Due to their broad application range, monoclonal antibodies mab are used worldwide in a variety. Monoclonal antibodies mabs are the fastestgrowing biological therapeutics with important applications ranging from cancers, autoimmunity diseases and metabolic disorders to. Antibody aggregation can occur at various stages including the up and downstream processes in manufacturing, formulation, and longterm storage 5, 6. Secretory leakage of igg1 aggregates from recombinant. Strategies for the assessment of protein aggregates in pharmaceutical biotech product development. Removing aggregates in monoclonal antibody purification. The control and avoidance of aggregation in the manufacturing process are needed because aggregates affect drug performance and safety97,98. The study focuses on very large scale processes in the region of five 5 tonnes per annum of bulk antibody. Monoclonal antibody mab products have emerged as an extremely important and valuable class of therapeutic products for the treatment of cancer and other diseases such. Monoclonal antibodies can have monovalent affinity. Clarification technologies for monoclonal antibody.
Are you considering how best to manage and remove aggregates across your template. Removing the aggregates of an acidic monoclonal antibody with. Humanized monoclonal antibody produced by mammalian cell culture may contain significant amounts of antibody dimers and smaller amounts of higher order aggregates. The stability of 11 purified monoclonal human igg1 and igg4 antibodies, including an igg1based bispecific crossmab, was compared in downscale mixing stress models. Aggregation mechanism of an igg2 and two igg1 monoclonal antibodies at low ph. Mab aggregates may form during protein expression, downstream processing, and storage, and may be caused. There is a clear need to reduce costs and manufacturing risk in order to. The agilent advancebio sec column, used with the agilent 1260 biolc system, proved capable of. Removing the aggregates of an acidic monoclonal antibody. Thus, values obtained from sec may not actually represent the true aggregate levels in the protein drug container, so there is a continued effort to. Quality attributes of monoclonal antibodies and effect of cell culture. The principles described in this document apply to monoclonal antibodies used as reagents, as well as monoclonal antibody related products, such as fragments, conjugates, and fusion proteins.
Individual antibodies vary widely in their propensity to form aggregates 5. For the final biopharmaceutical product approval and subsequent manufacturing processes, a comprehensive characterization of mab. In this talk, herb lutz, global consultant, will discuss the formation of protein. Nonsizebased membrane chromatographic separation and. Pdf aggregates in monoclonal antibody manufacturing processes. Antibodydrug conjugates adcs are monoclonal antibodies coupled to cytotoxic agents with stable linkers. This study demonstrated the suitability of cationexchange laterallyfed membrane chromatography lfmc for rapid and highresolution separation of monoclonal antibody mab aggregates. Imbaratto, cell culture manufacturing, covance biotechnology services, inc.
Various economic analyses estimate that process development and clinical manufacturing costs can. The structure of the monoclonal antibody should be justified with respect to its mechanism of action. In the creation of this study, we used the industry standard modelling software, biosolve process, to address these questions. Analysis of monoclonal antibodies and their fragments by size. Today, antibody concentrations of 35 gl are achieved routinely and some companies have attained up to 10 gl in fedbatch processes 5,6,8.
For some years, new methodologies have advanced with new production processes, allowing scaleup production and market introduction. Simultaneous removal of leached proteina and aggregates from monoclonal antibody using hydrophobic interaction membrane chromatography. Preparative separation of monoclonal antibody aggregates. The agilent advancebio sec column, used with the agilent 1260 biolc system, proved capable of delivering high resolution analysis of mab aggregates with short run times. Capto adhere is a multimodal ion exchanger and is designed to be used as a second or third step in monoclonal antibody mab puri.
