INTRODUCTION TO THERAPEUTIC ANTIBODIES
Over the past 20 years, therapeutic monoclonal antibodies (mAbs) have become increasingly important in the fight against various diseases. In several therapeutic areas such as oncology, haematology, and immunology, mAbs have become the treatment modality of choice (Lu et al., 2020). As of March 5, 2020, 93 therapeutic mAbs have been approved in the United States or Europe by the U.S. Federal Drug and Food Administration (FDA) or European Medicines Agency (EMA), respectively, of which more than one third are for the treatment of different cancers (‘The Antibody Society’, 2020). The first mAb approved for therapeutic use was in 1986, namely orthoclone OKT3 (Muromonab®), a murine monoclonal antibody targeting CD3, which was approved for the treatment of kidney transplantation rejection (Kung, Goldstein, Reinherz, & Schlossman, 1979; Ribatti, 2014). The latest FDA-approved mAb is isatuximab (Sarclisa®), approved in March 2020 for the treatment of multiple myeloma by targeting CD38. As of 2018, the global mAb market was valued at US$115.2 billion and is expected to continue growing at an increasing pace, reaching US$300 billion by 2025. Seven companies rule 87% of the market, i.e. Genentech, AbbVie, Johnson & Johnson, Bristol-Myers Squibb, Merck Sharpe & Dohme, Novartis and Amgen, with all other companies making up the remaining 13%. Adalimumab (Humira®), targeting TNFα, has reported the highest sales figures for a biopharmaceutical in history, with nearly $20 billion in sales in 2018 (Lu et al., 2020). Due to platform-based approaches, mAb products are easily adjusted for production and show a lower safety risk in clinical trials compared to other modalities. Thus, mAbs have become the go-to modality for first drug candidates against new targets, as they provide a rapid route for new therapeutics or proof-of-concept studies, and drive the continued growth of the mAb market (Ecker, Jones, & Levine, 2015).
The use and success of monoclonal antibodies for therapeutic applications is largely due to their high specificity, resulting from their complex glycoprotein structure. mAbs are immunoglobulins (Ig), of which there are 5 sub-classes: IgA, IgD, IgE, IgG and IgM, with IgG being the most relevant isotype for therapeutic use (Awwad & Angkawinitwong, 2018). IgGs have a molecular weight of approximately 150,000 Daltons (Da) and are Y-shaped molecules consisting of three equal-sized parts connected with a flexible hinge. IgG molecules consist of two heavy (H) chains, of approximately 50 kDa, and two light (L) chains, of 25 kDa. The two arms of the Y-shape end in so-called variable regions that vary between different mAbs and are responsible for antigen binding, termed the f ragment a ntigen b inding (Fab ) fragments. On the other hand, the stem of the Y, termed the constant region, is less variable and interacts with effector cells and molecules, commonly known as the Fc fragment (Figure 1).
With the development of hybridoma technology in 1975, Köhler and Milstein have paved the way for modern mAb technologies (Köhler & Milstein, 1975). In addition, display technologies such as phage display (Smith, 1985) or yeast display (Boder & Wittrup, 1998) are nowadays used for the generation of antibodies (J. K. H. Liu, 2014). Technological advances in the generation of mAbs are not within the scope of this review but have been recently reviewed by Lu et al. (2020). Drug discovery begins with millions of potential drug candidates. Nonetheless, resource-intensive steps, such as development of a manufacturing process are typically carried out for a single variant after extensive screening, characterization and in vitroand in vivo PK/PD experiments and animal safety studies (Jarasch et al., 2015).
While in theory mAbs are defined by a unique primary structure of amino acids, in reality a single dose of a mAb product represents a plethora of variants, inherent to the biotechnological procedure used to manufacture these drug products. Heterogeneity of monoclonal antibodies comes from different sources throughout process development, but variations start at the transcriptional level through errors during gene transcription and translation. The use of protein engineering and improved manufacturability techniques can be applied to minimize mAb variants and guarantee batch-to-batch consistency. Reduction of such variants is a crucial requirement as they may impair the activity, efficacy, safety and/or pharmacokinetic properties of an antibody, ultimately resulting in the failure of a product in pre-clinical and clinical trials. Manufacturing and formulation of lead candidates are critical aspects that are often overlooked in drug discovery and early drug development. This review serves to summarize challenges and recent technological advances used to minimize mAb heterogeneity during the manufacturing process and improve the formulability of a drug product.