17 results on '"Li, Yifeng"'
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2. Obtaining acidic and basic charge variants using a twin-column continuous chromatography system.
- Author
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Yuan G, Zhang X, Zhang Y, Dong W, and Li Y
- Subjects
- Chromatography, Ion Exchange methods, Cations chemistry, Sodium Chloride chemistry, Antibodies, Monoclonal chemistry
- Abstract
For recombinantly produced monoclonal antibody (mAb), charge variants including acidic and basic species are common heterogeneities. For characterization purpose, sufficient amount of acidic and basic species with high purity is needed. In this work, we developed an approach that allows for continuous separating and collecting of acidic and basic charge variants. First, with batch-mode cation exchange (CEX) chromatography, the load density and linear salt gradient elution conditions under which good separation of both charge variants can be achieved were determined. Next, a stepwise elution protocol was developed based on the linear gradient elution. Finally, acidic and basic charge variants were persistently produced under stepwise elution using a customized twin-column continuous chromatography system. This approach allows acidic and basic charge variants with high purity (i.e., >90%) to be efficiently generated in sufficient amount, which greatly facilitates the necessary characterization of these mAb variants., (Copyright © 2022 Elsevier Inc. All rights reserved.)
- Published
- 2023
- Full Text
- View/download PDF
3. Screening of six cation exchange resins for high binding capacity, monomer purity and step yield: A case study.
- Author
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Lu W, Zhang Z, Zhang S, Zhang T, Wan Y, and Li Y
- Subjects
- Cations chemistry, Chromatography, Ion Exchange methods, Kinetics, Antibodies, Monoclonal chemistry, Cation Exchange Resins chemistry
- Abstract
Cation exchange (CEX) chromatography has been widely used as a polishing step in downstream processing of therapeutic monoclonal antibodies (mAbs). It has been well documented that the performance of a particular CEX resin heavily relies on buffer type, pH and conductivity. In this work, with a case study, we screened six commercial CEX resins under different pHs and conductivities. Initial screening was conducted on Tecan to find conditions under which high binding capacity was achieved. Next, performance of each resin was further evaluated using a small column under the identified optimum binding conditions. Variations in binding capacity and purity of eluate were observed among these resins. The adopted approach allows for identification of resins exhibiting good binding capacity, aggregate clearance and recovery., (Copyright © 2022 Elsevier Inc. All rights reserved.)
- Published
- 2022
- Full Text
- View/download PDF
4. Two major mechanisms contributing to copurification of CHO host cell proteins and strategies to minimize their negative impact.
- Author
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Li Y
- Subjects
- Animals, CHO Cells, Cricetinae, Cricetulus, Antibodies, Monoclonal chemistry
- Abstract
For CHO expressed monoclonal antibodies (mAbs), host cell proteins (HCPs) represent a major class of process-related impurities and their removal is a key focus of downstream process development. HCPs are highly heterogeneous in nature, differing in molecular weight, isoelectric point and hydrophobicity, and some of them can be difficult to remove. Although Protein A affinity chromatography alone can typically remove more than 90% of HCPs in the clarified culture harvest, in many cases reducing HCPs in the final drug product to an acceptable level (e.g., <100 ppm) can still be a challenging task. The relative difficulty of HCP clearance is case dependent and in certain cases a small population of HCPs can persist throughout the downstream process. This article reviews the two major mechanisms that contribute to copurification of CHO HCPs, namely leaching from chromatin heteroaggregates and nonspecific HCP-mAb association. In addition, strategies that can minimize the impact of these two factors are briefly discussed., (Copyright © 2022 Elsevier Inc. All rights reserved.)
- Published
- 2022
- Full Text
- View/download PDF
5. Immunoglobulin-binding protein-based affinity chromatography in bispecific antibody purification: Functions beyond product capture.
