Formulation of Monoclonal Antibody Therapies: From Lab to Market

By Amal Ali Elkordy

Formulation of Monoclonal Antibody Therapies: From Lab to Market covers a wide range of topics about therapeutic monoclonal antibodies (mAbs) with a focus on formulation aspects. Therapeutic monoclonal antibodies are used for treatment of chronic diseases. It brings together a comprehensive knowledge in one accessible volume. Starting with foundational information on monoclonal antibodies, the book then discusses the importance of biopharmaceutical products, monoclonal antibodies, and biosimilars in treatment of chronic diseases, pharmaceutical aspects of mAbs, and how it can be administered. It also covers the industrial point of view and the clinical application of mAbs including in oncology, general medicine, rheumatology, hematology, dermatology, gastrointestinal tract, metabolic diseases, and dentistry. Formulation of Monoclonal Antibody Therapies: From Lab to Market is essential reading for researchers in biotechnology and biopharmaceutical fields, academics and pharmaceutical industrial scientists, and university students in pharmaceutical and biopharmaceutical sciences.

• Covers details of recent advances in using mAbs
• Examines how to overcome the challenges for formulations of therapeutic mAbs
• Includes clinical application of mAbs

• Language: English
• Publisher: Academic Press
• Release date: May 20, 2023
• ISBN: 9780128234280

Amal Ali Elkordy

His research is on the areas of formulation of macromolecules, solid dosage forms, gene therapy, niosomal nano-particulate systems and enhancement of dissolution of poorly-water soluble drugs and has received several awards and presented work at several national and international conferences. Research outcomes are reachable to non-specialist audiences as well as to experts in the field of pharmaceutics and are recognised both nationally and internationally as evidenced from the significantly publish of over 130 publications, including journal articles and conference papers, most of which are published in high-impact journals and well-cited. Dr Elkordy has extensive teaching experience incorporating research findings as “Research informs Teaching”. He designed MSc Pharmaceutical and BioPharmaceutical Formulations which is one of the few courses in the UK covering biopharmaceuticals as well as pharmaceuticals.

Book preview

Formulation of Monoclonal Antibody Therapies

From Lab to Market

Amal Ali Elkordy

School of Pharmacy and Pharmaceutical Sciences, Faculty of Health Sciences and Wellbeing, University of Sunderland, Sunderland, United Kingdom

Table of Contents

Cover image

Title page

Copyright

Contributors

Chapter One. Introduction about monoclonal antibodies

1.1. Introduction

1.2. What are therapeutic antibodies?

1.3. Classification, types, and structures of mAb-based therapeutics

1.4. Nomenclature of therapeutic monoclonal antibodies

1.5. Production of monoclonal antibodies using various technologies

1.6. Mechanism of mAbs action and examples for mAbs with their clinical uses

Chapter Two. Biosimilar antibodies

2.1. Introduction

2.2. Approval and market availability of biosimilar antibodies-based medicines

2.3. Differences between generic and biosimilar medicines

2.4. Importance of biosimilar mAbs

Chapter THREE. Pharmaceutical aspects of mAbs: formulation, characterization, and current route of administration of monoclonal antibody therapies

3.1. Introduction: pharmaceutical formulations of lyophilized powder and liquid mAb dosage forms

3.2. Formulation of lyophilized mAb powder dosage forms

3.3. Formulation/preparation of mAbs in liquid dosage forms

Chapter Four. Route of monoclonal antibodies administration

4.1. Introduction

4.2. Approaches for novel mAbs delivery systems to assist on mAb delivery by a route/s other than injections

4.3. Noninvasive routes for biologics including mAbs

Chapter Five. Industrial aspects of finished monoclonal antibody therapies

5.1. Introduction

5.2. Glass vials

5.3. Prefilled syringes

5.4. Autoinjectors

Chapter Six. Application of approved and marketed products of monoclonal antibody therapies

6.1. Introduction

6.2. Application of recently approved or in regulatory review mAb therapies in cancer

6.3. Application of recently approved or in regulatory review mAb therapies in noncancer diseases

Index

Copyright

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Contributors

Sohib Bashier Al-Abdulrazag,     School of Pharmacy and Pharmaceutical Sciences, Faculty of Health Sciences and Wellbeing, University of Sunderland, Sunderland, United Kingdom

