.5b SE-HPLC analytics of selected FT fractions. load step 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Load mab1 HMW 0.30 0.48 0.85 1.28 1.54 1.68 1.80 1.93 2.93 value [%] mab2 HMW 1.66 3.60 4.77 5.51 6.12 6.54 9.97 value [%] mab4 HMW 0.58 0.84 1.04 1.17 1.27 1.44 1.54 2.52 value [%] mab5 HMW 0.74 0.95 0.99 1.04 1.08 1.13 1.62 value [%] mab6 HMW 8.19 12.63 13.22 13.57 13.71 14.03 14.27 16.75 value [%] mab7 HMW 4.56 5.55 6.07 6.45 6.89 6.89 7.13 9.62 value [%] mab8 HMW 3.56 5.02 5.69 6.2 6.38 6.49 6.77 6.89 8.95 value [%]
TABLE-US-00014 TABLE X-1.5c HMW removal values. load step 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 mab1 HMW removal 89.76 83.62 70.99 56.31 47.44 42.66 38.57 34.13 value [%] mab2 HMW removal 83.35 63.89 52.16 44.73 38.62 34.40 value [%] mab4 HMW removal 76.98 66.67 58.73 53.57 49.60 42.86 38.89 value [%] mab5 HMW removal 54.32 41.36 38.89 35.80 33.33 30.25 value [%] mab6 HMW removal 51.10 24.60 21.07 18.99 18.15 16.24 14.81 value [%] mab7 HMW removal 52.60 42.31 36.90 32.95 28.38 28.38 25.88 value [%] mab8 HMW removal 60.22 43.91 36.42 30.73 28.72 27.49 24.36 23.02 value [%]
[0431] The HMW removal value was calculated with the following formula 1:
[0432] Example for calculation of the HMW removal value for mab2 in load step 10: [0433] In load step 10 the determined HMW value of the respective FT fraction was 1.66%. The HMW value of the load was 9.97%. Applying this to formula 1
Analysis
[0434] Either the HMW values or mainpeak values or the HMW removal values were plotted against the total loaded amount of antibody.
[0435]
[0436] The HMV removal values in Table X-1.5c show the actual HMW removal that was obtained in the respective load step, i.e. for the corresponding load fraction. However, a pool HMW removal value more closely reflects the actual large-scale process. This pool HMW removal value is the HMW removal obtained for a total loaded amount when applied in a single fraction. Pool HMW removal values were calculated for single, pool load amounts of 150 g/L, 250 g/L 350 g/L, 450 g/L and 550 g/L based on a logarithmic best fit line of the data in Table X-1.5c.
[0437] This procedure is explained exemplarily for mab2 below;
[0438]
[0439] For example, to calculate the pool HMW removal value for a single, pool loaded amount of, e.g., 150 g/L, the average was calculated based on the HMW removals of each calculated HMW removal value ?150 g/L. For small loaded amounts (5-50 g/L) the HMW removal was set to 100% as the calculation resulted in non-logic HMW removals >100%. Table X-1.6 shows the calculated HMW removals.
TABLE-US-00015 TABLE X-1.6 Example 1-calculation of HMW removal and pool HMW removal values. 70 mM Tris/Acetate, 1.5M Tris/Acetate, 100 mM (NH.sub.4).sub.2SO.sub.4, pH 8 pH 8 mab 2 HMW pool HMW pool loaded removal HMW removal HMW amount [%] using removal [%] using removal [g/L] best fit line value [%] best fit line value [%] 5 100 66 25 100 44 50 100 35 75 97 30 100 84 26 125 74 23 150 66 89 20 35 175 59 18 200 53 16 225 48 15 250 43 75 13 28 275 39 12 300 35 11 325 32 10 350 28 64 9 23 375 25 8 400 22 7 425 20 6 450 17 55 6 20 475 15 5 500 12 4 523 10 3 550 8 47 3 17
[0440] Additionally these calculations were performed for one hydrophobic mab (mab7).
[0441] In
Summary of Example 1
[0442] For hydrophilic mabs (mab1, mab2, mab4, mab5) the HMW value was reduced better when an antichaotropic salt was added to the load compared to a load without an antichaotropic salt at same conductivity. This is illustrated in
[0443] In contrast to that, for the hydrophobic mabs (mab6, mab7, mab8) HMW value reduction was similar for loads containing an antichaotropic salt and for loads without an antichaotropic salt (see
[0444] To calculate the HMW removal for FT pools, trend lines were introduced. In
Example 2
Robocolumn? Runs with Loads at pH 6 and 20 mS/Cm
[0445] RC-runs were performed with 7 mabs at pH 6 and a conductivity of 20 mS/cm using Tris/Citrate buffers in the absence as well as the presence of two antichaotropic salts, i.e. Na.sub.2SO.sub.4 and KCl. The respective references were loads in 1.0 M Tris/Citrate, pH 6 having same conductivity in the absence of any antichaotropic salt.
