Since the approval of the first therapeutic monoclonal antibody in 1986. 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. Monoclonal antibodies are expensive to develop and to manufacture. Aggregation mechanism of an igg2 and two igg1 monoclonal. 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. Introduction to antibody production and purification. Managing aggregates in your monoclonal antibody mab process. In custom antibody development, once target selection. Are you considering how best to manage and remove aggregates across your template. 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.
To investigate antibody stability and formation of modified species under upstream processing conditions. Monoclonal antibodies mab or moab are antibodies that are made by identical immune cells that are all clones of a unique parent cell. The term antibody production has both general and specific meanings. Aggregation and chemical modification of monoclonal. Adcs travel to target cells, where the antibody binds to its antigen expressed on. It has been more than a decade since the industry started establishing the process platform.
Stress factors in mab drug substance production processes. Taken together, these developments are driving the industry to explore alternative separation technologies as a future manufacturing strategy. Review aggregates in monoclonal antibody manufacturing processes mar. These include production cell culture, harvest, antibody capture by protein a, virus. Preparative separation of monoclonal antibody aggregates. Monoclonal antibodies can have monovalent affinity. Dec 16, 2016 understanding and managing aggregates are critical to your monoclonal antibody mab process. To demonstrate the analysis of monoclonal antibody mab aggregates. Protein aggregation remains a major area of focus in the production of monoclonal antibodies. 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.
Production processes for monoclonal antibodies intechopen. For example, we have seen issues in manufacturing, such as quality, process. Recovery and purification process development for monoclonal. Preparative separation of monoclonal antibody aggregates by. In the creation of this study, we used the industry standard modelling software, biosolve process, to address these questions. Largescale manufacturing of pharmaceutical antibodies is a complex activity that requires considerable effort in both process and analytical development. During the manufacturing process, the protein is exposed to multiple stress. Removing aggregates in monoclonal antibody purification. Removing the aggregates of an acidic monoclonal antibody with. Selecting the optimal resins for aggregate removal references he x et al. Guideline on development, production, characterisation and.
Due to their broad application range, monoclonal antibodies mab are used worldwide in a variety. This study demonstrated the suitability of cationexchange laterallyfed membrane chromatography lfmc for rapid and highresolution separation of monoclonal antibody mab aggregates. Jan 16, 20 to investigate antibody stability and formation of modified species under upstream processing conditions. Integrated flowthrough purification for therapeutic. Secretory leakage of igg1 aggregates from recombinant. Low ph elution can induce mab aggregation in protein a chromatography 7, 8. Monoclonal antibody production in commercial scale cell culture bioprocessing requires a thorough understanding of the engineering process and components used. Prediction and reduction of the aggregation of monoclonal antibodies. For the final biopharmaceutical product approval and subsequent manufacturing processes, a comprehensive characterization of mab.
The study of aggregation propensity of a monoclonal antibody mab and its. Aggregation kinetics for monoclonal antibody products. Analysis of monoclonal antibodies and their fragments by. Humanized monoclonal antibody produced by mammalian cell culture may contain significant amounts of antibody dimers and smaller amounts of higher order aggregates.
Mab aggregates may form during protein expression, downstream processing, and storage, and may be caused. Processing impact on monoclonal antibody drug products. Aggregation of mabs continues to be a major problem in their developability. In this talk, herb lutz, global consultant, will discuss the formation of protein. Review aggregates in monoclonal antibody manufacturing processes mara va. 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. The stability of 11 purified monoclonal human igg1 and igg4 antibodies, including an igg1based bispecific crossmab, was compared in downscale mixing stress models. This 31 st article in the elements of biopharmaceutical production series focuses. Simultaneous removal of leached proteina and aggregates from monoclonal antibody using hydrophobic interaction membrane chromatography. The structure of the monoclonal antibody should be justified with respect to its mechanism of action. There is a clear need to reduce costs and manufacturing risk in order to. As a result, aggregation has to be monitored throughout process development and manufacturing of a mab product.
Pdf monoclonal antibodies have proved to be a highly successful class of therapeutic products. Current trends in monoclonal antibody development and. Monoclonal antibodies mabs are the fastestgrowing biological therapeutics with important applications ranging from cancers, autoimmunity diseases and metabolic disorders to. After completing this chapter, students will be able to. Various economic analyses estimate that process development and clinical manufacturing costs can. Largescale production has revolutionized the market for monoclonal antibodies by boosting its production and becoming a more practical method of production. Nonsizebased membrane chromatographic separation and. The control and avoidance of aggregation in the manufacturing process are needed because aggregates affect drug performance and safety97,98. Strategies for the assessment of protein aggregates in pharmaceutical biotech product development. Dec 16, 2016 aggregates in monoclonal antibody manufacturing processes biotechnol bioeng 2011, 108 7, 1494508. The stability of 11 purified monoclonal human igg1 and igg4.
Extremes of ph, ionic strength, temperature, concentration, shear forces, and other processing conditions can lead to increased aggregation. Aggregates in monoclonal antibody manufacturing processes. But this method can provide misleading results because aggregates can dissociate or form during sec andor adsorb to the column resin. Purification is intended to remove impurities, such as protein aggregates, but some such operations may actually generate protein aggregation 1. Individual antibodies vary widely in their propensity to form aggregates 5. Trends in upstream and downstream process development for. 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. Pdf aggregates in monoclonal antibody manufacturing. This study demonstrated the suitability of cationexchange laterallyfed membrane chromatography lfmc for rapid and highresolution separation of monoclonal antibody.