- Author
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Li Y
- Subjects
- Antibodies, Bispecific chemistry, Antibodies, Bispecific metabolism, Antibodies, Monoclonal chemistry, Antibodies, Monoclonal metabolism, Bacterial Proteins metabolism, Humans, Immunoglobulin G chemistry, Immunoglobulin G metabolism, Immunoglobulin kappa-Chains chemistry, Immunoglobulin kappa-Chains isolation & purification, Immunoglobulin kappa-Chains metabolism, Immunoglobulin lambda-Chains chemistry, Immunoglobulin lambda-Chains isolation & purification, Immunoglobulin lambda-Chains metabolism, Lymphokines chemistry, Lymphokines metabolism, Protein Binding, Staphylococcal Protein A metabolism, Antibodies, Bispecific isolation & purification, Antibodies, Monoclonal isolation & purification, Bacterial Proteins chemistry, Chromatography, Affinity methods, Immunoglobulin G isolation & purification, Staphylococcal Protein A chemistry
- Abstract
In general, purification of bispecific antibody (bsAb) is more challenging than that of monospecific antibody due to the increased complexity in byproduct profile. Like in the case of monospecific antibody purification, immunoglobulin-binding protein-based affinity chromatography is an indispensable tool for bsAb purification. For example, Protein A affinity chromatography has been widely used to capture Fc-containing bsAbs whereas other affinity media such as Protein L and KappaSelect, which bind kappa light chain, are used to capture bsAbs that do not contain a Protein A-binding site. In fact, affinity chromatography also possesses the capability of removing certain product-related impurities in bsAb purification when it is conducted with suitable medium and under appropriate conditions. Fully exploring the potential of affinity chromatography in bsAb purification to achieve both product capture and byproduct removal is highly desirable, as this can greatly alleviate the purification burden on subsequent polishing steps and hence improves the overall robustness of the downstream process. This article briefly reviews the byproduct clearance potential of several commonly used affinity media under relevant bsAb purification scenarios., (Copyright © 2021 Elsevier Inc. All rights reserved.)
- Published
- 2021
- Full Text
- View/download PDF
6. Comparing the relative robustness of byproduct removal by wash and by elution in column chromatography for the purification of a bispecific antibody.
- Author
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Wan Y, Zhang T, Wang Y, and Li Y
- Subjects
- Animals, Antibodies, Bispecific immunology, Antibodies, Monoclonal immunology, CHO Cells, Cricetulus, Humans, Recombinant Proteins immunology, Staphylococcal Protein A chemistry, Antibodies, Bispecific isolation & purification, Antibodies, Monoclonal isolation & purification, Chromatography, Affinity methods, Immunoglobulin G chemistry, Recombinant Proteins isolation & purification
- Abstract
For recombinant antibody purification, removal of product-related impurities usually relies on the two polishing steps post Protein A chromatography. A certain impurity may bind weaker or tighter to a particular type of column than the target antibody, and this forms the basis for separation. For impurities that bind weaker, they can be removed by pre-elution wash under appropriate conditions. For impurities that bind stronger, they can be separated by using a suitable condition that selectively elutes the product. In this study, with a bispecific antibody case, we compared the relative robustness of byproduct removal by wash and by elution using two different types of chromatography. The data suggest that elution-enabled byproduct clearance is more robust than wash-enabled clearance, and the former approach provides consistent impurity clearance over a relatively wide range of loading density., (Copyright © 2020 Elsevier Inc. All rights reserved.)
- Published
- 2021
- Full Text
- View/download PDF
7. Antibody Aggregate Removal Using a Mixed-Mode Chromatography Resin.
- Author
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Chen T, Guo G, Tan G, Wang Y, and Li Y
- Subjects
- Chromatography, Gel, Chromatography, Ion Exchange, Antibodies, Monoclonal chemistry, Antibodies, Monoclonal isolation & purification, Protein Aggregates, Staphylococcal Protein A chemistry
- Abstract
In monoclonal antibody (mAb) production, aggregates represent a major class of product-related impurities that needs to be removed by the downstream process. Protein A chromatography is generally less effective at removing antibody aggregates under typical conditions, and in most cases aggregate removal relies on a subsequent polishing chromatography. Here we describe a procedure for effective removal of antibody aggregates using the mixed-mode chromatography resin Capto MMC ImpRes. Clearance of aggregates was confirmed by analytical size-exclusion chromatography (SEC) and native gel electrophoresis.
- Published
- 2021
- Full Text
- View/download PDF
8. Removing light chain-missing byproducts and aggregates by Capto MMC ImpRes mixed-mode chromatography during the purification of two WuXiBody-based bispecific antibodies.