[0446]
Summary of Example 2
[0447] For hydrophilic mabs HMW reduction was significantly improved when an antichaotropic salt was added to the load compared to a load in the same buffer without antichaotropic salt at same conductivity (see
Example 3
Robocolumn? Runs with Loads at pH 6 or 8 and 10 mS/Cm
[0448] RC-runs were performed with one hydrophilic and one hydrophobic mab in the presence of an antichaotropic salt and in the absence (i.e. without) an antichaotropic salt in the buffer. In both cases, the conductivity of the loads was identical (10 mS/cm). These experiments were performed at pH 6 as well as at pH 8. Up to 350 g.sub.protein/L.sub.resin were loaded on the RCs. The effect of five antichaotropic salts was analyzed. The runs with a load comprising 400 mM Tris/Acetate, pH 8 and a load comprising 300 mM Tris/Citrate, pH 6 but without antichaotropic salt are the references, respectively.
[0449]
Summary of Example 3
[0450]
Part II: Impact of Antichaotropic Salt Molarity on HMW Reduction
Example 4
Robocolumn? Runs with Mab2 at pH 8
[0451] RC-runs with mab2 containing loads at pH 8 with antichaotropic salt concentrations in the range of 0-500 mM were performed. The procedure for the RC-runs that was used was already outlined in detail in Example 1. The following salts were used: Na.sub.2SO.sub.4, NaCl, (NH.sub.4).sub.2SO.sub.4, KCl, K.sub.2SO.sub.4. Flowthrough (FT) fractions were collected and analyzed.
[0452]
Summary of Example 4
[0453] In Example 4 the impact of an increasing antichaotropic salt molarity (and conductivity) was investigated for five antichaotropic salts at pH 8 with hydrophilic mab2. The following antichaotropic salts were used: sodium sulfate (see
[0454] The effect of increasing salt molarity was also seen for three mabs (hydrophilic and hydrophobic) and four salts, (NH.sub.4).sub.2SO.sub.4, KCl, Gua/HCl and Urea, using Kp screens (see Example 5).
Example 5
Kp Screens (pH 5.5-8.0)
[0455] The methodology of Kp screens is described in detail in this example.
Preparation of Mab Solutions
[0456] The antibody containing solutions were concentrated with Amicon Ultra Centrifugal Filters and buffer exchanged to 10 mM Tris/Acetate, pH 6.5 with Slide-A-Lyzer Dialysis Cassettes. The protein concentrations were in the range of 67 g/L-89 g/L. The total loaded amount for Kp screen was 150 g/L. The loads were 0.2 ?m filtered and protein content was determined (OD 280-320).
Preparation of the Filter Plate
[0457] A 50% slurry of the Capto? adhere ImpRes resin in water was produced in a tube using a centrifuge for rapid settlement. Then the resin was transferred to a shaker placed on the Hamilton Microlab STARlet roboter. Per well of the filter plate 50 ?L resin Capto? adhere ImpRes were added.
Preparation of Buffers
[0458] For the preparation of buffer plate and load plate the following materials were used: [0459] high salt buffers; [0460] low salt buffers; [0461] 10 mM Tris/Acetate, pH 6.5 (0.6 mS/cm); [0462] protein stock solution in 10 mM Tris/Acetate, pH 6.5 in an appropriate concentration; [0463] strip buffer.
[0464] For Kp screens a buffer plate and a load plate were produced by the robotic system (Tecan Freedom EVO 200) using high salt buffers as well as low salt buffers. The high and low salt stock solutions were prepared by weighing in Tris and the required amount of salt. Then the pH was adjusted with acetic acid.
[0465] Table X-2.1 and X-2.2 summarize the low and high salt buffers used to prepare the equilibration and load plates.