Protein aggregation remains a major area of focus in the production of monoclonal antibodies. Current trends in monoclonal antibody development and manufacturing will provide readers with an understanding of what is currently being done in the industry to develop, manufacture, and release. After completing this chapter, students will be able to. Here, we explore the potential of predicting and reducing the aggregation propensity of monoclonal antibodies. Removal of leached protein a, antibody dimers and aggregates da, host cell proteins hcp, viruses and. Aggregates in mab therapeutics pose a risk to patients. In the broad sense, it refers to the entire process of creating a usable specific antibody, including steps of immunogen preparation, immunization, hybridoma creation, collection, screening, isotyping, purification, and labeling for direct use in a particular method. Largescale manufacturing of pharmaceutical antibodies. The small molecules attached to the mab are often relatively hydrophobic. Size exclusion chromatography analysis of antibody drug. Analysis of monoclonal antibodies and their fragments by. Monoclonal antibody mab products have emerged as an extremely important and valuable class of therapeutic products for the treatment of cancer and other diseases such as rheumatoid arthritis, autoinflammatory disease, allergic asthma, and multiple sclerosis 1, 2.
Clarification technologies well established in other industries, such as flocculation and precipitation are increasingly considered as a viable solution to address this bottleneck in antibody processes. This study demonstrated the suitability of cationexchange laterallyfed membrane chromatography lfmc for rapid and highresolution separation of monoclonal antibody. Integrated flowthrough purification for therapeutic. Monoclonal antibody production in commercial scale cell culture bioprocessing requires a thorough understanding of the engineering process and components used. Evolution of the monoclonal antibody purification platform. Extremes of ph, ionic strength, temperature, concentration, shear forces, and other processing conditions can lead to increased aggregation. Aggregates in monoclonal antibody manufacturing processes.
The primary objective of this project was to use the experimental analysis together with the knowledge of chemical kinetics to build a kinetic model for protein. Aggregates in the final drug product are undesirable for two major reasons. These include production cell culture, harvest, antibody capture by protein a, virus. A read is counted each time someone views a publication summary such as the title, abstract, and list of authors, clicks on a figure, or views or downloads the fulltext. Largescale production has revolutionized the market for monoclonal antibodies by boosting its production and becoming a more practical method of production. Aggregates in monoclonal antibody manufacturing processes ifm. To investigate antibody stability and formation of modified species under upstream processing conditions. Watch our experts discuss which steps in the biomanufacturing process are involved in the formation or removal of aggregates and industryleading approaches.
Usp manufacturing processes which generate higher titers still take. Predicting aggregation propensity and monitoring aggregates. Adcs travel to target cells, where the antibody binds to its antigen expressed on. As a result, aggregation has to be monitored throughout process development and manufacturing of a mab product. See how oncolumn aggregation is dependent on the type of resin used and how one particular highresolution cation exchange resin allows for the lowest proportion of aggregate formation. Adcs travel to target cells, where the antibody binds to its antigen expressed on the cell surface. In custom antibody development, once target selection. Guideline on development, production, characterisation and.
Selecting the optimal resins for aggregate removal references he x et al. Aggregates have been characterized in mab purification and formulation. Preparative separation of monoclonal antibody aggregates by. Current trends in monoclonal antibody development and manufacturing will provide readers with an understanding of what is currently being done in the industry to develop, manufacture, and. Low ph elution can induce mab aggregation in protein a chromatography 7, 8. Schematic manufacturing process of monoclonal antibodies from cell. Stress factors in mab drug substance production processes. Monoclonal antibodies mab or moab are antibodies that are made by identical immune cells that are all clones of a unique parent cell. Monoclonal antibodies mabs are a successful class of therapeutic products used increasingly in the past 1520 years, but the manufacture of safe and effective mab drug products provides many challenges to downstream molecule purification. This 31 st article in the elements of biopharmaceutical production series focuses on evolution of the purification platform for manufacturing of mab therapeutics.
The current trend for cell culture processes is to increase. Protein a chromatography pac is commonly used as an efficient capture step in monoclonal antibody mab separation processes. For the final biopharmaceutical product approval and subsequent manufacturing processes, a comprehensive characterization of mab purity, aggregate forms, and charge variants is required by the regulatory agency. Factors influencing biotherapeutic monoclonal antibody. It has been more than a decade since the industry started establishing the process platform. Protein a chromatography increases monoclonal antibody aggregation rate during subsequent low ph virus inactivation hold. Aggregates in monoclonal antibody manufacturing processes marava. Taken together, these developments are driving the industry to explore alternative separation technologies as a future manufacturing strategy. A systematic framework to optimize and control monoclonal. Largescale manufacturing of pharmaceutical antibodies is a complex activity that requires considerable effort in both process and analytical development. The term antibody production has both general and specific meanings. Dec 16, 2016 aggregates in monoclonal antibody manufacturing processes biotechnol bioeng 2011, 108 7, 1494508.