- Author
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Wan Y, Zhang T, Wang Y, Wang Y, and Li Y
- Subjects
- Chromatography, Liquid, Antibodies, Bispecific chemistry, Antibodies, Bispecific isolation & purification, Antibodies, Monoclonal chemistry, Antibodies, Monoclonal isolation & purification, Immunoglobulin Fab Fragments chemistry, Immunoglobulin Fab Fragments isolation & purification, Immunoglobulin G chemistry, Immunoglobulin G isolation & purification, Protein Multimerization
- Abstract
WuXiBody is a novel bispecific antibody (bsAb) platform developed by WuXi Biologics. Its key feature is the replacement of one parental antibody's CH1/CL constant region with the T cell receptor (TCR) constant domain, a design aimed at promoting cognate heavy chain (HC)-light chain (LC) pairing. BsAbs based on WuXiBody can adopt either asymmetric or symmetric format. LC-missing species, a byproduct frequently associated with bsAb containing sequence engineered Fab arm, is also identified during WuXiBody production. Nevertheless, WuXiBody's unique design greatly facilitates removal of this type of impurity, which can otherwise be difficult to clear. In this work, with two concrete cases (WuXiBodies with asymmetric and symmetric designs, respectively), we showed that Capto MMC ImpRes mixed-mode chromatography can effectively remove LC-missing byproducts as well as aggregates, demonstrating that this resin is a powerful tool for WuXiBody purification., (Copyright © 2020 Elsevier Inc. All rights reserved.)
- Published
- 2020
- Full Text
- View/download PDF
9. Removing a difficult-to-separate byproduct by Capto L affinity chromatography during the purification of a WuXiBody-based bispecific antibody.
- Author
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Wang Y, Chen X, Wang Y, and Li Y
- Subjects
- Antibodies, Bispecific chemistry, Antibodies, Bispecific isolation & purification, Antibodies, Monoclonal chemistry, Antibodies, Monoclonal isolation & purification, Bacterial Proteins chemistry, Chromatography, Affinity, DNA-Binding Proteins chemistry, Immunoglobulin G chemistry, Immunoglobulin G isolation & purification, Protein Multimerization
- Abstract
For IgG-like bispecific antibodies (bsAbs) whose construction involves sequence engineering to promote desired heavy chain-light chain pairing, ¾ antibody (antibody lacking one light chain) is a frequent byproduct during their recombinant production. As this byproduct shares high similarity in physicochemical properties with the target bsAb, its removal poses a challenge to downstream purification. Capto L is an affinity resin based on Protein L, which binds to the variable region of kappa light chain without interfering with the antigen binding site. In this work, we demonstrated that Capto L provides a convenient means for separating ¾ antibody from the bsAb product., (Copyright © 2020 Elsevier Inc. All rights reserved.)
- Published
- 2020
- Full Text
- View/download PDF
10. Protein L chromatography: A useful tool for monitoring/separating homodimers during the purification of IgG-like asymmetric bispecific antibodies.
- Author
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Chen X, Wang Y, Wang Y, and Li Y
- Subjects
- Chromatography, Liquid, Antibodies, Bispecific chemistry, Antibodies, Bispecific isolation & purification, Antibodies, Monoclonal chemistry, Antibodies, Monoclonal isolation & purification, Bacterial Proteins chemistry, DNA-Binding Proteins chemistry, Immunoglobulin G chemistry, Immunoglobulin G isolation & purification, Protein Multimerization
- Abstract
Asymmetric IgG-like bispecific antibodies (bsAbs) are normally derived from two parental mAbs with different origin. Despite the implementation of heterodimerization-promoting strategy in the design, homodimerization can still occur at a low level during the recombinant production of these molecules. In general, monitoring and removal of homodimers pose great challenges to analytical and purification teams, respectively, as these byproducts share high similarity in physicochemical properties with the target heterodimeric bsAb. Protein L is a bacterial surface protein that binds to the variable region of kappa light chain without interfering with the antigen binding site. In this work, we first showed that different antibodies bind Protein L-conjugated resin with varied strength, and then based on this observation we further demonstrated that Protein L chromatography can be a useful tool for monitoring/separating homodimers during the purification of asymmetric bsAbs., (Copyright © 2020 Elsevier Inc. All rights reserved.)
- Published
- 2020
- Full Text
- View/download PDF
11. Processing of high-salt-containing protein A eluate using mixed-mode chromatography in purifying an aggregation-prone antibody.
- Author
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Zhang Y, Cai L, Wang Y, and Li Y
- Subjects
- Antibodies, Monoclonal chemistry, Durapatite chemistry, Polyethylene Glycols chemistry, Protein Aggregates, Antibodies, Monoclonal isolation & purification, Chlorides chemistry, Chromatography, Ion Exchange methods, Staphylococcal Protein A chemistry
- Abstract
We previously developed a method that can significantly improve Protein A chromatography's capability of removing antibody aggregates. That particular method requires polyethylene glycol (PEG) and 400 mM or more of calcium chloride/sodium chloride to be added to wash and elution buffers. Consequently, Protein A chromatography performed using this method has relatively high concentration of salt in its eluate. The high salt content prevents the neutralized eluate from binding to ion exchange columns without conductivity adjustment. In the current study we demonstrated that mixed-mode chromatography can be used as a subsequent step to Protein A chromatography with high-salt-containing eluate. As mixed-mode ligand mediates salt-tolerant adsorption, it allows the neutralized Protein A eluate to be directly loaded without the need of conductivity adjustment, and thus enables a smooth and convenient connection between capture and polishing steps. In this work we also showed that the mixed-mode chromatography, performed in bind-elute mode, removed most of PEG in the Protein A eluate., (Copyright © 2019 Elsevier Inc. All rights reserved.)