TABLE-US-00016 TABLE X-2.1 Example 5-low salt buffers for Kp Screen conductivity low salt buffers pH [mS/cm] 70 mM Tris/Acetate, pH 5.5 5.50 3.7 70 mM Tris/Acetate, pH 7.0 7.07 3.4 70 mM Tris/Acetate, pH 8.0 8.08 2.0
TABLE-US-00017 TABLE X-2.2 Example 5-high salt buffers for Kp Screen conductivity high salt buffers pH [mS/cm] 70 mM Tris/Acetate, 5.56 137.7 1M (NH.sub.4).sub.2SO.sub.4, pH 5.5 70 mM Tris/Acetate, 7.06 136.8 1M (NH.sub.4).sub.2SO.sub.4, pH 7.0 70 mM Tris/Acetate, 8.00 135.3 1M (NH.sub.4).sub.2SO.sub.4, pH 8.0 70 mM Tris/Acetate, 5.42 111.4 1M KCl, pH 5.5 70 mM Tris/Acetate, 6.96 111.7 1M KCl, pH 7.0 70 mM Tris/Acetate, 7.94 111.2 1M KCl, pH 8.0 70 mM Tris/Acetate, 5.49 3.6 1M Urea, pH 5.5 70 mM Tris/Acetate, 6.93 3.5 1M Urea, pH 7.0 70 mM Tris/Acetate, 7.99 2.3 1M Urea, pH 8.0 70 mM Tris/Acetate, 5.56 83.1 1M Gua/HCl, pH 5.5 70 mM Tris/Acetate, 6.87 83.2 1M Gua/HCl, pH 7.0 70 mM Tris/Acetate, 7.89 82.9 1M Gua/HCl, pH 8.0
[0466] The load and equilibration plates were pipetted by the robot as shown in Table X-2.3. The molarities of the four salts were in the range of 10 mM up to ?800 mM.
TABLE-US-00018 TABLE X-2.3 Example 5plate layout of KpScreen (NH.sub.4).sub.2SO.sub.4 KCl Urea Gua/HCl salt pH pH pH pH pH pH pH pH pH pH pH pH molarity 5.5 7.0 8.0 5.5 7.0 8.0 5.5 7.0 8.0 5.5 7.0 8.0 [mM] A 10 B 75 C 150 D 225 E 300 F 450 G 650 H ~800
[0467] The concentrated protein stock solution was pipetted by the robot into the load plate. To neglect a shift in pH and conductivity of each well condition, the protein stock solution was available in 10 mM Tris/Acetate, pH 6.5 and at a high protein concentration to minimize the pipetting volume.
Execution of Kp Screen
[0468] The total loaded amount for the Kp screens was set to 150 g/L and split into two loading steps of each 75 g/L.
[0469] The Kp screen method consisted of the following steps: [0470] removal of storage buffer; [0471] equilibration 1+2: transfer of 300 ?L equilibration buffer, incubation of 5 min. on Shaker (1100 rpm) and centrifugation to remove the equilibration buffer (2,500 rpm, 600 sec); [0472] loading 1+2: transfer of 300 ?L load, incubation of 60 min. on Shaker (1100 rpm) and centrifugation to collect the FT (2,500 rpm, 600 sec) on a FT plate; [0473] strip 1+2: transfer of 300 ?L strip buffer, incubation of 5 min. on Shaker (1100 rpm) and centrifugation to remove the strip buffer (2,500 rpm, 600 sec).
[0474] SE-HPLC analytics of the FT plate was performed.
[0475] The HMW removal was calculated as followed:
[0476] HMW.sub.well is the HMW value [%] measured in a well of the FT plate, HMW.sub.load is the HMW level [%] of the protein stock solution.
[0477] The protein concentration of the load plate and FT plate were determined using the Infinite M200 plate reader.
[0478]
[0479] The effect of (NH.sub.4).sub.2SO.sub.4 on HMW reduction is shown in
Summary of Example 5
[0480] The effect of four salts (two antichaotropic and two chaotropic salts) was shown with two hydrophilic and one hydrophobic mab in the pH range of pH 5.5-8.0 and a salt molarity up to 800 mM. The chaotropic salts (Gua/HCl and urea) were chosen to determine HMW reduction when hydrophobic interactions were weakened.
[0481] Depending on the hydrophobicity of the mab, differences in HMW reduction were observed. For the hydrophilic mabs (mab2 and mab4) the addition of an antichaotropic salt (ammonium sulfate,
[0482] In summary, HMW reduction was improved for the hydrophilic mabs in the presence of an antichaotropic salt. For the hydrophobic mab, no improved HMW reduction could be seen in the presence of an antichaotropic salt. Furthermore, an improved HMW reduction was not obtained in the presence of chaotropic salts.
