Patent Application
20210380638
The present invention relates generally to a combination and a method of removing antibody aggregate by Protein A chromatography.
In general, Protein A chromatography under typical conditions is less effective at removing aggregates. Although aggregates are known to bind more strongly than monomer (D. Yu, Y. Song, R. Y. Huang, et al., Molecular perspective of antibody aggregates and their adsorption on Protein A resin, J. Chromatogr. A, 2016, 1457, 66-75), they are often co-eluted with the latter and adjusting elution pH alone usually is not sufficient for good separation. Consequently, in many cases aggregate removal relies on a single polishing chromatography post Protein A. However, relying on a single step for aggregate removal is unfavorable as such design decreases the robustness of the entire downstream process.
In certain case the yield of the step dedicated to aggregate removal needs to be greatly sacrificed in order to meet the purity requirement. This relying on single-step design is especially problematic for projects with higher-than-average aggregate content. It is desirable to have a complementary aggregate-removing step that can share the burden and partially clear the aggregates at an early stage.
The present invention provides a combination for use in Protein A chromatography comprising a component A which is at least one type of polyethylene glycol (PEG) polymer and a component B which is at least one Hofmeister series salt (e.g. chaotropic salts or kosmotropic salts).
In one embodiment, the combination consists of a component A which comprises or preferably is at least one type of polyethylene glycol (PEG) polymer and a component B which comprises or preferably is at least one Hofmeister series salt.
In one embodiment, ratio of the PEG and the salt ranges from 1 g:2.5 mmol to 1 g:100 mmol, preferably from 1 g:10 mmol to 1 g:25 mmol.
In one embodiment, the component of combination such as component A or component B may be formulated separately. In one embodiment, the component of combination such as component A or component B may be formulated as a homogenous composition.
In one embodiment, the molecular weight of PEG polymer ranges from about 200 Dalton to about 10,000,000 Dalton, preferably from about 400 Dalton to about 6000 Dalton. For example, PEG 200 Dalton, PEG 400 Dalton, PEG 600 Dalton, PEG 800 Dalton, PEG 1000 Dalton, PEG 1500 Dalton, PEG 2000 Dalton, PEG 3000 Dalton, PEG3350 Dalton, PEG 4000 Dalton, PEG 6000 Dalton and PEG 8000 Dalton. The PEG that is able to improve Protein A chromatography's antibody aggregate removal together with a Hofmeister series salt is within the scope of the invention.
In one embodiment, the Hofmeister series salt is composed of a combination of Hofmeister series of cations and inions, preferably is one salt selected from the group consisting of calcium chloride, sodium chloride, magnesium chloride, and potassium chloride.
In one embodiment, Protein A chromatography is used to improve protein sample aggregate removal, wherein the protein sample comprises any type of protein which contains an Fc region recognizable by Protein A. Such protein comprises antibodies and Fc-fusion proteins. The antibody could be a monoclonal antibody, or a polyclonal antibody. The antibody could be monospecific, bispecific or multi-specific. The antibody could be a mouse antibody, a chimeric antibody, a humanized antibody or a human antibody. An Fc-fusion protein is composed of an Fc region of an antibody and a genetically linked active protein.
In other aspect, inventors provide a composition or a kit, wherein the combination or the kit further comprises a component C, the component C is one buffer selected from the group consisting of wash buffer solution and elution buffer solution, wherein the wash buffer solution or elution buffer solution comprises NaAc and/or HAc, for example. It will be understood by those skilled in the art that in the present invention PEG and Hofmeister series salts can be dissolved in any background buffer as long as the buffer can be used for wash or elution.
