Advances in Monoclonal Antibody Purification and Analysis

Progress in antibody therapeutics in recent years has been revolutionary. Although the US Food and Drug Administration approved the first monoclonal antibody (mAb) therapeutic in 1986, only 13 more had been approved by the end of 2002. Over the next 20 years, more than 100 mAbs would be approved (figure 1).1 This growing interest in antibody therapeutics has propelled the growth of the global market, which had a value of about $200 billion in 2022.2 Figure 1: Antibody therapies approved by the US Food and Drug Administration by year. Gray indicates conventional antibodies and blue indicates engineered antibodies, including antibody-drug conjugates, bispecific antibodies, and Fc-fusion scaffolds. Source: Reference 1. INTRODUCTION THE STATE OF MONOCLONAL ANTIBODY MANUFACTURING Conventional antibodies Engineered antibodies 4 Antibody therapeutics provide cell-specific recognition customized for a patient and the potential to modulate an immune response. They offer the ability to treat cancers and autoimmune diseases with fewer side effects than traditional treatments such as chemotherapy. Many mAb therapeutics are made from socalled conventional antibodies, Y-shaped proteins with specific binding sites at the end of each arm of the Y. As the scientific understanding of traditional mAb therapeutics has advanced, researchers have moved into engineering proteins to create next-generation antibody modalities. Antibody fragments, bispecific antibodies, antibody-drug conjugates (ADCs), and Fc-fusion scaffolds have all received FDA approval since 2019.1 Innovations in the field of mAb therapeutics are accompanied by advances in the biotechnologies designed to develop and manufacture them. Engineered antibodies are produced in cultured cells. Research and development teams must show that their entire biomanufacturing process is reliable before receiving approval (figure 3). This effort includes instituting measures to ensure purity and confirming that analytical techniques effectively test for contamination, activity, and other quality concerns. As the saying goes: the process is the product. Figure 2: While most antibody-based therapeutics are conventional antibodies (left), an increasing number of engineered antibodies have received regulatory approval. Engineered antibodies include antibody-drug conjugates, bispecific antibodies that have two different recognition sites, and various antibody fragments. Credit: C&EN BrandLab Figure 3: The process of antibody manufacturing involves cell culture, separation, purification, and filling. Credit: Thermo Fisher Scientific Conventional antibodies Antibody-drug conjugates Bispecific antibodies Engineered antibodies Fragments Fab single domain antibodies Cell culture development Scale up to production Harvest and collection Purification Bulk storage and final fill 5 The purification step of antibody manufacturing provides an example of the need for technology evolution. A common purification technique involves a bacterial protein called protein A. Protein A binds to the stem, or Fc region, of immunoglobulin G (IgG) antibodies, which are the antibody type most used for therapeutics. By preparing a chromatographic resin with protein A, it is possible to separate mAbs from other cellular materials. Protein A affinity chromatography remains a popular and effective tool for purifying conventional mAbs. But protein A is ineffective at purifying newer antibody-derived therapies, such as fragments or bispecifics.3 For this increasingly diverse class of biotherapeutics, scientists must find alternative methods to meet the purity requirements without losing yield. Now, pharmaceutical manufacturers that produce mAb-based therapeutics face a complex set of scientific and business challenges. Part of the commercial success of antibody-derived therapeutics depends on efficient purification and analytical techniques. Companies that implement state-ofthe-art technology can improve their time to market, reduce the chance of quality issues, and lower manufacturing costs. This e-book addresses common purification, analysis, and quality evaluation challenges when manufacturing modern antibody-derived therapies. Tools such as affinity chromatography, quantitative polymerase chain reaction (qPCR), and single-domain antibodies provide effective and flexible solutions for improving the efficiency and reliability of antibody biomanufacturing practices. References: 1. Xiaochen Lyu et al., “The Global Landscap