Monoclonal Antibodies: Physicochemical Analysis

Monoclonal antibodies (mAbs) are naturally occurring complex biomolecules. New engineering methods have turned mAbs into a leading therapeutic modality for addressing immunotherapeutic challenges and led to the rise of mAbs as the dominant class of protein therapeutics. mAbs have already demonstrated a great potential in developing safe and reliable treatments for complex diseases and creating more affordable healthcare alternatives. Developing mAbs into well-characterized antibody therapeutics that meet regulatory expectations, however, is extremely challenging. Obstacles to overcome include the determination and development of physiochemical characteristics such as aggregation, fragmentation, charge variants, identity, carbohydrate structure, and higher-order structure (HOS).

This book dives deep into mAbs structure and the array of physiochemical testing and characterization methods that need to be developed and validated to establish a mAb as a therapeutic molecule. The main focus of this book is on physiochemical aspects, including the importance of establishing quality attributes such as glycosylation, primary sequence, purity, and HOS and elucidating the structure of new antibody formats by mass spectrometry. Each of the aforementioned quality attributes has been discussed in detail; this will help scientists in researching and developing biopharmaceuticals and biosimilars to find practical solutions to physicochemical testing and characterization.

• Describes the spectrum of analytical tests and characterization methods necessary for developing and releasing mAb batches
• Details antibody heterogeneity in terms of size, charge, and carbohydrate content
• Gives special focus to the structural analysis of mAbs, including mass spectrometry analysis
• Presents the basic structure of mAbs with clarity and rigor
• Addresses regulatory guidelines - including ICH Q6B - in relation to quality attributes
• Lays out characterization and development case studies including biosimilars and new antibody formats

• Language: English
• Publisher: Academic Press
• Release date: Aug 3, 2021
• ISBN: 9780128223192

Book preview

Chapter 1

Overview of monoclonal antibodies

Harleen Kaur,    Analytical Sciences, Aurobindo Biologics (Unit-XVII) (CurateQ Biologics), Hyderabad, India

Abstract

Monoclonal antibodies (mAbs) are immunoglobulins (Igs) produced from single B-cell clone and are highly specific for an epitope on the antigen surface. Given their characteristic to bind any foreign substance that enters inside human body, mAbs have become an indispensable tool in therapeutic and diagnostic industry. In the last few decades, mAbs have demonstrated promising therapeutic potential, have become a critical part of healthcare systems, and consequently led to surge in the regulatory approvals of mAbs as therapeutic drugs for a wide range of indications. In this chapter, the history of mAbs development from their discovery to therapeutic applications will be presented. In addition, the molecular structure of the IgG antibody and functions of different regions/domains will be discussed. This work also covers a brief description of the FDA-approved therapeutic mAbs that are currently available in the market and their biosimilars in the treatment of various diseases.

Keywords

Monoclonal antibodies; heavy chain; light chain; therapeutics; Nobel Prize; CDR; hinge region

1.1 Introduction

Antibodies also referred to as immunoglobulins (Ig) are large Y-shaped soluble proteins naturally produced by the immune system in response to the invading foreign particles or abnormal agents (antigens) such as harmful pathogens and viruses. Antibodies are one of the first lines of defense in higher organisms that recognize and bind these antigens to remove them from the body by phagocytosis or complement lysis. The part of the antibody that binds with antigens is known as paratope (or antigen-binding region) or complementarity-determining regions (CDRs). The paratope is located on the variable region of both the antibody’s heavy chain (HC) and light chain (LC). The part of the antigen that is recognized by the antibody is referred to as epitope or antigenic determinant. With the antibody’s ability to bind with high affinity and high specificity to the antigen surface, they have been widely explored as therapeutic and diagnostic tools for the treatment of diseases and the detection of antigens (analyte). Although very successful in targeting pathogens and increasingly being used in the clinic for treatment of several diseases for few decades, they are still compared against their rival molecules termed as Aptamers or often called as chemical antibodies in therapeutics and diagnostics industry (Kaur & Yung, 2012; Kaur, Li, Bay, & Yung, 2013; Kaur, Bruno, Kumar, & Sharma, 2018). Aptamers exhibit significant advantages over the antibodies in terms of small size, nonimmunogenic nature, less batch to batch variability and sensitivity to temperature and pH. However, with antibodies having roots in the immune system and extensive information already known about their structure, binding, and function, for the time being therapeutic antibodies are heavily dominating the biologics therapeutic market and are expected to grow at the compound annual growth rate of around 12.5% between 2017 and 2023 with revenue generation of USD 218.97 billion by the end of 2023 (https://www.globenewswire.com/news-release/2018/04/10/1467446/0/en/Global-Monoclonal-Antibody-Therapeutics-Market-Will-Reach-USD-218-97-Billion-by-2023-Zion-Market-Research.html).

