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.