In one specific embodiment, ratio of weight of PEG polymer relative to the volume of the wash buffer solution or elution buffer solution is from about 10 g:1 L to about 100 g:1 L, preferably from about 20 g:1 L to about 50 g:1 L., that is, percentage of weight of PEG polymer in the volume of the wash buffer solution or elution buffer solution is from about 1 w/v % to about 10 w/v %, preferably from about 2 w/v % to about 5 w/v %, such as 1 w/v %, 2 w/v %, 3 w/v %, 4 w/v %, 5 w/v %, 6 w/v %, 7 w/v %, 8 w/v %, 9 w/v %, 10 w/v %; the effective PEG concentration depends on the molecular weight of the particular PEG being used. For example, the required percentage of weight of PEG3350 in the volume of the wash buffer solution or elution buffer solution is from about 3.5 w/v % to about 5 w/v %. A lower percentage is sufficient for PEG polymer with higher molecular weight (e.g., PEG 6000) whereas a higher percentage is required for PEG with lower molecular weight (e.g., PEG 600).
In one specific embodiment, ratio of molar mass of the Hofmeister series salt relative to the volume of the wash buffer solution or elution buffer solution is about 250 mmol:1 L and more, preferably is from about 250 mmol:1 L to about 1 mol:1 L, more preferably is from about 500 mmol:1 L to 750 mmol:1 L, that is, percentage of molar mass of the Hofmeister series salt such as calcium chloride, or sodium chloride, or magnesium chloride, or potassium chloride in the volume of the wash buffer solution or elution buffer solution is about 250 mM and more, preferably is from about 250 mM to about 1 M, more preferably is from about 500 mM to about 750 mM, such as 200 mM, 300 mM, 400 mM, 500 mM, 600 mM, 700 mM, 800 mM, 900 mM and 1 M.
In another aspect, the invention provides aforementioned combination or composition or kit for use in protein sample purification by Protein A chromatography, wherein the combination improves monomer-aggregate resolution on Protein A chromatography column, allowing effective removal of antibody aggregates.
The invention provides the use of the aforementioned combination for preparation of the wash buffer and/or the elution buffer for the Protein A column. In particular, PEG and Hofmeister series salts are used together as wash and/or elution buffer additives to achieve the resolution enhancing effect.
In one embodiment, the component of combination such as component A or component B may be formulated separately. In one embodiment, the component of combination such as component A or component B may be formulated as a homogenous composition.
In one embodiment, the molecular weight of PEG polymer ranges from about 200 Dalton to about 10,000,000 Dalton, preferably from about 400 Dalton to about 6000 Dalton. For example, PEG 200 Dalton, PEG 400 Dalton, PEG 600 Dalton, PEG 800 Dalton, PEG 1000 Dalton, PEG 1500 Dalton, PEG 2000 Dalton, PEG 3000 Dalton, PEG3350 Dalton, PEG 4000 Dalton, PEG 6000 Dalton and PEG 8000 Dalton. The PEG that is able to improve Protein A chromatography's protein sample such as Fc-region containing antibody aggregate removal together with a Hofmeister series salt is within the scope of the invention.
In one embodiment, the Hofmeister series salt is composed of a combination of Hofmeister series of cations and inions, preferably is one salt selected from the group consisting of calcium chloride, sodium chloride, magnesium chloride, and potassium chloride.
In one embodiment, the protein sample comprises any type of protein which contains an Fc region recognizable by Protein A. Such protein comprises antibodies and Fc-fusion proteins. The antibody could be a monoclonal antibody, or a polyclonal antibody. The antibody could be monospecific, bispecific or multi-specific. The antibody could be a mouse antibody, a chimeric antibody, a humanized antibody or a human antibody. An Fc-fusion protein is composed of an Fc region of an antibody and a genetically linked active protein.
In one embodiment, the aforementioned combination further comprises a component C, the component C is one buffer selected from the group consisting of wash buffer solution and elution buffer solution, wherein the wash buffer solution or elution buffer solution comprises NaAc and/or HAc, for example. It will be understood by those skilled in the art that in the present invention PEG and Hofmeister series salts can be dissolved in any background buffer as long as the buffer can be used for wash or elution.