Example 6
Kp Screen with Mab2 (pH 4-9)
[0483] For the Kp screen with one hydrophilic mab (mab2) and the two buffer systems i) 25 mM Tris/Acetate comprising (10 to 850) mM Na.sub.2SO.sub.4 and ii) (25 to 975) mM Tris/Acetate the following stock solutions were prepared (see Table X-2.4):
TABLE-US-00019 TABLE X-2.4 Example 6-low and high salt buffers for Kp Screen high salt buffers high salt buffers low salt buffers (Na.sub.2SO.sub.4) (Tris/Acetate) 25 mM Tris/Acetate, 25 mM Tris/Acetate, 1.4M Tris/Acetate, pH 4.0 1.4M Na.sub.2SO.sub.4, pH 4.0 pH 4.0 25 mM Tris/Acetate, 25 mM Tris/Acetate, 1.4M Tris/Acetate, pH 5.0 1.4M Na.sub.2SO.sub.4, pH 5.0 pH 5.0 25 mM Tris/Acetate, 25 mM Tris/Acetate, 1.4M Tris/Acetate, pH 6.0 1.4M Na.sub.2SO.sub.4, pH 6.0 pH 6.0 25 mM Tris/Acetate, 25 mM Tris/Acetate, 1.4M Tris/Acetate, pH 7.0 1.4M Na.sub.2SO.sub.4, pH 7.0 pH 7.0 25 mM Tris/Acetate, 25 mM Tris/Acetate, 1.4M Tris/Acetate, pH 8.0 1.4M Na.sub.2SO.sub.4, pH 8.0 pH 8.0 25 mM Tris/Acetate, 25 mM Tris/Acetate, 1.4M Tris/Acetate, pH 9.0 1.4M Na.sub.2SO.sub.4, pH 9.0 pH 9.0
[0484] The total loaded amount was 150 g/L and splitted into two loading steps. [0485] Buffer conditions: 2 buffer systems were investigated as shown in Table X-2.5: [0486] 25 mM Tris/Acetate+(10-850) mM Na.sub.2SO.sub.4 (pH 4.0-9.0); Na.sub.2SO.sub.4 molarities: 10, 75, 150, 225, 300, 450, 650, 850 mM [0487] 25-975 mM Tris/Acetate (pH 4.0-9.0); Tris molarities: 25,100,175, 250, 350, 500, 750, 975 mM
TABLE-US-00020 TABLE X-2.5 Example 6plate layout KpScreen mo- larity molarity Na.sub.2SO.sub.4 Tris/Acetate Tris/ Na.sub.2SO.sub.4 pH pH pH pH pH pH pH pH pH pH pH pH Acetate [mM] 4.0 5.0 6.0 7.0 8.0 9.0 4.0 5.0 6.0 7.0 8.0 9.0 [mM] 10 25 75 100 150 175 225 250 300 350 450 500 650 750 850 975
[0488]
Summary of Example 6
[0489] Example 6 shows that in the presence of an antichaotropic salt HMW reduction of mab2 containing loads is increased. An increasing Na.sub.2SO.sub.4 molarity (see
Part III: Comparison of HMW Removal with Other Resins/Chromatography Material
Example 7
Kp Screen with Mixed Mode AEX Resins
[0490] The HMW reduction of loads comprising a hydrophilic mab (mab2) and the following mixed mode AEX resins was determined: Capto? adhere ImpRes, Capto? adhere and Nuvia aPrime4A. These 3 resins exhibit anionic and hydrophobic moieties.
[0491] The Kp screens were executed corresponding Example 5.
[0492]
Summary of Example 7
[0493] In this Example HMW reduction on three mixed mode resins with anion exchange and hydrophobic interaction were compared. Capto? adhere ImpRes flowthrough contour plots (
[0494] In general, for all salts the contour plots of Capto? adhere, Nuvia aPrime and Capto? adhere ImpRes showed comparable effects. With increasing (NH.sub.4).sub.2SO.sub.4 and KCl molarity, all three mixed mode resins showed an improved HMW reduction. The Capto? adhere, Nuvia aPrime and Capto? adhere ImpRes contour plots showed good comparability. For the chaotropic salts no improved HMW reduction was observed with the 3 resins.
Example 8
Kp Screen with MMAEX, AEX, HIC, MMCEX Resins
[0495] The contour plots for one hydrophilic mab and one hydrophobic mab were determined with the following resins: [0496] a mixed mode anion exchange resin (Capto? adhere ImpRes) (MMAEX); [0497] an anion exchange resin (Q Sepharose FF) (AEX); [0498] a hydrophobic resin (Phenyl Sepharose 6 FF (high sub)) (HIC); [0499] a mixed mode cation exchange resin (Capto? MMC ImpRes) (MMCEX).
[0500] The total loaded amount for the resins was 150 g/L except for Capto? MMC ImpRes with a total loaded amount of 75 g/L.
[0501] The Kp screens were executed corresponding Example 5.