Antibodies may be polyclonal or monoclonal depending on their distinct properties and the way they are generated (Pohanka, 2009). Polyclonal antibodies are heterogeneous mixture of Igs produced by different clones of B-cells against a specific antigen but can bind to different epitopes of the same antigen. On the other hand, monoclonal antibodies (mAbs) are homogenous population of antibodies that are produced from single clone of B-cells and interact with specific epitope on the antigen surface. Derivation from a single clone of cells and subsequently targeting single epitope on the antigen surface is what differentiates mAbs from polyclonal antibodies. The high specificity of the mAb for an epitope reduces the possibility of the cross-reactivity, batch to batch variability, allows to conduct structural studies to understand the conformational changes, and provides concise and accurate quantitative analytical assay results which may not be the case for the naturally producing polyclonal antibodies. However, compared to polyclonal antibodies, the process to produce mAbs is expensive, laborious, and time consuming, a drawback that needs to be overcome.

Over the last few decades, the rise in the applications of mAbs has been remarkable. They have become important tools in basic research and have taken center stage in the area of therapeutics and diagnostics. The therapeutic market for mAb has witnessed the accelerated growth in the biopharmaceutical sector with over 80 mAbs granted marketing approval by regulatory agencies for treatment of several disorders like oncology, respiratory diseases, autoimmune, and inflammatory diseases. In addition, the introduction of biosimilars aimed at decreasing the cost of medication while increasing patient access to healthcare presents an attractive opportunity which has boosted the market growth in emerging and developed countries.

This chapter titled Overview of Monoclonal Antibodies is divided into three sections—the first section (Section 1.1) will provide an overview of the historical milestones in the discovery of antibodies, and the second section (Section 1.2) will present the chemical structure of antibodies with special focus on the IgG structure which is the most abundant antibody (75%) in body fluids and the most approved Ig as a therapeutic drug. The relative abundance of IgG in serum is 8–16 mg mL−1. Finally, the third section (Section 1.3) will highlight the importance of mAbs in biopharmaceutical industry and their therapeutic applications.