In one specific embodiment, ratio of weight of PEG polymer relative to the volume of the wash buffer solution or elution buffer solution is from about 10 g:1 L to about 100 g:1 L, preferably from about 20 g:1 L to about 50 g:1 L, that is, percentage of weight of PEG polymer in the volume of the wash buffer solution or elution buffer solution is from about 1 w/v % to about 10 w/v %, preferably from about 2 w/v % to about 5 w/v %, such as 1 w/v %, 2 w/v %, 3 w/v %, 4 w/v %, 5 w/v %, 6 w/v %, 7 w/v %, 8 w/v %, 9 w/v %, 10 w/v %; the effective PEG concentration depends on the molecular weight of the particular PEG being used. For example, the required percentage of weight of PEG3350 in the volume of the wash buffer solution or elution buffer solution is from about 3.5 w/v % to about 5 w/v %. A lower percentage is sufficient for PEG polymer with higher molecular weight (e.g., PEG 6000) whereas a higher percentage is required for PEG with lower molecular weight (e.g., PEG 600).
In one specific embodiment, ratio of molar mass of the Hofmeister series salt relative to the volume of the wash buffer solution or elution buffer solution is about 250 mmol:1 L and more, preferably is from about 250 mmol:1 L to about 1 mol:1 L, more preferably is from about 500 mmol:1 L to 750 mmol:1 L, that is, percentage of molar mass of the Hofmeister series salt such as calcium chloride, or sodium chloride, or magnesium chloride, or potassium chloride in the volume of the wash buffer solution or elution buffer solution is about 250 mM and more, preferably is from about 250 mM to about 1 M, more preferably is from about 500 mM to about 750 mM, such as 200 mM, 300 mM, 400 mM, 500 mM, 600 mM, 700 mM, 800 mM, 900 mM and 1 M.
In further aspect, the invention provides a method for removing antibody aggregates by Protein A chromatography, comprising the following steps:
1) loading a protein sample onto a Protein A chromatography column,
2) washing the column with a wash buffer, wherein the wash buffer comprises at least one type of PEG polymer and at least one Hofmeister series salt, and
3) eluting the column with an elution buffer, wherein the elution buffer comprises at least one type of PEG polymer and at least one Hofmeister series salt.
In the method, the PEG polymer has a molecular weight of from about 200 Dalton to about 10,000,000 Dalton, preferably from about 400 Dalton to about 6000 Dalton. For example, PEG 200 Dalton, PEG 400 Dalton, PEG 600 Dalton, PEG 800 Dalton, PEG 1000 Dalton, PEG 1500 Dalton, PEG 2000 Dalton, PEG 3000 Dalton, PEG3350 Dalton, PEG 4000 Dalton, PEG 6000 Dalton, and PEG 8000 Dalton. The PEG that is able to improve Protein A chromatography's protein sample such as Fc-region containing antibody aggregate removal together with a Hofmeister series salt is within the scope of the invention.
In one embodiment, the Hofmeister series salt is composed of a combination of Hofmeister series of cations and inions, preferably is one salt selected from the group consisting of calcium chloride, sodium chloride, magnesium chloride, and potassium chloride.
In one embodiment, the protein sample comprises any type of protein which contains an Fc region recognizable by Protein A. Such protein comprises antibodies and Fc-fusion proteins. The antibody could be a monoclonal antibody, or a polyclonal antibody. The antibody could be monospecific, bispecific or multi-specific. The antibody could be a mouse antibody, a chimeric antibody, a humanized antibody or a human antibody. An Fc-fusion protein is composed of an Fc region of an antibody and a genetically linked active protein.
In one embodiment, the aforementioned combination further comprises a component C, the component C is one buffer selected from the group consisting of wash buffer solution and elution buffer solution, wherein the wash buffer solution or elution buffer solution comprises NaAc and/or HAc, for example. It will be understood by those skilled in the art that in the present invention PEG and Hofmeister series salts can be dissolved in any background buffer as long as the buffer can be used for wash or elution.