[0502] For the Kp screen for the Q Sepharose FF, Phenyl Sepharose 6FF (high sub) and Capto? MMC ImpRes a Na.sub.2SO.sub.4 buffer system was used: 25 mM Tris/Acetate+(5 to 850) mM Na.sub.2SO.sub.4. The following stock solutions were prepared (see X-3.1):
TABLE-US-00021 TABLE X-3.1 Example 8-low and high salt buffers low salt buffers high salt buffers 25 mM 25 mM Tris/Acetate, Tris/Acetate, pH 4.0 1.4M Na.sub.2SO.sub.4, pH 4.0 25 mM 25 mM Tris/Acetate, Tris/Acetate, pH 5.0 1.4M Na.sub.2SO.sub.4, pH 5.0 25 mM 25 mM Tris/Acetate, Tris/Acetate, pH 6.0 1.4M Na.sub.2SO.sub.4, pH 6.0 25 mM 25 mM Tris/Acetate, Tris/Acetate, pH 7.0 1.4M Na.sub.2SO.sub.4, pH 7.0 25 mM 25 mM Tris/Acetate, Tris/Acetate, pH 8.0 1.4M Na.sub.2SO.sub.4, pH 8.0 25 mM 25 mM Tris/Acetate, Tris/Acetate, pH 9.0 1.4M Na.sub.2SO.sub.4, pH 9.0
[0503] The following Na.sub.2SO.sub.4 containing buffers were investigated as shown in Table X-3.2: [0504] 25 mM Tris/Acetate+ (5-850) mM Na.sub.2SO.sub.4 (pH 4.0-9.0); Na.sub.2SO.sub.4 molarities: 10, 75, 150, 225, 300, 450, 650, 850 mM
TABLE-US-00022 TABLE X-3.2 Example 8plate layout of KpScreen molarity mab2 mab6 Na.sub.2SO.sub.4 pH pH pH pH pH pH pH pH pH pH pH pH [mM] 4.0 5.0 6.0 7.0 8.0 9.0 4.0 5.0 6.0 7.0 8.0 9.0 5 75 150 225 300 450 650 850
[0505]
Summary of Example 8
[0506] With one hydrophilic mab (mab2) and one hydrophobic mab (mab6) the HMW reduction in the flowthrough of the following resins was determined: a mixed mode anion exchange resin (Capto? adhere ImpRes), an anion exchange resin (Q Sepharose FF), a hydrophobic resin (Phenyl Sepharose 6 FF) and a mixed mode cation exchange resin (Capto? MMC ImpRes).
[0507] For the AEX resin Q Sepharose FF no effect of an antichaotropic salt regarding HMW reduction was observed for both mabs (
[0508] In contrast thereto, for the mixed mode resins HMW reduction for the hydrophilic and hydrophobic mab were different. The MMAEX resin showed an improved HMW reduction for mab2 (
[0509] It has been shown that an increased HMW reduction for a hydrophilic mab by addition of an antichaotropic salt could be attributed to the combination of ionic and hydrophobic interaction. For both ionic mixed mode resins, the Capto? adhere ImpRes (with anionic and hydrophobic moieties) and the Capto? MMC ImpRes (with cationic and hydrophobic moieties), an increased HMW reduction was achieved with increasing salt molarity, but only for the hydrophilic mab.
Part IV: ?KTA Column Runs
Example 9
Runs at Same Conductivity and Different Na.sub.2SO.sub.4 Molarities
[0510] Two loads were prepared with the same conductivity, but different molarities of Na.sub.2SO.sub.4.
[0511] Load 1 and load 2 were prepared using the same affinity chromatography pool (column 1 pool) of mab2. [0512] Load 1: A column 1 pool of mab2 was adjusted to pH 8.0 with 1.5 M Tris-base, depth filtered, then conductivity was adjusted to 9 mS/cm using 1 M Na.sub.2SO.sub.4 solution. Then the load was applied to a Capto? adhere ImpRes column. The conductivity of load 1 was 9 mS/cm, the Na.sub.2SO.sub.4 molarity was 39 mM. [0513] Load 2: A column 1 pool of mab2 was adjusted to pH 8.0 with 1.5 M Tris-base, depth filtered, then pH was adjusted to pH 5.6 with acetic acid, followed by a readjustment to pH 8.0 with 1.5 M Tris. Thereafter conductivity was adjusted to 9 mS/cm with 1 M Na.sub.2SO.sub.4 solution and the solution applied to the column. The conductivity of load 2 was 9 mS/cm, the Na.sub.2SO.sub.4 molarity was 19 mM.
[0514] Table X-4.1 summarizes the load adjustment.
