Aggregates in monoclonal antibody manufacturing processes ifm. Various economic analyses estimate that process development and clinical manufacturing costs can. Quality attributes of monoclonal antibodies and effect of cell culture. These include production cell culture, harvest, antibody capture by protein a, virus.
Adcs travel to target cells, where the antibody binds to its antigen expressed on the cell surface. 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. Table 1 summarizes potential sources of aggregate formation during. Integrated flowthrough purification for therapeutic. Removing aggregates in monoclonal antibody purification.
Pdf aggregates in monoclonal antibody manufacturing. For some years, new methodologies have advanced with new production processes, allowing scaleup production and market introduction. Introduction to antibody production and purification. Preparative separation of monoclonal antibody aggregates. The study of aggregation propensity of a monoclonal antibody mab and its. 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. In custom antibody development, once target selection. The small molecules attached to the mab are often relatively hydrophobic. The formation of aggregates is a common but highly undesirable occurrence during monoclonal antibody purification. Largescale manufacturing of pharmaceutical antibodies is a complex activity that requires considerable effort in both process and analytical development. Largescale production has revolutionized the market for monoclonal antibodies by boosting its production and becoming a more practical method of production.
Schematic manufacturing process of monoclonal antibodies from cell. Selecting the optimal resins for aggregate removal references he x et al. 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. Humanized monoclonal antibody produced by mammalian cell culture may contain significant amounts of antibody dimers and smaller amounts of higher order aggregates. Biomanufacturing of monoclonal antibodies mab involves a number of unit operations, including cell culture in a bioreactor followed by chromatography and filtration. Size exclusion chromatography analysis of antibody drug conjugates using the agilent 1260 infinity ii bioinert lc system. Aggregate content of cht xt eluate mab s significant reduction of the mab s aggregate was achieved unosphere supra eluate. 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.
Protein a chromatography increases monoclonal antibody aggregation rate during subsequent low ph virus inactivation hold. Removing the aggregates of an acidic monoclonal antibody with. Monoclonal antibodies mabs are the fastestgrowing biological therapeutics with important applications ranging from cancers, autoimmunity diseases and metabolic disorders to. Secretory leakage of igg1 aggregates from recombinant. Mab aggregates may form during protein expression, downstream processing, and storage, and may be caused. Aggregation mechanism of an igg2 and two igg1 monoclonal. To investigate antibody stability and formation of modified species under upstream processing conditions.
Antibody aggregation could be triggered by partial unfolding of its domains, leading to monomermonomer. Usp manufacturing processes which generate higher titers still take. In the broad sense, it refers to the entire process of creating a usable specific antibody, including steps of. After completing this chapter, students will be able to. Prediction and reduction of the aggregation of monoclonal antibodies. 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. As a result, aggregation has to be monitored throughout process development and manufacturing of a mab product. During the manufacturing process, the protein is exposed to multiple stress.
Mar 25, 2018 aggregates in monoclonal antibody manufacturing processes. For the final biopharmaceutical product approval and subsequent manufacturing processes, a comprehensive characterization of mab. 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. Due to their broad application range, monoclonal antibodies mab are used worldwide in a variety. Aggregation and chemical modification of monoclonal. 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. 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. 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. Low ph elution can induce mab aggregation in protein a chromatography 7, 8.
The control and avoidance of aggregation in the manufacturing process are needed because aggregates affect drug performance and safety97,98. The structure of the monoclonal antibody should be justified with respect to its mechanism of action. Analysis of monoclonal antibodies and their fragments by. 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 demonstrate the analysis of monoclonal antibody mab aggregates.
This hydrophobicity can be problematic during manufacturing and storage in terms of aggregate formation as well as in analytical characterization of the latter1. Simultaneous removal of leached proteina and aggregates from monoclonal antibody using hydrophobic interaction membrane chromatography. It has been more than a decade since the industry started establishing the process platform. Purification is intended to remove impurities, such as protein aggregates, but some such operations may actually generate protein aggregation 1. Reliable, timely and costeffective monoclonal antibody production and purification programs. 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. Current trends in monoclonal antibody development and. Factors influencing biotherapeutic monoclonal antibody. Removing the aggregates of an acidic monoclonal antibody. This 31 st article in the elements of biopharmaceutical production series focuses on evolution of the purification platform for manufacturing of mab therapeutics. Removal of leached protein a, antibody dimers and aggregates da, host cell proteins hcp, viruses and. 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. Lang2 1manufacturing science and technology, lonza biologics porrin. Secretory leakage of igg1 aggregates from recombinant chinese.