In one specific embodiment, ratio of weight of PEG polymer relative to the volume of the wash buffer solution or elution buffer solution is from about 10 g:1 L to about 100 g:1 L, preferably from about 20 g:1 L to about 50 g:1 L, that is, percentage of weight of PEG polymer in the volume of the wash buffer solution or elution buffer solution is from about 1 w/v % to about 10 w/v %, preferably from about 2 w/v % to about 5 w/v %, such as 1 w/v %, 2 w/v %, 3 w/v %, 4 w/v %, 5 w/v %, 6 w/v %, 7 w/v %, 8 w/v %, 9 w/v %, 10 w/v %; the effective PEG concentration depends on the molecular weight of the particular PEG being used. For example, the required percentage of weight of PEG3350 in the volume of the wash buffer solution or elution buffer solution is from about 3.5 w/v % to about 5 w/v %. A lower percentage is sufficient for PEG polymer with higher molecular weight (e.g., PEG 6000) whereas a higher percentage is required for PEG with lower molecular weight (e.g., PEG 600).
In one specific embodiment, ratio of molar mass of the Hofmeister series salt relative to the volume of the wash buffer solution or elution buffer solution is about 250 mmol:1 L and more, preferably is from about 250 mmol:1 L to about 1 mol:1 L, more preferably is from about 500 mmol:1 L to 750 mmol:1 L, that is, percentage of molar mass of the Hofmeister series salt such as calcium chloride, or sodium chloride, or magnesium chloride, or potassium chloride in the of volume the wash buffer solution or elution buffer solution is about 250 mM and more, preferably is from about 250 mM to about 1 M, more preferably is from about 500 mM to about 750 mM, such as 200 mM, 300 mM, 400 mM, 500 mM, 600 mM, 700 mM, 800 mM, 900 mM and 1 M.
The inventors have generated a combination and a method of removing antibody aggregates by Protein A chromatography. Protein A's antibody aggregate removing capability is improved significantly by using the combination comprising PEG and Hofmeister series salt such as calcium chloride or sodium chloride. This new method, by allowing the majority of aggregates to be removed at the Protein A capture step, significantly alleviates the burden on subsequent polishing steps and hence improves the overall robustness of downstream process.
In order that the present invention may be more readily understood, certain terms are first defined. Additional definitions are set forth throughout the detailed description.
The term “Polyethylene glycol/PEG” as used in this disclosure, refers to an oligomer or polymer of ethylene oxide. PEG is also known as polyethylene oxide (PEO) or polyoxyethylene (POE), depending on its molecular weight. The structure of PEG is commonly expressed as H—(O—CH2—CH2)n-OH. PEGs are commercially available over a wide range of molecular weight from 200 g/mol to 10,000,000 g/mol. For example, molecular weight of the PEG used in this invention ranges from about 400 to about 6000.
The term “protein sample” employed in the present invention refers to a protein which contains an Fc region recognizable by Protein A. Such protein comprises antibodies and Fc-fusion proteins. The antibody could be a monoclonal antibody, or a polyclonal antibody. The antibody could be monospecific, bispecific or multi-specific. The antibody could be a mouse antibody, a chimeric antibody, a humanized antibody or a human antibody. The antibody could be a natural antibody or a recombinant antibody. An Fc-fusion protein is composed of an Fc region of an antibody and a genetically linked active protein.
The term “Fc region” employed in the present invention refers to the fragment crystallizable region of an antibody. Fc region is derived from the constant domains of the antibody's heavy chains. The “Fc region” can be recognized and bound by Protein A.