1.2 Discovery of monoclonal antibodies

The discovery of the antibodies dates back to 1890s when Emil Adolf von Behring and Kitasato Shibasaburōs work into serum therapy showed that serum from an animal immunized with diphtheria or tetanus toxin could be used to neutralize the effect of fatal doses of toxin in another animal (https://www.globenewswire.com/news-release/2018/04/10/1467446/0/en/Global-Monoclonal-Antibody-Therapeutics-Market-Will-Reach-USD-218-97-Billion-by-2023-Zion-Market-Research.html; Pohanka, 2009). This discovery was one of the remarkable developments in the field of medicine at the time that saved many lives and opened up new therapeutic possibilities. Behring and Kitasato also introduced the terminology antitoxic for the serum component present in the animals that were immunized with bacterial toxins and acquired immunity against the toxin (Behring & Kitasato, 1890; Grundbacher, 1992). The antitoxins are commonly referred to as antibodies now. Behring won the Nobel Prize in Physiology or Medicine in 1901 for his work on serum therapy particularly the discovery of a diphtheria antitoxin (https://www.nobelprize.org/prizes/medicine/1901/summary/). In 1897 bacteriologist Paul-Ehrlich proposed the side-chain theory of toxicity which became basis of immunological research at the time. This theory postulated that cells have side chains present on their surface for the adsorbance of the nutrients. These side chains can also bind to foreign substances like toxins (diphtheria/tetanus toxin or microorganism) in a manner similar to lock and key model for enzymes and their substrates (Bosch & Rosich, 2008; Winau, Westphal, & Winau, 2004). A large number of these side chains would be released into the bloodstream and act as antibodies or antitoxins to neutralize the toxin and thus preventing the binding of the toxin to other toxins in the blood. In the following years, Ehrlich extended his side-chain research and imagined side chains to be receptives that bind to toxins and introduced the term receptor. Later, he collaborated with John Newport Langley who was investigating the effects of the alkaloids on muscle cells and nerves, and together they developed the receptor theory and extended the concept of receptor to chemoreceptor to understand the interaction of the drugs with the cells (Bennett, 2000). Ehrlich also contributed to the field of cancer research and introduced chemotherapy for the treatment of trypanosoma infections. In 1908 Nobel Prize in Physiology or Medicine was awarded to Paul-Ehrlich jointly with Elias Metschnikow for their ground-breaking work on immunity (https://www.nobelprize.org/prizes/medicine/1908/summary/).

Ehrlich’s side-chain theory was rejected when experimental work led by Landsteiner demonstrated that upon injection of haptens (chemical groups which were covalently linked to the animal protein), antibodies specific for the haptenic group were produced and the production of the possible antibodies in an animal would be limited by the possible number of antigens (Landsteiner, 1945). With considerable amount of immunological data generated in addition to Landsteiners work, it was proven that any foreign substance that enters the body is an antigen and it was considered impossible that so many side chains of different specificities would be required by the body for absorbance of nutrients. The dismissal of side-chain theory led to the development of new hypothesis to understand the mechanism of antibody formation. Linus Paulings direct template theory assumed globulin as a single-polypeptide chain which folds around the antigen molecules resulting in formation of a high-specificity antibody molecule with stable configuration (Pauling, 1940). The specificity of the final antibody molecule is dependent on the complementarity with the antigen that serves as a template in the process of formation of an antibody molecule.

In 1955 Niels K. Jerne—a Danish immunologist—proposed the natural selection theory to explain the phenomenon of antibody synthesis and challenged the existing template theories. The natural selection theory suggested that globulins (antibodies) are continuously synthesized inside the body and few of the circulating globulins referred to as natural antibodies will have affinity toward an antigen. When an antigen enters the body, globulins will selectively bind to the antigens surface and few of them more or less fit well to the antigen resulting in an antigen–antibody complex which is engulfed by the phagocytic cell (Jerne, 1955). Upon entering the cell, the globulins dissociate from the antigen which plays no further part and antibodies stimulate the cell to produce more antibodies of the same kind. In addition to natural selection theory, Jerne’s developed the plaque method and postulated the network theory to explain the antibody production process. The plaque method could help to identify and count the number of single antibody-producing cells among a mixture of cell population (Jerne & Nordin, 1963). Results from the experiments using mouse spleen cells and rabbit lymph node cells demonstrated that the number of obtained plaques on the Petri plate is directly proportional to the number of the lymphoid cells which eventually indicate the activity of the individual cells. Jerne’s network theory was based on the idea that antibodies recognize and attach themselves not only to an antigen but also to other antibodies and interact with each other (antibodies) as part of a network and both antigen and antibodies can bind to the same site of the antibody (Jerne, 1974). In 1984 Jerne’s was awarded the Nobel Prize jointly with George J.F. Kohler and César Milstein for their theories that explain the specificity in development and control of the immune system and the principle for production of mAbs (https://www.nobelprize.org/prizes/medicine/1984/summary/). The contributions of Kohler and Milstein will be discussed later in this section. Subsequently, in 1957 Frank Macfarlane Burnet introduced the clonal selection theory which was a modification of the Jerne’s natural selection theory (Burnet, 1976). The clonal selection theory explains when an antigen enters the body, it binds to the lymphocytes cells that carry reactive sites corresponding to the antigenic determinants. This results in the activation of lymphocyte cells that further proliferate into a large number of clone cells (identical cells) to produce antibody against the foreign antigen.