Adcs travel to target cells, where the antibody binds to its antigen expressed on the cell surface. Aggregate content of cht xt eluate mab s significant reduction of the mab s aggregate was achieved unosphere supra eluate. 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. For some years, new methodologies have advanced with new production processes, allowing scaleup production and market introduction. 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. Evolution of the monoclonal antibody purification platform. The small molecules attached to the mab are often relatively hydrophobic. Antibody aggregation could be triggered by partial unfolding of its domains, leading to monomermonomer. Lang2 1manufacturing science and technology, lonza biologics porrin. The agilent advancebio sec column, used with the agilent 1260 biolc system, proved capable of. 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. Review aggregates in monoclonal antibody manufacturing processes marava.
Schematic manufacturing process of monoclonal antibodies from cell. Reliable, timely and costeffective monoclonal antibody production and purification programs. Quality attributes of monoclonal antibodies and effect of cell culture. Size exclusion chromatography analysis of antibody drug conjugates using the agilent 1260 infinity ii bioinert lc system.
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. Table 1 summarizes potential sources of aggregate formation during. 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. The current trend for cell culture processes is to increase. Capto adhere is a multimodal ion exchanger and is designed to be used as a second or third step in monoclonal antibody mab puri. 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. Overall purification performance of cht xt chromatography is summarized in table 1. Antibody aggregation can occur at various stages including the up and downstream processes in manufacturing, formulation, and longterm storage 5, 6.
This hydrophobicity can be problematic during manufacturing and storage in terms of aggregate formation as well as in analytical characterization of the latter1. Antibodydrug conjugates adcs are monoclonal antibodies coupled to cytotoxic agents with stable linkers. In the broad sense, it refers to the entire process of creating a usable specific antibody, including steps of. Usp manufacturing processes which generate higher titers still take. Given the high cell densities achievable in both mammalian cell culture and microbial processes, primary recovery can be a. Today, antibody concentrations of 35 gl are achieved routinely and some companies have attained up to 10 gl in fedbatch processes 5,6,8. Factors influencing biotherapeutic monoclonal antibody.
The formation of aggregates is a common but highly undesirable occurrence during monoclonal antibody purification. Aggregates in mab therapeutics pose a risk to patients. Monoclonal antibodies have proved to be a highly successful class of therapeutic products. Aggregates in the final drug product are undesirable for two major reasons. Imbaratto, cell culture manufacturing, covance biotechnology services, inc. Size exclusion chromatography analysis of antibody drug. A systematic framework to optimize and control monoclonal. 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. Protein a chromatography pac is commonly used as an efficient capture step in monoclonal antibody mab separation processes. Aggregates have been characterized in mab purification and formulation. Aggregates in monoclonal antibody manufacturing processes ifm.
Removing the aggregates of an acidic monoclonal antibody. Protein a chromatography increases monoclonal antibody aggregation rate during subsequent low ph virus inactivation hold. This 31 st article in the elements of biopharmaceutical production series focuses on evolution of the purification platform for manufacturing of mab therapeutics. The study focuses on very large scale processes in the region of five 5 tonnes per annum of bulk antibody. Predicting aggregation propensity and monitoring aggregates. Aggregation mechanism of an igg2 and two igg1 monoclonal antibodies at low ph. Trends in upstream and downstream process development. Analysis of monoclonal antibodies and their fragments by size. Jan 09, 2018 the formation of aggregates is a common but highly undesirable occurrence during monoclonal antibody purification. Largescale manufacturing of pharmaceutical antibodies.
Here, we explore the potential of predicting and reducing the aggregation propensity of monoclonal antibodies. Howe ver, their applicability will be determined on a case bycase basis, based on their specific properties, and may be addressed in specific annexes. Removal of leached protein a, antibody dimers and aggregates da, host cell proteins hcp, viruses and. Understanding and managing aggregates are critical to your monoclonal antibody mab process. 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.
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. Clarification technologies for monoclonal antibody. Pdf aggregates in monoclonal antibody manufacturing processes. One of these molecules was further evaluated in realistic bioreactor stress models and in cell culture fermentations. Watch our experts discuss which steps in the biomanufacturing process are involved in the formation or removal of aggregates and industryleading approaches. Monoclonal antibodies mabs are the fastestgrowing biological therapeutics with important applications ranging from cancers, autoimmunity diseases and metabolic disorders to emerging infectious diseases. Aggregates in monoclonal antibody manufacturing processes biotechnol bioeng 2011, 108 7, 1494508. In this talk, herb lutz, global consultant, will discuss the formation of protein aggregates and their.
Secretory leakage of igg1 aggregates from recombinant chinese. 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. 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. Aggregates in monoclonal antibody manufacturing processes marava. Upon binding, the full adc can be internalized by a process called receptormediated endocytosis. Impact of media and antifoam selection on monoclonal. The outcomes of this study will benefit scientists and engineers who develop biologic product manufacturing processes by providing a better understanding of drug product. Introduction to antibody production and purification thermo. Hic as a complementary, confirmatory tool to sec for the.
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