Overall purification performance of cht xt chromatography is summarized in table 1. Strategies for the assessment of protein aggregates in pharmaceutical biotech product development. Recovery and purification process development for monoclonal. Are you considering how best to manage and remove aggregates across your template. Antibodydrug conjugates adcs are monoclonal antibodies coupled to cytotoxic agents with stable linkers. Preparative separation of monoclonal antibody aggregates by. Processing impact on monoclonal antibody drug products. A systematic framework to optimize and control monoclonal. Monoclonal antibody production in commercial scale cell culture bioprocessing requires a thorough understanding of the engineering process and components used. 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. Capto adhere is a multimodal ion exchanger and is designed to be used as a second or third step in monoclonal antibody mab puri. Dec 16, 2016 aggregates in monoclonal antibody manufacturing processes biotechnol bioeng 2011, 108 7, 1494508. Antibody aggregation can occur at various stages including the up and downstream processes in manufacturing, formulation, and longterm storage 5, 6. Trends in upstream and downstream process development.
Extremes of ph, ionic strength, temperature, concentration, shear forces, and other processing conditions can lead to increased aggregation. This study demonstrated the suitability of cationexchange laterallyfed membrane chromatography lfmc for rapid and highresolution separation of monoclonal antibody. Review aggregates in monoclonal antibody manufacturing processes mar. Protein aggregation remains a major area of focus in the production of monoclonal antibodies. Production processes for monoclonal antibodies intechopen. Adcs travel to target cells, where the antibody binds to its antigen expressed on. Aggregation kinetics for monoclonal antibody products. 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. Clarification technologies for monoclonal antibody. Trends in upstream and downstream process development for. Today, antibody concentrations of 35 gl are achieved routinely and some companies have attained up to 10 gl in fedbatch processes 5,6,8. Given the high cell densities achievable in both mammalian cell culture and microbial processes, primary recovery can be a. Aggregates have been characterized in mab purification and formulation. In this talk, herb lutz, global consultant, will discuss the formation of protein aggregates and their.
Managing aggregates in your monoclonal antibody mab process. Aggregates in the final drug product are undesirable for two major reasons. The term antibody production has both general and specific meanings. The current trend for cell culture processes is to increase. Aggregates in monoclonal antibody manufacturing processes marava. In the creation of this study, we used the industry standard modelling software, biosolve process, to address these questions. This study demonstrated the suitability of cationexchange laterallyfed membrane chromatography lfmc for rapid and highresolution separation of monoclonal antibody mab aggregates. 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. Monoclonal antibodies can have monovalent affinity. Pdf aggregates in monoclonal antibody manufacturing processes.
This 31 st article in the elements of biopharmaceutical production series focuses. Predicting aggregation propensity and monitoring aggregates. Monoclonal antibodies are expensive to develop and to manufacture. The agilent advancebio sec column, used with the agilent 1260 biolc system, proved capable of. Pdf monoclonal antibodies have proved to be a highly successful class of therapeutic products. Individual antibodies vary widely in their propensity to form aggregates 5.
Dec 16, 2016 understanding and managing aggregates are critical to your monoclonal antibody mab process. Understanding and managing aggregates are critical to your monoclonal antibody mab process. Jan 16, 20 to investigate antibody stability and formation of modified species under upstream processing conditions. Imbaratto, cell culture manufacturing, covance biotechnology services, inc. Aggregates in mab therapeutics pose a risk to patients. 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. Impact of media and antifoam selection on monoclonal. Hic as a complementary, confirmatory tool to sec for the. Analysis of monoclonal antibodies and their fragments by size. Size exclusion chromatography analysis of antibody drug. Aggregation mechanism of an igg2 and two igg1 monoclonal antibodies at low ph. Largescale manufacturing of pharmaceutical antibodies. Watch our experts discuss which steps in the biomanufacturing process are involved in the formation or removal of aggregates and industryleading approaches.
The stability of 11 purified monoclonal human igg1 and igg4 antibodies, including an igg1based bispecific crossmab, was compared in downscale mixing stress models. One of these molecules was further evaluated in realistic bioreactor stress models and in cell culture fermentations. But this method can provide misleading results because aggregates can dissociate or form during sec andor adsorb to the column resin. Protein a chromatography pac is commonly used as an efficient capture step in monoclonal antibody mab separation processes. Upon binding, the full adc can be internalized by a process called receptormediated endocytosis. The study focuses on very large scale processes in the region of five 5 tonnes per annum of bulk antibody. Since the approval of the first therapeutic monoclonal antibody in 1986. Introduction to antibody production and purification thermo. 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. Evolution of the monoclonal antibody purification platform. 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. Guideline on development, production, characterisation and. The stability of 11 purified monoclonal human igg1 and igg4.