Mark Carlile,     School of Pharmacy and Pharmaceutical Sciences, Faculty of Health Sciences and Wellbeing, University of Sunderland, Sunderland, United Kingdom

Cheng Shu Chaw,     School of Pharmacy and Pharmaceutical Sciences, Faculty of Health Sciences and Wellbeing, University of Sunderland, Sunderland, United Kingdom

Amal Ali Elkordy,     School of Pharmacy and Pharmaceutical Sciences, Faculty of Health Sciences and Wellbeing, University of Sunderland, Sunderland, United Kingdom

Moustafa Elsayed,     School of Pharmacy and Pharmaceutical Sciences, Faculty of Health Sciences and Wellbeing, University of Sunderland, Sunderland, United Kingdom

Marc Faltes,     School of Pharmacy and Pharmaceutical Sciences, Faculty of Health Sciences and Wellbeing, University of Sunderland, Sunderland, United Kingdom

Rita Haj-Ahmad

School of Pharmacy and Pharmaceutical Sciences, Faculty of Health Sciences and Wellbeing, University of Sunderland, Sunderland, United Kingdom

Well Pharmacy, Nottingham, United Kingdom

Amerah Parveen,     School of Pharmacy and Pharmaceutical Sciences, Faculty of Health Sciences and Wellbeing, University of Sunderland, Sunderland, United Kingdom

Kamalinder K. Singh,     School of Pharmacy and Biomedical Sciences, Faculty of Clinical and Biomedical Sciences, University of Central Lancashire, Preston, United Kingdom

Zeinab Moataz Zarara,     Silver and Charlton Dental Practice, Sunderland, United Kingdom

Chapter One: Introduction about monoclonal antibodies

Amal Ali Elkordy, and Mark Carlile     School of Pharmacy and Pharmaceutical Sciences, Faculty of Health Sciences and Wellbeing, University of Sunderland, Sunderland, United Kingdom

Abstract

Therapeutic antibodies are the fastest growing class of biopharmaceutical in development. They are highly specific for target recognition and accordingly produce highly selective results after their administration. This chapter will cover a brief history of monoclonal antibodies (mAbs), their types, classification, structures, mechanism of actions, and clinical uses. In addition, this chapter will demonstrate the uniqueness of mAb characteristics leading to mAbs use for abroad range of targets, such as cancer, autoimmune disease, gastrointestinal, rheumatologically, hematological, dermatological, metabolic, cardiovascular, central nervous system, ophthalmic, dental and infectious diseases. The dominant areas of mAb therapeutic application are in oncology, immunology, and hematology. Also, the recent potential role of mAbs to treat coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), will be reported.

Keywords

Biopharmaceutical products; COVID-19; History of mAbs; Immunoglobulins; mAbs production techniques; Mechanism of mAbs action; Monoclonal antibodies (mAbs)

1.1. Introduction

The introduction will cover a brief history of monoclonal antibody (mAb) therapeutics. Monoclonal antibodies, also known as immunoglobulins, drugs are therapeutic glycoproteins. At least 570 mAbs have already been progressed to the clinic by commercial companies and to-date 100 mAbs have received approval from the Food and Drug Administration (FDA) for a range of clinical applications. The first mAb to be approved was muromonab-CD3, (tradename Orthoclone OKT3) in 1986 as a kidney transplant immunosuppressant (Liu, 2014; Smith, 1996). Orthoclone OKT3 was latter voluntarily withdrawn from the U.S. market due to the levels of side effects and the introduction of better-tolerated competitor molecules.