Given the high cell densities achievable in both mammalian cell culture and microbial processes, primary recovery can be a. One of these molecules was further evaluated in realistic bioreactor stress models and in cell culture fermentations. Review aggregates in monoclonal antibody manufacturing processes mara va. Review aggregates in monoclonal antibody manufacturing processes marava. In the broad sense, it refers to the entire process of creating a usable specific antibody, including steps of. Antibody aggregation could be triggered by partial unfolding of its domains, leading to monomermonomer. To demonstrate the analysis of monoclonal antibody mab aggregates. Impact of media and antifoam selection on monoclonal. During the manufacturing process, the protein is exposed to multiple stress. Trends in upstream and downstream process development for. Monoclonal antibodies are expensive to develop and to manufacture. Lang2 1manufacturing science and technology, lonza biologics porrin. Overall purification performance of cht xt chromatography is summarized in table 1. Recovery and purification process development for monoclonal.
Jan 09, 2018 the formation of aggregates is a common but highly undesirable occurrence during monoclonal antibody purification. For example, we have seen issues in manufacturing, such as quality, process. Purification is intended to remove impurities, such as protein aggregates, but some such operations may actually generate protein aggregation 1. Trends in upstream and downstream process development. Introduction to antibody production and purification. This hydrophobicity can be problematic during manufacturing and storage in terms of aggregate formation as well as in analytical characterization of the latter1. The study of aggregation propensity of a monoclonal antibody mab and its. Current trends in monoclonal antibody development and. Secretory leakage of igg1 aggregates from recombinant chinese.
Understanding and managing aggregates are critical to your monoclonal antibody mab process. The stability of 11 purified monoclonal human igg1 and igg4. Aggregation mechanism of an igg2 and two igg1 monoclonal. In this talk, herb lutz, global consultant, will discuss the formation of protein aggregates and their. Table 1 summarizes potential sources of aggregate formation during. Mar 25, 2018 aggregates in monoclonal antibody manufacturing processes. Biomanufacturing of monoclonal antibodies mab involves a number of unit operations, including cell culture in a bioreactor followed by chromatography and filtration. Since the approval of the first therapeutic monoclonal antibody in 1986. Hic as a complementary, confirmatory tool to sec for the. Given the high cell densities achievable in both mammalian cell culture and microbial processes, primary recovery can be a significant challenge at lab, pilot and commercial manufacturing scales. But this method can provide misleading results because aggregates can dissociate or form during sec andor adsorb to the column resin. Monoclonal antibody production a report of the committee on methods of producing monoclonal antibodies institute for laboratory animal research national research council national academy press washington, dc 1999. Upon binding, the full adc can be internalized by a process called receptormediated endocytosis.
Aggregation kinetics for monoclonal antibody products. Aggregate content of cht xt eluate mab s significant reduction of the mab s aggregate was achieved unosphere supra eluate. Size exclusion chromatography analysis of antibody drug conjugates using the agilent 1260 infinity ii bioinert lc system. Aggregates in monoclonal antibody manufacturing processes biotechnol bioeng 2011, 108 7, 1494508. Aggregation of mabs continues to be a major problem in their developability. Managing aggregates in your monoclonal antibody mab process. Pdf monoclonal antibodies have proved to be a highly successful class of therapeutic products. Reliable, timely and costeffective monoclonal antibody production and purification programs. Howe ver, their applicability will be determined on a case bycase basis, based on their specific properties, and may be addressed in specific annexes. Review aggregates in monoclonal antibody manufacturing processes mar. Monoclonal antibodies have proved to be a highly successful class of therapeutic products. Processing impact on monoclonal antibody drug products. The formation of aggregates is a common but highly undesirable occurrence during monoclonal antibody purification. Monoclonal antibodies mabs are the fastestgrowing biological therapeutics with important applications ranging from cancers, autoimmunity diseases and metabolic disorders to emerging infectious diseases.
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