TABLE-US-00023 TABLE X-4.1 Example 9-load adjustment Load 1 Load 2 (containing ~40 mM (containing ~20 mM Na.sub.2SO.sub.4 Na.sub.2SO.sub.4) 1. pH adjustment to pH 8.0 1. pH adjustment to pH 8.0 with 1.5M Tris with 1.5M Tris 2. depth filtration 2. depth filtration 3. conductivity adjusted 3. back titration to pH 5.6 to 9 mS/cm 4. adjustment again to 4. applied to Capto adhere pH 8.0 ImpRes column 5. conductivity adjusted to 9 mS/cm using 1M Na.sub.2SO.sub.4 6. applied to Capto adhere ImpRes column Load 1 properties Load 2 properties molarity Na.sub.2SO.sub.4: 39 mM molarity Na.sub.2SO.sub.4: 19 mM load concentration: 20.34 g/L load concentration: 18.73 g/L load pH: 8.08 load pH: 8.00 load conductivity: 9.02 mS/cm load conductivity: 9.10 mS/cm
[0515] The buffers used in example 9 are listed in Table X-4.2:
TABLE-US-00024 TABLE X-4.2 Example 9-buffers conductivity buffer pH [mS/cm] 70 mM Tris/Acetate, 7.96 8.73 40 mM Na.sub.2SO.sub.4, pH 8 1.5M Tris 11.02 0.196 1M Na.sub.2SO.sub.4 5.88 93.8
[0516] The column volume of the Capto? adhere ImpRes column was 6.84 mL with a column diameter of 0.66 cm. After equilibration of the column with 70 mM Tris/Acetate, 40 mM Na.sub.2SO.sub.4, pH 8 the load was applied to the column. The load capacity was ?150 g/l and the flow rate was 150 cm/h. The Capto? adhere ImpRes FT was fractionated. Protein concentration of the fractions was measured and SE-HPLC was performed.
[0517]
Summary of Example 9
[0518] Two loads of mab2 were prepared with the same conductivity of 9 mS/cm, but different molarities of Na.sub.2SO.sub.4 (about 40 mM and about 20 mM). The FT fractions of the load with the higher Na.sub.2SO.sub.4 molarity had higher mainpeak values compared to the load with lower Na.sub.2SO.sub.4 molarity (see
Example 10
Runs at Different Na.sub.2SO.sub.4 Molarities and Conductivities
[0519] The impact of increasing salt molarity (and conductivity) was determined with mab2. Column runs were performed at pH 7 and pH 8 with different Na.sub.2SO.sub.4 molarities and conductivities on a Capto? adhere ImpRes column (column volume=6.84 mL; d=0.66 cm). The FT was fractionated and the load capacity was ?150 g/L.
[0520] The chromatographic conditions are shown in Table X-4.3:
TABLE-US-00025 TABLE X-4.3 Example 10 - chromatography steps step buffer CV flow [cm/h] Equilibration 70 mM Tris/Acetate, 7 150 x mM Na.sub.2SO.sub.4, (pH 7/pH 8) Load ~150 g/L 150
TABLE-US-00026 TABLE X-4.4 Example 10-buffers buffer conductivity buffer pH [mS/cm] 70 mM Tris/Acetate, pH 8.0 7.96 2.4 70 mM Tris/Acetate, 8.04 5.7 20 mM Na.sub.2SO.sub.4, pH 8.0 70 mM Tris/Acetate, 8.00 8.9 40 mM Na.sub.2SO.sub.4, pH 8.0 70 mM Tris/Acetate, 8.00 11.8 60 mM Na.sub.2SO.sub.4, pH 8.0 70 mM Tris/Acetate, 6.92 9.8 40 mM Na.sub.2SO.sub.4, pH 7.0 70 mM Tris/Acetate, 6.95 12.5 60 mM Na.sub.2SO.sub.4, pH 7.0 1.5M Tris 11.02 0.2 1M Na.sub.2SO.sub.4 5.88 93.8
[0521] The following loads were obtained after adjustment with 1.5 M Tris and 1 M Na.sub.2SO.sub.4 (see Table X-4.5):
TABLE-US-00027 TABLE X-4.5 Example 10load conditions Run 1 Run 2 Run 3 Run 4 Run 5 Run 6 load pH 8.0 8.0 8.1 8.1 7.1 7.1 load conductivity 4.9 6.2 9.2 12.3 9.2 12.1 [mS/cm] load concentration 10.7 24.5 23.3 21.8 23.6 23.5 [mg/mL] load Na.sub.2SO.sub.4 0 10 35 60 34 55 molarity [mM]
[0522]
[0523]
[0524] Pools were calculated in the following way:
[0525] The mainpeak value of pools was calculated using the average mainpeak value of the fractions. Wash fractions (following the load step) were not included in the FT pools. Table X-4.6 summarizes the run conditions and mainpeak values.