Currently as of August 2, 2021, 100 therapeutic mAbs have been accepted and therefore approved by the FDA and are currently on the market, this is in addition to 17 mAbs under review by the FDA (Antibody therapeutics approved or in regulatory review in the EU or US, 2021). Also, there are many novel mAbs therapeutics in development. Those protein therapeutics show high efficacy and good safety profiles hence they progress through clinical trials very quickly. Accordingly, mAbs are the most rapidly growing class of biopharmaceutical products. Being complex quaternary glycoproteins, mAbs have unique structural features that provide target specificity and downstream cellular signaling activities. They are in use for treatment of complex and difficult to treat diseases such as rheumatoid arthritis, cancer, Crohn's disease, psoriasis, transplant rejection, autoimmune, asthma, infectious diseases, migraine headaches and most recently they are used to treat Alzheimer's diseases (Aduhelm (aducanumab) approved for the treatment of Alzheimer's disease, 2021).

As the pharmaceutical market in the United States and the rest of the world continues to expand, biopharmaceutical products have taken on increasing importance in the treatment of disease. Sales of monoclonal antibody products have grown from approximately $50 billion in 2010 to almost $90 billion in 2015, an approximately 1.8-fold increase and represent approximately 58% of biopharmaceutical sales. As more and more exciting monoclonal antibody products for treatment of cancer, autoimmune diseases, cardiovascular disease, and others are introduced, sales from new products approved in the coming years will drive the world-wide sales of monoclonal antibody products to approximately $150 billion by 2021 (Insight Pharma Reports, 2020).

There are different laboratory production techniques for mAbs (as will be explained later in this Chapter); however, each technique presents challenges in terms of product expression format, yield, purity, complexity, and heterogeneity. These challenges are compounded at the formulation stages, wherein, aggregate formation is commonplace and may lead to unwanted immunogenicity. For example, muromonab-CD3 and other early-mAbs were generated via murine models. These purely mouse-derived proteins were not tolerated by patients for extended administration periods due to induced immunogenicity. The development of hybridoma and transgenic humanized mouse expression techniques has helped in the manufacture of more clinically useful antibodies (for recombinant mAbs production technique refer to Section 1.5 below, and for overcoming the formulation challenges refer to Chapters 3 and 4).

The immune system is crudely dived into the innate and the adaptive immune response components and is mobilized against foreign agents and infectious organisms through the generation of antibodies from the adaptive immune system (as shown in Fig. 1.1A and B). The immune system is constantly responding to all diseases and thus to a wide diversity of pathogens and antigens. Therefore, part of the body's normal immune response to an external toxin or substance is the composition and production of antibodies (Abs). The Abs production is via B-cells and sustained for later mobilization via plasma cells (their origin is bone marrow–derived B lymphocyte cells and the process requires help from T cells (Kaunitz, 2017; Kugelberg, 2016) (Fig. 1.1B(ii)).

The power and specificity of the immune system to target specific antigens and select the best antibody candidate generated via unique B cell generation gave rise to traditional antibody therapies, usually from animal serum. These crude therapies were generated via repeated immunization of experimental animals with an antigen followed by subsequent purification of the serum to isolate the antibody fractions (against the antigen) (Fig. 1.2). However, the administration of purified animals' sera preparations can produce allergic reactions in many patients.

Figure 1.1a  Representation on how immune system works. (i) Cellular immunity—adaptive immunity part 1: https://www.youtube.com/watch?v=nqRn5fN22t4. (ii) Humoral immunity—adaptive immunity part 2: https://www.youtube.com/watch?v=rAepZG_ChyQ.

Figure 1.1b  Link to videos for (i) cellular immunity; (ii) humoral immunity.

The first Nobel Prize in Medicine in 1901 was won by Emil von Behring. This was for his work on serum therapy (with other scientists in the late 1800s and early 1900s) that not only emphases that blood is a very unusual fluid but also led to discovery of antibodies in protection against diseases and this discovery denotes the dawn of immunological science (Nunes-Alves, 2016; Bordon, 2016).

Passive antibody therapy or serum therapy has been used as a preventative of infection as well as postinfection treatment. Famously, obtaining serum filled with antibodies from people who have recovered from the disease—was used during the 1918 Spanish influenza pandemic, this was convalescent serum therapy. More recently serum therapy was used during the Ebola and SARS epidemics (First Nobel Prize in Medicine and the Coronavirus (COVID-19), 2020). Nevertheless, human serum therapy can be the only available treatment for some diseases, exemplified by the 2019 global pandemic outbreak. Serum therapy was used recently in 2020 against the SARS2-COVID-19 virus i.e., as a treatment for the coronavirus (COVID-19), which seems unusual with all the available advanced technology to produce medicines. It involves injecting serum from COVID-19 recovered patients into patients with COVID-19 virus.