Exemplary antibodies that could be used in the present invention include Adalimumab, Bezlotoxumab, Avelumab, Dupilumab, Durvalumab, Ocrelizumab, Brodalumab, Reslizumab, Olaratumab, Daratumumab, Elotuzumab, Necitumumab, Infliximab, Obiltoxaximab, Atezolizumab, Secukinumab, Mepolizumab, Nivolumab, Alirocumab, Evolocumab, Dinutuximab, Bevacizumab, Pembrolizumab, Ramucirumab, Vedolizumab, Siltuximab, Alemtuzumab, Trastuzumab, Pertuzumab, Infliximab, Obinutuzumab, Brentuximab, Raxibacumab, Belimumab, Ipilimumab, Denosumab, Ofatumumab, Besilesomab, Tocilizumab, Canakinumab, Golimumab, Ustekinumab, Certolizumab, Catumaxomab, Eculizumab, Ranibizumab, Panitumumab, Natalizumab, Catumaxomab, Bevacizumab, Omalizumab, Cetuximab, Efalizumab, Ibritumomab, Fanolesomab, Tositumomab, Alemtuzumab, Trastuzumab, Gemtuzumab, Infliximab, Palivizumab, Necitumumab, Basiliximab, Rituximab, Capromab, Satumomab, Muromonab, etc.
Exemplary Fc-fusion proteins that could be used in the present invention include Etanercept, Alefacept, Abatacept, Rilonacept, Romiplostim, Belatacept, Aflibercept, etc.
The term “chromatography” refers to any kind of technique which separates an analyte of interest (e.g., an Fc region containing protein such as an immunoglobulin) from other molecules present in a mixture. Usually, the analyte of interest is separated from other molecules as a result of differences in rates at which the individual molecules of the mixture migrate through a stationary medium under the influence of a moving phase, or in bind and elute processes.
The term “Protein A” employed in the present invention encompasses Protein A recovered from a native source, Protein A produced synthetically (e.g., by peptide synthesis or by recombinant techniques), and functional variants thereof. Protein A exhibits high affinity for an Fc region. Protein A can be purchased commercially from Repligen, Pharmacia and Fermatech. Protein A is generally immobilized on a solid phase support material. The term “Protein A” also refers to an affinity chromatography resin or column containing chromatographic solid support matrix to which Protein A is covalently attached.
The term “Hofmeister series salt” refers to salt composed of Hofmeister series of cations (e.g., NH4+, K+, Na+, Li+, Mg2+, Ca2+, guanidinium+) and inions (e.g., SO42−, HPO42−, acetate−, citrate−, Cl−, NO3−, Br−, I−, ClO4−, SCN−). Various Hofmeister series salts which may be used in the buffers described herein include, but are not limited to, acetate (e.g. sodium acetate), citrate (e.g. sodium citrate), chloride (e.g. sodium chloride), sulphate (e.g. sodium sulphate), or a potassium salt.
A “buffer” is a solution that resists changes in pH by the action of its acid-base conjugate components. Various buffers which can be employed depending, for example, on the desired pH of the buffer are described in “Buffers. A Guide for the Preparation and Use of Buffers in Biological Systems, Gueffroy, D., ed. Calbiochem Corporation, 1975”. In some steps of the methods of the claimed invention, a buffer has a pH in the range from 2.0 to 4.0, or from 2.8 to 3.8. In other steps of the claimed invention, a buffer has a pH in the range of 5.0 to 9.0. In other steps of the claimed invention, a buffer has a pH in the range of 4.0 to 6.5. In yet other steps of the methods of the claimed invention, a buffer has a pH lower than 4.0. Non-limiting examples of buffers that will control the pH in this range include IVIES, MOPS, MOPSO, Tris, HEPES, phosphate, acetate, citrate, succinate, and ammonium buffers, as well as combinations of these.
The term “wash buffer” refers to the buffer used to wash the chromatography column post sample loading and prior to elution.
The term “elution buffer” refers the buffer used to elute the target protein from the solid phase. The conductivity and/or pH of the elution buffer is/are usually such that the target protein is eluted from the chromatography resin.