- Published
- 2019
- Full Text
- View/download PDF
12. Sodium caprylate induced precipitation post Protein A chromatography as an effective means for host cell protein clearance.
- Author
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Wan Y, Zhang T, Chen T, Wang Y, and Li Y
- Subjects
- Animals, Antibodies, Monoclonal analysis, CHO Cells, Chemical Precipitation, Chromatography, Gel methods, Chromatography, High Pressure Liquid methods, Cricetulus, Antibodies, Monoclonal isolation & purification, Caprylates chemistry, Staphylococcal Protein A chemistry
- Abstract
In downstream processing of monoclonal antibody (mAb), post Protein A neutralization and subsequent intermediate depth filtration are critical steps for host cell protein (HCP) clearance. Previous studies have shown that adding caprylic acid (CA) during neutralization can further improve HCP removal by promoting their precipitation. In this study, we replaced CA with its sodium salt - sodium caprylate (SC). For the five mAbs studied, SC has been shown to be equally effective as CA at precipitating HCPs. As the salt form has a higher solubility, SC stock solution with relatively high concentration can be easily prepared, which facilitates its adding to the Protein A elution pool. Thus, this study not only confirms the effectiveness of CA/SC-induced HCP precipitation but also provides a more convenient way to integrate this method into the downstream process., (Copyright © 2019 Elsevier Inc. All rights reserved.)
- Published
- 2019
- Full Text
- View/download PDF
13. Major cause of antibody artifact bands on non-reducing SDS-PAGE and methods for minimizing artifacts.
- Author
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Zhang Y, Wang Y, and Li Y
- Subjects
- Alkylating Agents chemistry, Hot Temperature, Iodoacetamide chemistry, Oxidation-Reduction, Protein Denaturation, Urea chemistry, Antibodies, Monoclonal analysis, Electrophoresis, Polyacrylamide Gel methods
- Abstract
When monoclonal antibodies (mAbs) are analysed by non-reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), method-induced artifacts are a frequent phenomenon. Previous studies suggested that incomplete denaturation and disulfide-bond scrambling are two main causes of artifact bands. Thus, in practice samples are normally heated and treated with alkylating agent iodoacetamide (IAM) before loading to promote denaturation and block free sulfhydryl groups, respectively. In this work, we further studied the major cause of artifact bands on non-reducing SDS-PAGE and ways of eliminating artifacts with two purified mAbs. In both cases, it was found that artifact bands on non-gradient Tris-glycine gels are mainly caused by incomplete denaturation under typical gel conditions. In general, heating minimizes artifact bands due to incomplete denaturation but it also generates some extra bands. Combining heating with IAM treatment achieved slightly better results than heating alone. As an alternative to heating, treating the samples with 8 M urea also allows close to complete denaturation of samples and thus minimizes artifact bands. In addition, we learned that untreated samples (samples that are not heated or treated with urea) may look different on Bis-Tris gel depending on gel composition (non-gradient vs. gradient) and the buffer used (MES vs. MOPS). In certain case, the apparent lack of artifact bands on gradient Bis-Tris gel may be in fact due to insufficient resolution. In conclusion, this study further confirmed that full-denaturation of sample is critical for minimizing/avoiding artifact bands on non-reducing SDS-PAGE., (Copyright © 2019 Elsevier Inc. All rights reserved.)
- Published
- 2019
- Full Text
- View/download PDF
14. The adequate amount of sodium chloride in Protein A wash buffer for effective host cell protein clearance.
- Author
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Han J, Yang J, Wang Y, and Li Y
- Subjects
- Animals, Buffers, CHO Cells, Cricetinae, Cricetulus, Antibodies, Monoclonal chemistry, Antibodies, Monoclonal isolation & purification, Chromatography, Affinity, Sodium Chloride chemistry, Staphylococcal Protein A chemistry
- Abstract
Post-load column wash in Protein A chromatography can effectively improve host cell protein (HCP) clearance. A commonly used wash additive for this purpose is sodium chloride. However, the adequate amount of sodium chloride required for effective HCP clearance is less consistent in literature. In this study we investigated the impact of different amounts of sodium chloride on HCP clearance with five monoclonal antibodies (mAbs). For each mAb, elution pool HCP levels from runs under different wash conditions are compared. For all five mAbs, the data suggested that 250 mM would be an adequate amount for the salt to largely achieve its HCP reducing effect. The same conclusion is also reached for calcium chloride, a less commonly used but equally effective Protein A wash additive for HCP clearance., (Copyright © 2019 Elsevier Inc. All rights reserved.)