Figure 1.2  Serum therapy steps.

Due to the complexity of antibodies, biological systems, i.e., cells or entire organisms capable of immunological response are the only viable option for generating antibodies against specific targets. However, scientists have developed laboratory methods (recombinant DNA/gene technologies) to engineer therapeutic antibodies to finetune and improve their application to specific diseases. When treating a complex or difficult to treat diseases (such as cancer, autoimmune diseases) that impair or compromise the immune system, supplementation or augmentation of the immune resposne is still possible through the use of engineered antibody molecules that may have been engineered in the laboratory (mAb-based therapeutics).

However, the laboratory production and commercial manufacture of antibodies is not an easy process and biological systems are still needed for mass production beyond the initial molecule discovery and screening activities. Many mAbs used in the clinic originated via the use of hybridoma technologies for mAb generation and selection (e.g., Fig. 1.3). The expression of these mAbs is then moved to a recombinant expression system (Fig. 1.3) for clinical manufacture and commercial supply. More information can be found here (Frank Lectures, 2017, Hybridoma Technology: Production of Monoclonal Antibodies (FL-Immuno/55)—YouTube) (Askonas et al., 1970; Kaunitz, 2017; Köhler and Milstein, 1975). Also for general explanation refers to this link (Khan Academy, 2016 for https://www.youtube.com/watch?v=5ffl-0OYVQU).

Therapeutic mAbs are routinely generated in traditional recombinant expression systems, such as yeast, insect, animal, or human cell lines. Some modified mAb fragments can be generated using microbial expression systems. The choice of the system is based mainly on the glycosylation capacity of the system, the efficiency of expression, the biological efficacy, and the yield requirements for the mAb. The choice of expression system can impact the downstream purification process unit operations and the final formulation of the drug molecule and its application. Kaunitz reported in detail the dawn of mAbs rule, where the commencing of mAbs revolution stated (Kaunitz, 2017).

Figure 1.3  Schematic presentation of hybridoma mAb production and recombinant monoclonal antibody (mAb) technology.

1.2. What are therapeutic antibodies?

Therapeutic monoclonal antibodies (mAbs) are a class of therapeutic glycoproteins namely immunoglobulins (Igs) that are produced from a single clonal lymphocyte in response to a foreign agent. Cloning a single lymphocyte is very challenging to produce homogenous immunoglobulins in large quantities. Many of the first therepeutic antibodies have been produced via the fusion of a single B lymphocyte and a single tumor cell—termed hybridoma technology. This technology has been used successfully against many disease-causing and diagnostic antigens (Fig. 1.3), also refer to Section 1.5, Fig. 1.10.

1.2.1. General structure of proteins

Proteins are large molecules (macromolecules), consisting of one or more chains of amino acid residues joined by peptide bonds. Nature utilizes 20 different L-amino acids for protein generation. The peptide bonds form a polyamide backbone (known as protein primary structure) with the specific amino acid side chains contributing to the protein folding, final conformation, and function of the protein. The final three-dimensional conformation of a protein is achieved through interactions between the side chains of the amino acids and the specific physicochemical properties of these side chains (Fig. 1.4).

The tertiary structure refers to the three-dimensional arrangement of the folded polypeptide chains. Proteins with the quaternary structures are characterized by a combination of more than two polypeptide chains. Tertiary and quaternary structures are stabilized by covalent and noncovalent interactions. The interactions include hydrogen bonds, disulfide linkage, van der Waals forces, and electrostatic interactions. Proteins are unique in their molecular masses, based on the number of amino acids and any modifications that are made to the protein (e.g., glycosylation), and in their properties, more information on basic protein structures and characteristics can be found in Florence and Attwood (2015), Vella (1992) and Brändén et al. (1999); and information on protein therapeutics, in general, can be found in (Leader et al., 2008).

