Introduction
Monoclonal antibodies (mAbs) are a mixture of analogous antibody molecules having
monovalent affinity towards a defined antigen. These are synthesized via hybridoma
technology that allows the production of mAbs at large scale with increased purity.
Hybridoma technique involves the fusion of normal B-cell (desired antibody-producing
splenocytes) to myeloma cell (immortal, cancerous B cells), ultimately generating a pool of
single cell type secreting the identical antibody. A selection media, hypoxanthine
aminopterin thymidine (HAT), is then used where only hybridoma cells can grow and further
screened for the desired mAb. Orthoclone OKT3 (muromonab-CD3) was the first licensed
monoclonal antibody, released in 1986 to prevent kidney transplant rejection [1]. The mAbs
bind only to a particular epitope on the antigen which contrasts with polyclonal antibodies
that bind to many epitopes on an antigen [2]. This makes mAbs functionally advantageous
over polyclonal antibodies in terms of specificity and reproducibility. On the other hand,
recombinant antibodies (rAbs) have also emerged which are in vitro generated mAbs from
genes expressed in high efficiency expression vectors. In contrast to mAbs that are
produced using conventional hybridoma-based technologies, rAbs do not require
hybridomas and animals in their production [3]. The mAbs are widely employed in fields like
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research and diagnostics; therapeutic solutions for cancers and immunological disorders;
and pharmaceuticals resulting in high market demand [4]. The human trials of mAbs have
shown their immensely improved biological compatibility and reduced adverse effects
(immunogenicity) [5]. Successful experimental trials of mAbs have extended their use from
immune disorders and oncology to other ailments like migraine, infectious, and genetic
disorders.
Besides the therapeutic use of mAbs, they can be used for diagnostic purposes (biochemical
analysis, diagnostic imaging) and protein purification. The mAbs are very potent biological
agents to evaluate various diagnostic assays, which include immunohistochemistry (IHC),
enzyme-linked immunosorbent assay (ELISA), western immunoblotting, immunofluorscent
antibody test (IFAT), flow cytometry, and radioimmuno assay (RIA) [5,6]. The mAbs are also
used for molecular imaging in various pathologies, such as oncology, autoimmune diseases,
and cardiovascular diseases, where mAbs target the imaging agents to the diseased sites in
vivo. Moreover, mAbs are used for protein purification through the immunoaffinity
chromatography (IAC) technique where the stationary phase comprises of mAbs as they
have distinctive specificity for the desired protein, ultimately minimizing the contamination
by unwanted molecules [6]. Several glutamylated polypeptides were identified by using mAb
GT335 (glutamylated tubulin). At present, nearly 80 mAbs have been approved by
regulatory agencies like the United States Food and Drug Administration (USFDA) and
European Medicines Agency (EMA) [5,6]. Consequently, a rise of 7.1% in the compound
annual growth rate (CAGR) of the mAbs global market is expected since the year, 2020. The
demand for analytical methodologies optimized for rigorous characterization of mAbs has
grown as the number of certified mAbs in the pharmaceutical industry has expanded with
simultaneous entry of the potent biosimilars hitting the market. In this work we have
attempted to give an overview of major analytical techniques for mAb characterization
which will be useful to students, researchers, and personnel from the biopharmaceutical
industry.
2. Structure of mAbs
Monoclonal antibodies are basically glycoproteins of the Ig (immunoglobulin) superfamily,
and its five isotypes are categorized as: IgA, IgD, IgE, IgG and IgM. Among these isotypes,
the IgGs are frequently used for therapeutic applications. The IgGs are high molecular
weight (~150 kDa) complex glycoproteins and comprise of two identical light and heavy
chains of molecular weight of ~25 kDa and ~50 kDa, respectively, that are joined by
disulfide bonds and non-covalent bonding at their pivotal point (Figure 1). In this way, the
formed tetramer creates Y-like shapes with two identical halves. Constant and variable
domains are formed by the intra-chain disulfide bonds in the polypeptide chains [7]. The
antibody can be divided into two main regions: the Fab (antigen binding fragment) that
recognizes the antigen; and the Fc (crystallizable fragment) that interacts with other
elements of the immune system, such as phagocytes to promote removal of the antigen.
The Fab consists of two variable and two constant domains. The variable domain known as
variable fragment (Fv) provides specificity for the antigen whereas the constant domain acts
as a structural framework. Each variable domain consists of three hypervariable loops,
known as complementarity determining regions (CDRs), evenly distributed between four less
variable framework (FR) regions