Calcium chloride dihydrate, sodium acetate trihydrate, sodium chloride, sodium hydroxide and Tris (hydroxymethyl) aminomethane were purchased from Merck (Darmstadt, Germany). Arginine hydrochloride and acetic acid were purchased from J. T. Baker (Phillipsburg, N.J., USA). Polyethylene Glycol (PEG) 3350 and urea were purchased from Sigma-Aldrich (St. Louis, Mo., USA). Mab Select SuRe LX and Tricorn 5/200 column (inner diameter: 5 mm, length: 20 mm) were purchased from GE Healthcare (Uppsala, Sweden). The three antibodies used are intact Immunoglobulin G (IgG). The one used to confirm calcium chloride's effect is IgG4 and the other two are IgG1. All three antibodies used were expressed in CHO—K1 cells grown in HyClone ActiPro culture medium supplemented with Cell Boost 7a and 7b (the medium and feeding supplements are from GE Healthcare) as previously described (X Zhang, T Chen, Y. Li, A parallel demonstration of different resins' antibody aggregate removing capability by a case study, Protein Expr. Purif., 2019, 153, 59-69). For the case used for method development and demonstration, the clarified harvest contains greater than 20% aggregates.
An AKTA pure 150 system installed with Unicorn software version 6.3 (GE Healthcare, Uppsala, Sweden) was used for all chromatographic runs. pH and conductivity was measured using SevenExcellence S470 pH/Conductivity meter (Mettler-Toledo, Columbus, Ohio, USA). Protein concentration was measured using a NanoDrop One spectrophotometer (Thermo Fisher Scientific, Waltham, Mass., USA). An Agilent 1260 liquid chromatography instrument (Agilent Technologies, Santa Clara, Calif., USA) was used for SEC-HPLC analysis.
Protein a Chromatography
Mab Select SuRe LX (Protein A affinity medium) was packed in a 0.5 cm diameter column with 15 cm bed height. The column volume (CV) is approximately 3 ml. Recipes of critical buffers for each run are listed in Table 1 (A1: equilibration/wash 1 buffer, A2: wash 2 buffer, B: elution buffer). Protein A load is the culture harvest clarified by depth filtration. For all runs, the column was loaded at 25 mg/ml and run in bind-elute mode. The antibody (IgG) with high percentage of aggregate was eluted with linear (0-100% B over 20 CV) or stepwise gradient. For all runs, after sample loading the column was washed with buffer A1 and A2 each for 3 CV prior to elution. For all chromatographic runs, the system was run at a flow rate of 180 cm/hr (residence time: 5 min). All chromatograms were recorded by monitoring UV absorbance at 280 nm. Elution from selected runs was collected in fractions and analyzed by SEC-HPLC for monomer purity.
aThe numbers are solely used to distinguish different runs and the actual experiments were not necessarily performed in this order.
bThis series of experiments was also conducted with another antibody to confirm the observed trend.
cStepwise elution.
All samples (Protein A elution fractions and elution pool) were analysed using a Tosoh TSKgel G3000SWx1 stainless steel column (7.8×300 mm). 100 μg of sample was injected per run. The mobile phase consisted of 50 mM sodium phosphate, 300 mM sodium chloride at pH 6.8. Each sample was eluted isocratically for 20 min at a flow rate of 1.0 ml/min. Protein elution was monitored by UV absorbance at 280 nm. The peaks corresponding to the monomer and aggregates were integrated to calculate the percentage of each species.
In this study, we first investigated PEG's effect on Protein A elution profile by adding different amounts of PEG (i.e., 1.5%, 3%, 5% and 10%) to wash and elution buffers. With increasing PEG concentration, retention of the aggregation-prone antibody was slightly increased and the elution peak became sharper (
The inventors designed experiments to explore the effect of calcium chloride on monomer-aggregate resolution as a mobile phase additive. For the case under study, different amounts of calcium chloride (i.e., 250 mM, 500 mM, 750 mM and 1 M) were added to Protein A wash and elution buffers.