Proteins carry out a variety of physiological functions, for example, enzymes (they are proteins that control the biochemical reactions needed to sustain life processes); hormones (they are proteins that control cellular physiology, growth, and differentiation); transport and storage proteins (e.g., serum albumin) and antibodies (they are proteins for the body immune responses. Accordingly, deficiency or abnormality of the body's endogenous proteins can lead to pathophysiology; hence, the therapeutic proteins are being clinically used to treat various diseases.

Figure 1.4  The primary, secondary, tertiary, and quaternary structures of proteins (ProteinStructure.jpg (546 × 800), n.d.) ProteinStructure.jpg (546 × 800) (bu.edu).

Recombinant proteins form the majority of the therapeutic proteins on the market, and in clinical trials for a variety of intended uses, for example treating cancers, immune disorders, infections, and other diseases. Dimitrov (2012) reviewed the types of therapeutic proteins and classified them according to their pharmacological activity into five different groups. The first group is the proteins used to replace a deficiency or abnormalities of the endogenous proteins e.g., insulin in diabetes mellitus Type I. The second group encompasses the proteins that augment an existing physiological pathway such as erythropoietin in anemia caused by renal failure (Dimitrov, 2012). In addition, therapeutic proteins may provide a novel function or activity as in the case of Botulinum Toxin Type A when it is used as a drug of choice for patients suffering from muscle dystonia (Cloud and Jinnah, 2010). Moreover, some proteins are given to the patients targeted for a particular activity by interfering with a molecule or organisms such as monoclonal antibodies to treat immunity disorders. Finally, some proteins are being used as delivering vehicles for other medications or proteins, e.g., gemtuzumab ozogamicin is used as a conjugate for the treatment of de novo CD33-positive acute myeloid leukemia (AML).

Monoclonal antibodies are complex glycoproteins with distinctive structural features that contribute to their variable and diverse biological functions. Specific carbohydrates can also have an influence on the biological activity of mAbs. For example, by looking into Fig. 1.5, insulin (a peptide hormone) is a small protein that consists of 51 amino acids and its molecular mass is 5.8 kDa; erythropoietin is a larger protein hormone consists of 165 amino acids and its molecular weight is 30.4 kDa, while mAbs or immunoglobulins are larger globular proteins, IgG1 has a molecular mass of about 150 kDa with more than 660 amino acids. Also, the hinge region of IgG1 consists of 15 amino acids and is very flexible. IgG2 and IgG4 have shorter hinges than IgG1, with 12 amino acid residues (Vidarsson et al., 2014). The hinge region joins the Fab, of the heavy chain, and Fc fragment and allows some flexibility for the Fab arms (Figs. 1.6 and 1.7). Hence, mAbs are proteins but have different unique features compared to other proteins.

Figure 1.5  Schematic presentation to show the large size of the mAbs. Insulin: By Theislikerice—Own work, CC BY-SA 4.0, mAbs (Monoclonal antibodies—all you need to know about antibody generation | tebu-bio's blog, 2018).

1.3. Classification, types, and structures of mAb-based therapeutics

Monoclonal antibodies are immunoglobulins (Igs), they present in five class forms or isotypes (IgA, IgD, IgE, IgG, and IgM) (Figs. 1.6 and 1.7).

IgG is the most clinically used antibodies as therapeutics, mainly due to their characteristics (Table 1.1) (Antibody Basics, n.d.) and because they are the most common type of antibodies found in the blood and additionally based on literature they may live longer in the blood. IgGs are globular glycoproteins and are highly site specific. IgG contains two identical light chains of about 25 kDa and two identical γ, gamma, heavy chains of about 50 kDa (Fig. 1.7), thus a tetrameric quaternary structure. The two heavy chains are connected to each other and to a light chain each by disulfide bonds (Fig. 1.7). The resulting tetramer has two similar halves, which together form the Y-like shape. The Fc regions of IgGs bear a highly conserved N-glycosylation site (Cobb, 2020; Parekh et al., 1985).

Figure 1.6  Class identity that is determined by class-specific sequences in the Fc region of the heavy chain which are: alpha-IgA, delta-IgD, epsilon-IgE, gamma-IgG, mu-IgM. Light chains are common among immunoglobulins and occur as two types—kappa, k or lambda, λ (Monoclonal antibodies—all you need to know about antibody generation | tebu-bio's blog, 2018).