TABLE-US-00028 TABLE X-4.6 Example 10-run conditions and mainpeak values of FT pools conductivity load molarity loaded mainpeak load Na.sub.2SO.sub.4 amount (FT Pool) pH [mS/cm] [mM] [g/L] [%] 8.0 5 0 159 96.96 8.0 6 10 157 97.67 8.0 9 35 163 98.69 8.0 12 60 153 99.09 7.0 9 34 166 96.87 7.0 12 55 165 97.41
[0526]
Summary of Example 10
[0527] The effect of Na.sub.2SO.sub.4 molarity on HMW removal for pH 7 and pH 8 has been shown. These data support the data obtained with the robotic systems. The mainpeak values of the FT fractions raised with increasing Na.sub.2SO.sub.4 molarity at pH 7 (see
Examples 11 and 12
KpScreen: Impact of Antichaotropic Salt and Mab Hydrophobicity on RVLP Removal
Kp Screens (pH 5.0-8.0)
[0528] The methodology of Kp screens in connection with RVLP removal is described in detail in this example.
Preparation of Mab Solutions
[0529] The antibody containing solutions (column 2 pools) were concentrated with Amicon Ultra Centrifugal Filters and buffer exchanged to 10 mM Tris/Acetate, pH 6.5 and concentrated with Amicon Ultra Centrifugal Filters. The protein concentrations were in the range of 61 g/L-72 g/L. The total loaded amount for Kp screen was 150 g/L. The loads were 0.2 ?m filtered and protein content was determined (OD 280-320).
Preparation of the Filter Plate
[0530] A 50% slurry of the Capto? adhere ImpRes resin in water was produced in a tube using a centrifuge for rapid settlement. Then the resin was transferred to a shaker placed on the Hamilton Microlab STARlet roboter. Per well of the filter plate 50 ?L resin Capto? adhere ImpRes were added.
Preparation of Buffers
[0531] For the preparation of buffer plate and load plate the following materials were used: [0532] high salt buffers; [0533] low salt buffers; [0534] 10 mM Tris/Acetate, pH 6.5 (0.6 mS/cm); [0535] protein stock solution in 10 mM Tris/Acetate, pH 6.5 in an appropriate concentration; [0536] strip buffer.
[0537] For Kp screens a buffer plate and a load plate were produced by the robotic system (Tecan Freedom EVO 200) using high salt buffers as well as low salt buffers. The high and low salt stock solutions were prepared by weighing in Tris and the required amount of salt. Then the pH was adjusted with acetic acid.
[0538] Table X-5.1 and X-5.2 summarize the low and high salt buffers used to prepare the equilibration and load plates for Example 11.
TABLE-US-00029 TABLE X-5.1 Example 11-low salt buffers for Kp Screen conductivity low salt buffers pH [mS/cm] 25 mM Tris/Acetate, pH 5.0 5.01 1.52 25 mM Tris/Acetate, pH 7.0 7.02 1.42 25 mM Tris/Acetate, pH 8.0 8.01 0.83
TABLE-US-00030 TABLE X-5.2 Example 11-high salt buffers for Kp Screen conductivity high salt buffers pH [mS/cm] 25 mM Tris/Acetate, 5.01 111.6 1.4M Na.sub.2SO.sub.4, pH 5.0 25 mM Tris/Acetate, 6.96 116.7 1.4M Na.sub.2SO.sub.4, pH 7.0 25 mM Tris/Acetate, 8.00 111.1 1.4M Na.sub.2SO.sub.4, pH 8.0
[0539] Table X-5.3 and X-5.4 summarize the low and high salt buffers used to prepare the equilibration and load plates for Example 12.
TABLE-US-00031 TABLE X-5.3 Example 12-low salt buffers for Kp Screen conductivity low salt buffers PH [mS/cm] 25 mM Tris/Acetate, pH 5.0 5.01 1.52 25 mM Tris/Acetate, pH 7.0 7.02 1.42 25 mM Tris/Acetate, pH 8.0 8.01 0.83
TABLE-US-00032 TABLE X-5.4 Example 12-high salt buffers for Kp Screen conductivity high salt buffers pH [mS/cm] 1.4M Tris/Acetate, pH 5.0 5.02 111.6 1.4M Tris/Acetate, pH 7.0 7.03 116.7 1.4M Tris/Acetate, pH 8.0 8.04 111.1
[0540] Before pipetting the high salt and low salt buffers, 10 ?l of an RVLP stock solution were pipetted to each well of the load plate by the robot for Example 11 and 12. The load and equilibration plates were pipetted by the robot as shown in Table X-5.5. For Example 11 the molarities of sodium sulfate were in the range of 25 mM up to 400 mM.