Adding calcium chloride to the mobile phase showed appreciable but not significant impact on both resolution and retention time (
It is interesting that calcium chloride improves resolution only at medium concentrations (i.e., 500 mM and 750 mM). It shows no effect on resolution at lower or higher concentrations (i.e., 250 mM and 1 M, respectively). It seems that at low concentration calcium chloride exhibits weak kosmotropic effect and therefore slightly increases retention time. At increased concentrations (i.e., 500 mM and 750 mM), calcium chloride exhibits chaotropic effect and reduces retention time. At these two concentrations, calcium chloride improves monomer-aggregate resolution. At 1 M calcium chloride concentration, the resolution observed at 500 mM and 750 mM diminished and the protein retention time was not further reduced. This suggest that calcium chloride at this high concentration may cause some changes to the target antibody and/or the Protein A ligand, which prevent the interaction between antibody and Protein A from being further weakened.
Although calcium chloride at 500 mM and 750 mM improves monomer-aggregate resolution, separation of the two species is far from complete under these conditions. Thus, the inventors next tried PEG/calcium chloride combination. Since PEG itself had little effect on the elution profile at different concentrations, in this study the inventors arbitrarily chose 5% PEG to combine with different amounts of calcium chloride. At low calcium chloride concentrations (i.e., 150 mM and 250 mM), this combination showed no obvious effect and the elution profile is almost identical to that of the run with 5% PEG only (
The data suggest that PEG starts to show an enhancing effect when calcium chloride reaches a concentration that improves resolution. Whereas calcium chloride at this concentration can weaken antibody binding to Protein A ligand, its role cannot be replaced by other interaction-weakening agents such as urea or arginine (
After observing the synergistic effect of PEG and calcium chloride, the inventors also studied the effect of PEG/sodium chloride combination, and received a similar result (
The inventors had learned that PEG alone had no major effect on resolution (
We further confirmed the effect of PEG/sodium chloride combination on resolution enhancement with another case. In this case, the load contains approximately 10% of aggregates. As shown in
aLinear gradient elution.
bStepwise elution.
In general Protein A chromatography does not provide good aggregate clearance under typical conditions. The present invention showed that PEG/calcium chloride and PEG/sodium chloride combination, when added to the mobile phase, significantly improves Protein A chromatography's aggregate removal capability. For the case used for method development and demonstration, the optimized procedure allows aggregates in Protein A elution pool to be reduced from 20% (control run) to approximately 3-4%.
In this case, the two different species need to be separated are monomer and aggregates, and the latter are known to bind tighter. In this study, the extent to which calcium chloride improves resolution between different species is less than that observed in a previous study. Nevertheless, the inventors learned that the calcium chloride mediated resolution-improving effect can be significantly enhanced by the presence of 5% PEG (
The two salts (i.e., calcium chloride and sodium chloride) achieve resolution-enhancing effect through similar mechanisms. In either case, the salt affects monomer and aggregates to a different extent, resulting in improved resolution. PEG, although showed no effect on resolution by itself at up to 10%, can significantly improve chaotropic/kosmotropic salt mediated resolution-enhancing effect, allowing near-complete separation of monomer from aggregates.
In conclusion, the inventors developed a novel method that significantly improves Protein A chromatography's aggregate removing capability. This new method, by allowing the majority of aggregates to be removed at the Protein A capture step, significantly alleviates the burden on subsequent polishing steps and hence improves the overall robustness of downstream process.
| Number | Date | Country | Kind |
|---|---|---|---|
| PCT/CN2018/122748 | Dec 2018 | CN | national |
The present application is a U.S. National Stage entry of PCT Application No. PCT/CN2019/127022, filed on Dec. 20, 2019, which claims priority to Application No. PCT/CN2018/122748, filed on Dec. 21, 2018, all of which are incorporated in reference herein.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2019/127022 | 12/20/2019 | WO | 00 |