Figure 1.7  A schematic representation of a monoclonal antibody (mAb) IgG structure. The N-terminal domain of an IgG consists of a variable (V) region with the complementarity-determining region (CDR) that binds to a specific epitope on antigens. CH, constant domain, heavy chain; CL, constant domain, light chain; COO − , carboxy terminal; Fab, fragment antigen-binding; Fc, fragment crystallisable region, NH 3 + , amino terminal end; S–S, disulfide bond; VH, variable domain, heavy chain; VL, variable domain, light chain.

The heavy chain is divided into a Fab portion, which is at the amino terminal (the arm of the Y) and an Fc portion, which is at the carboxyl terminal (the base of the Y) (Fig. 1.7). Carbohydrate chains are attached to the Fc portion of the molecule, specific carbohydrate chains are attached to the Fc portion of the molecule and can affect the biological activity of the mAb. The Fc portion of the Ig molecule, specifically IgG and IgM regions can bind to receptors on the surface of immunomodulatory cells such as macrophages and stimulate the release of cytokines that regulate the immune response. The Fc region contains determinants unique to the individual classes (Figs. 1.6 and 1.7) as well as protein sequences common to all Igs. These regions are known as the constant regions because they do not vary greatly among different Ig molecules within the same class. The Fab portion of the Ig molecule contains both light and heavy chains joined together by a single disulfide bond. One light and one heavy chain pair combine to form the antigen-binding site of the antibody. Each Ig monomer is able to bind two antigen molecules (Goebl et al., 2008; Shuptrine et al., 2012).

Table 1.1

a  Light chains are present in all Immunoglobulin classes. In humans, k. chains are found 67% of the time, and λ chains are found 33% of the time.

Adopted from Antibody Basics, n.d. Sigma-Aldrich. https://www.sigmaaldrich.com/technical-documents/articles/biology/antibody-basics.html. (Accessed 3 May 2021).

IgG class (Fig. 1.7) presents in four subclasses (IgG1, IgG2, IgG3, and IgG4) (Vidarsson et al., 2014) that are differentiated on the basis of the position of interchain disulfide bonds, size of the hinge region, and molecular weight. The subclasses also vary in their ability to activate complement. Hence, each subclass has unique characteristics, e.g., the ability to target and interfere with cell signaling as well as stimulating the release of CDC (complement-dependent cytotoxicity), ADCC (antibody-dependent cell-mediated cytotoxicity), and ADPh (Antibody-dependent cellular phagocytosis) (Hudis, 2007; Schneider-Merck et al., 2010).

Refer to Table 1.1 for human immunoglobulin properties. Fig. 1.5 shows IgG1 mAbs in its intact folded three-dimensional structure and Fig. 1.7 exhibits the schematic presentation of IgG in some detail.

In cancer therapy, it is becoming increasingly evident that the antitumor effects of antibodies are driven both by the properties of their Fc domains and their antigen-binding regions. The Fc domain (Fig. 1.7) can bind with Fc receptors (FcR) to cause effector functions as will be described later under the mechanism of mAbs actions, Section 1.6. Posttranslational modification of the Fc region can also influence the function of antibodies (Kubota et al., 2009).

Different subclasses of IgG vary in their abilities to facilitate antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). An example includes human IgG2 which can recruit myeloid cells for ADCC but do not activate complement (Schneider-Merck et al., 2010). Human IgG1 can activate complement and recruit immune effector cells for ADCC (Hudis, 2007; Weiner et al., 2010), while human IgG4 does not activate ADCC or CDC.

The Noble Prize was won by Porter and Edelman (1972) for elucidating the structure of antibodies by proteolytic digestion using thiol proteases, e.g., papain. The IgG molecule is cleaved into two Fabs and an Fc (Fig. 1.8) with the use of papain. The Fc is unable to block binding to a specific antigen, while Fab can bind to a specific antigen (Zhao et al., 2009). Fabs have been used in the determination of antibody–antigen interactions, they have better tissue penetration than full-length IgGs. This allows interactions with enzyme sites