Through the decades, a lot of significant discoveries were made and researchers continued to work on antibodies; however, the structure and its correlation to its function was still not clear. Due to the high-molecular weight of γ-globulin, it was not feasible to directly deduce its chemical structure and relate to its biological function. In order to tackle the structural problems of antibodies, R.R. Porter in 1959 attempted to use papain enzyme to cleave the γ-globulin molecule (attributed as antibody today) (Porter, 1959). The enzymatic treatment split the γ-globulin into three fragments with two of them constituting the antigen-binding sites, molecular weight around 50 kDa and demonstrating similar chemical and biological activity and no precipitating power. On the other hand, the third fragment is readily crystallizable with a molecular weight of around 80 kDa and no antibody activity. The two fragments with antigen-binding sites were Fab (fragment antigen binding), and the fragment that crystallizes was constant Fc (crystallizable fragment) domain. In the same year, Gerald M. Edelman intrigued by the structure of antibodies demonstrated that γ-globulin dissociate in the presence of denaturing agents indicating that γ-globulin subunits are linked by disulfide bonds though the possibility of the presence of other linkages was not eliminated (Edelman, 1959). In 1972 both Porter and Edelman were jointly awarded the Nobel Prize in Medicine or Physiology for their discoveries about the chemical structure of antibodies (https://www.nobelprize.org/prizes/medicine/1972/summary/).

A decade later, Edelman established the entire amino acid sequence of a human γG1 Ig including the location of disulfide linkages and arrangement of LC and HC (Edelman et al., 1969). The data from the study showed that variable regions of LC and HC are homologous and similar in length. While the constant regions of the HC is composed of three homologous regions of similar length—CH1, CH2, and CH3 which are also closely homologous to the constant region of the LC. However, the variable region of the chains is not homologous to the constant regions. Each variable and constant region is composed of one intrachain disulfide bond, and HC are linked via interchain disulfide bonds. The hinge region which lies in the middle of the HC (residue 221–233) contains two interchain disulfide bonds, rich in proline and cysteine residues and has no homology to any other portion of the antibody. The antibody structure will be discussed later in detail in Section 1.3.

mAbs were firstly isolated by the Argentinian biochemist César Milstein and his German postdoctoral fellow Georges Köhler back in the 1970s. Köhler while working at Milsteins laboratory was interested in finding the answer to the problem of finding the source of mAbs with predefined specificity. Along with the skills in studying myeloma in mice, Milstein’s lab utilized a technique developed by Potter to establish a culture of rapidly proliferating tumor cells that produced antibodies or Igs (Potter, 1972). They successfully established and proved the technique to fuse the antibody-producing cells with myeloma cells. Kohler decided to fuse one myeloma cell bearing a selection marker with one mouse spleen cells, antibody-producing B-cells (Kohler & Milstein, 1975, 1976). A mutant myeloma cell line deficient in the enzyme hypoxanthine phosphoribosyl transferase was employed for the work. In order to identify the positive fused cells with hypoxanthine phosphoribosyl transferase activity, the fused cells were cultured in a medium containing hypoxanthine, aminopterine, and thymidine (HAT). Only the hybrid cells (also referred as hybridoma cells) survived and were selected as the enzyme contribution in the hybrid cells came from the healthy spleen cells (Fig. 1.1). These hybridoma cells survived as they inherited the immortality from myeloma cells and selective resistance from B-lymphocytes. For the first time, pure antibody specific to single antigen was produced in large quantities. This development of such an important method that demonstrates the fusion of antibody-producing cells from different origins was never patented by Kohler and Milstein, allowing the use of this brilliant technology by biopharmaceutical industry and academicians for basic therapeutic research and bringing potential future therapeutic products for patients and also in diagnostic applications. For this breakthrough discovery, Milsten and Köhler along with Niels K. Jerne won a Nobel Prize in Medicine or Physiology in 1984 for their work on theories concerning the specificity in development and control of the immune system and the discovery of the principle for production of mAbs (https://www.nobelprize.org/prizes/medicine/1984/summary/).