TABLE-US-00033 TABLE X-5.5 Example 11plate layout of KpScreen mab1 mab2 mab9 mab7 Na.sub.2SO.sub.4 pH pH pH pH pH pH pH pH pH pH pH pH molarity 5.0 7.0 8.0 5.0 7.0 8.0 5.0 7.0 8.0 5.0 7.0 8.0 [mM] A 25 B 40 C 50 D 75 E 100 F 200 G 300 H 400
[0541] For Example 12 the load and equilibration plates were pipetted by the robot as shown in Table X-5.6. For example 12 the molarities of Tris were in the range of 25 mM up to 1100 mM.
TABLE-US-00034 TABLE X-5.6 Example 12plate layout of KpScreen mab1 mab2 mab9 mab7 Tris pH pH pH pH pH pH pH pH pH pH pH pH molarity 5.0 7.0 8.0 5.0 7.0 8.0 5.0 7.0 8.0 5.0 7.0 8.0 [mM] A 25 B 100 C 200 D 400 E 600 F 800 G 1000 H 1100
[0542] For Example 11 and 12 the concentrated protein stock solution was pipetted by the robot into the load plate. To neglect a shift in pH and conductivity of each well condition, the protein stock solution was available in 10 mM Tris/Acetate, pH 6.5 and at a high protein concentration to minimize the pipetting volume.
Execution of Kp Screen
[0543] The total loaded amount for the Kp screens was set to 150 g/L and split into two loading steps of each 75 g/L.
[0544] The Kp screen method consisted of the following steps: [0545] removal of storage buffer; [0546] equilibration 1+2: transfer of 300 ?L equilibration buffer, incubation of 5 min. on Shaker (1100 rpm) and centrifugation to remove the equilibration buffer (2,500 rpm, 600 sec); [0547] loading 1+2: transfer of 300 ?L load, incubation of 60 min. on Shaker (1100 rpm) and centrifugation to collect the FT (2,500 rpm, 600 sec) on a FT plate; [0548] strip 1+2: transfer of 300 ?L strip buffer, incubation of 5 min. on Shaker (1100 rpm) and centrifugation to remove the strip buffer (2,500 rpm, 600 sec).
[0549] RNA analytics were performed for the FT plate.
[0550] The RVLP removal (RNA log reduction) was calculated with the RNA concentrations as followed:
[0551] RNA concentration.sub.load is the average RNA concentration [copies/?L] measured in selected wells of the load plate. For Example 11 the average RNA concentration of the load wells was 182357 [copies/?L]. For Example 12 the average RNA concentration of the load wells was 84250 [copies/?L].
[0552] RNA concentration.sub.well is the RNA concentration [copies/?L] measured in each well of the FT plate for Example 11 and 12.
[0553]
[0554]
[0555]
[0556]
[0557]
Summary of Example 11 and 12
[0558] The effect of mab hydrophobicity and the presence of an antichaotropic salt was shown with two hydrophilic and two hydrophobic mabs in the pH range of pH 5.0-8.0. In Example 11 (
[0559] Depending on the hydrophobicity of the mab and the presence of an antichaotropic salt, different RNA reduction values were measured.
[0560] For Example 12 (no antichaotropic salt) the investigated mabs show a RNA log reduction range of 4-5 only for a low Tris/Acetate molarity <100 mM (corresponding to a conductivity of <3 mS/cm) independent of the mab hydrophobicity (no significant difference between hydrophilic and hydrophobic mabs). For the hydrophilic mabs 1 and 2 a log reduction value of 4-5 was measured only for salt molarities <25 mM (conductivity <1.5 mS/cm).
[0561] In contrast to Example 12, an improved RNA reduction was observed for the hydrophilic mabs in the presence of Na.sub.2SO.sub.4 (Example 11). For hydrophilic mab1 a RNA log reduction range of 4-5 was observed up to a salt molarity of 100 mM Na.sub.2SO.sub.4 at pH 5, corresponding to a conductivity of <9.4 mS/cm. For pH 8 a log reduction value of 4-5 was measured for molarities <40 mM (corresponding to a conductivity of 4 mS/cm). For hydrophilic mab2 a RNA log reduction range of 4-5 was observed up to a salt molarity of 225 mM, corresponding to a conductivity of <19 mS/cm. For the hydrophobic mabs no significant increase in RNA reduction was observed in the presence of an antichaotropic salt.