- Published
- 2019
- Full Text
- View/download PDF
15. The application of caprylic acid in downstream processing of monoclonal antibodies.
- Author
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Li Y
- Subjects
- Albumins chemistry, Animals, Anti-Infective Agents pharmacology, Caprylates pharmacology, Chromatography, Affinity methods, Humans, Protein Stability drug effects, Virus Inactivation drug effects, Viruses drug effects, Viruses growth & development, Anti-Infective Agents chemistry, Antibodies, Monoclonal chemistry, Caprylates chemistry, Chemical Precipitation drug effects, Immunoglobulin G chemistry
- Abstract
Caprylic acid (CA), a naturally occurring eight-carbon fatty acid, has long been used as albumin stabilizer, non-IgG fraction precipitant and bactericidal agent in pharmaceutical industry. The mechanisms through which CA achieves its effects have been correlated with the molecule's protein/lipid binding capacity conferred by its octyl moiety. This article, following an initial review of CA's historical applications, introduces CA's relatively new application in downstream processing of monoclonal antibodies (mAbs). By taking advantage of CA mediated impurity precipitation and virus inactivation, it might be possible to develop a two-column purification process in replacement of the standard three-column process without compromising product quality., (Copyright © 2018 Elsevier Inc. All rights reserved.)
- Published
- 2019
- Full Text
- View/download PDF
16. A parallel demonstration of different resins' antibody aggregate removing capability by a case study.
- Author
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Zhang X, Chen T, and Li Y
- Subjects
- Animals, Antibodies, Monoclonal biosynthesis, Antibodies, Monoclonal chemistry, CHO Cells, Chromatography, Ion Exchange instrumentation, Chromatography, Liquid instrumentation, Cricetulus, Humans, Hydrophobic and Hydrophilic Interactions, Polyethylene Glycols chemistry, Antibodies, Monoclonal isolation & purification, Durapatite chemistry, Ion Exchange Resins chemistry, Protein Aggregates, Resins, Synthetic chemistry
- Abstract
Aggregation is a major concern for therapeutic monoclonal antibody (mAb), as aggregates reduce drug efficacy and safety. In addition to aggregation control, aggregate removal by downstream processing is crucial. Hydrophobic and mixed-mode resins are widely used for aggregate removal in different cases, but they are seldom compared side by side. In this study, the aggregate removing capability of eight resins belonging to different chromatographic types was demonstrated by a case study. This work, by providing multiple options for aggregate removal, allows more flexibility to be gained in downstream processing., (Copyright © 2018 Elsevier Inc. All rights reserved.)
- Published
- 2019
- Full Text
- View/download PDF
17. Effective strategies for host cell protein clearance in downstream processing of monoclonal antibodies and Fc-fusion proteins.
- Author
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Li Y
- Subjects
- Animals, Cell Line, Humans, Antibodies, Monoclonal biosynthesis, Antibodies, Monoclonal genetics, Antibodies, Monoclonal isolation & purification, Gene Expression, Immunoglobulin Fc Fragments biosynthesis, Immunoglobulin Fc Fragments genetics, Immunoglobulin Fc Fragments isolation & purification, Recombinant Fusion Proteins biosynthesis, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins isolation & purification
- Abstract
Recombinant therapeutic proteins are typically produced through cell culture process. Host cell proteins (HCPs) are endogenous proteins derived from the host cells used for such bioproduction. HCPs form a major class of process-related impurities and even at low levels they can potentially compromise the safety and efficacy of biopharmaceuticals. Therefore, they need to be adequately removed via the downstream process. HCPs are complex mixtures with diverse physiochemical properties, and certain subpopulations can bind to the intended product. Hence reducing them to the generally accepted level can be challenging. This article reviews effective HCP removing strategies at different stages of downstream process for monoclonal antibodies and Fc-fusion proteins. When used in combination, these strategies can greatly enhance the chance of meeting the drug substance specifications for residual HCP., (Copyright © 2017 Elsevier Inc. All rights reserved.)
- Published
- 2017
- Full Text
- View/download PDF
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