Figure 1.1 Monoclonal antibody production.

1.3 Structure of monoclonal antibody

The basic structure of human mAb is a Y-shaped structure composed of two identical HC and two identical LC, of either kappa (κ) or Lambda (λ) type linked by disulfide bonds (Valentine & Green, 1967). The arms of the Y region of the antibody is the antigen-binding site and is commonly referred to as Fab (fragment, antigen-binding region). On the other hand, the tail of the Y region of the antibody is called the Fc (fragment, crystallizable) region which is always located on the C-terminal domain of the same antibody class.

The individual HC in the antibody has a molecular weight of 50,000 Daltons (Da), whereas the weight of each LC is 25,000 Daltons (Da). Individual LC is about 222 residues long with variable region (VL) composed of 110 residues and constant region of the same length. The LC are covalently linked to the HC by disulfide bonds. Each HC and LC is made up of a number of globular domains that have variable (V) and constant (C) amino acid sequences. The bulk of the antibody molecule, three-fourths of the HC, and one-half of the LC is composed of the highly conserved constant regions. The HC has one variable region followed by several constant regions (CH1, CH2, and CH3). On the other hand, the LC has one variable domain (VL) and one constant domain (CL). The variable domains (VH and VL) and constant domains (CH1, CH2, CH3, and CL) of HC and LC are similar to each other in three dimensional conformation.

The variable region is part of the antigen-binding site and antibody specificity is determined by the combination of the variable segments of the HC and LC. The variable regions of HC and LC fold together to form the hypervariable loops that form the antigen-binding site or previously mentioned as paratope. The variable region of the HC is directly connected to the HC CH1 domain. The CH1 and CH2 domain of the HC constitutes one of the most fascinating regions in the antibody structure, also called as the Hinge Region (or Interdomain Region) (Adlersberg, 1976). The hinge region is an amino acid stretch rich in cysteine and proline residues located in the central region of the HC that links the two HC by two disulfide bonds in an IgG molecule. The hinge region has no similarity to any other region in the antibody structure and is an extremely variable amino acid sequence from one IgG class to another. The hinge region is the primary mediator to the flexibility of the antibody—the larger the hinge region, the more flexible is the antibody, thus allowing better antigen–antibody interactions and good effector activity (Roux, 1997; Vidarsson, Dekkers, & Rispens, 2014). The upper hinge region connects the Fab arms to the middle hinge region and is responsible for the Fab–Fab segmental flexibility with length of the upper hinge region significantly influencing the Fab segmental flexibility (Roux, 1997; Schumaker, 1991). On the contrary, the middle hinge region which is rich is in cysteine and proline residues typically with the sequence –Cys–Pro–Pro–Cys– is relatively inflexible and serve as spacer that separates Fab from Fc arm in length-dependent manner (Matsunaga, 1991). Lastly, the lower hinge region which is less flexible compared to the upper region is responsible to connect the Fc to middle hinge region and contributes to Fab-Fc flexibility and Fc tail wagging movement required for Fc receptor binding. Compared to the rigid middle hinge region which is rich in cysteine and proline residues, the upper and lower regions are rich in highly flexible peptide segments. Roux et al. has defined different modes of flexibility in the hinge region-folding, rotation along the long axis, conical wagging, and translation (in- and out- motion) (Roux, 1997). Additionally, results from another study demonstrate that the hinge region particularly the upper hinge region undergoes an extensive degree of internal motion as obtained using high-resolution nuclear magnetic resonance analysis (Kim, 1994). The data from the same study have also shown that longitudinal interactions between CH2 domain and CH3 domain remain unaffected by the deletion of the Fab segment, cleavage of interchain disulfide bonds, and deletion of the most of the hinge region which is in alignment with the previously reported work about the protein A binding to IgG1 myeloma proteins DoB and Lec (Arata, 1980; Klein et al.,