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Introduction to Biotherapeutics

Jennie R. Lill

Department of Microchemistry, Proteomics & Lipidomics, Genentech Inc., South San Francisco, CA, USA

Abbreviations

ADAs

antidrug antibodies

ADC

antibody-drug conjugate

ADCC

antibody-dependent cell-mediated cytotoxicity

CDR

complementary-determining region

Fab

antigen binding fragment

Fc

cystallizable fragment

NMR

nuclear magnetic resonance

PEG

polyethyleneglycol

PTM

posttranslational modification

1.1 Introduction

Biotherapeutics, also known as biologics, include protein-based and nucleic acid-based drugs that are commonly derived by recombinant expression in living organisms although a few are made by chemical synthesis. This book focuses on the characterization of protein-based biotherapeutics, exploring the various analytical technologies that have enabled in-depth molecular characterization while discussing current triumphs and limitations.

The first human protein therapeutic derived from recombinant DNA technology was human insulin (Humulin®) created at Genentech, developed by Eli Lilly, and approved by the US Food and Drug Administration (FDA) in 1982. Since that time, major advancements in both recombinant DNA technology and recombinant protein production have contributed to the development of several hundred biotherapeutics [1] including relatively simple molecules such as interferons, insulin, and the human growth hormone to more complexly engineered moieties including ADCs such as trastuzumab emtansine [2] and brentuximab vedotin [3].

Unlike conventional small molecule (chemical) drugs such as aspirin, antibiotics, and various chemo-therapeutics, the manufacturing process for biotherapeutics is typically far more cumbersome as they are larger compounds with more complex structures and their production can be extremely sensitive to changes in fermentation and environmental conditions. In addition, biotherapeutics are often less stable than many small molecules and can be prone to aggregation [4] or deamidation, oxidation, and other modifications [5]. Since the manufacturing of biotherapeutics is often dependent upon the host cells of living organisms, complex process development is required to ensure reproducible fermentations, isolation, and characterization [6].

1.2 Types of Biotherapeutics and Manufacturing Systems

There are several different types of marketed biotherapeutics including antibody-based drugs, anticoagulants, blood factors, bone morphogenetic proteins, engineered protein scaffolds, enzymes, Fc (cystallizable fragment) fusion proteins, growth factors, hormones, interferons, interleukins, and thrombolytics (Figure 1.1).

Figure 1.1 Various categories of the main types of biotherapeutics currently marketed.

Source: Carter [7]. Reproduced with permission of Elsevier.

Antibody-based drugs represent the largest and most rapidly expanding class of biotherapeutics [1]. Figure 1.2 shows the diverse mechanisms by which the antibody structure can be modified to increase its biotherapeutic potential.

Figure 1.2 Monoclonal antibody (mAb) structure can be modified on the basis of the desired mechanism of action. Immunoglobulin G1 (IgG1) is the most effective naturally occurring human IgG isotype at mediating antibody-dependent cell-mediated cytotoxicity (ADCC). Glycomodified afucosylated mAbs (part a) (such as Obinutuzumab) demonstrate enhanced binding to IgG Fc receptors (Fc?Rs) and enhanced ADCC. In addition antibody-dependent cellular phagocytosis, a process mediated by macrophages, can also occur [8]. Afucosylated mAbs are produced using cell lines that lack the enzymes responsible for fucosylation. Modifying the amino acid sequence of mAb Fc (part b), as was done to produce ocaratuzumab [9], can also result in enhanced binding to Fc?Rs and enhanced ADCC. For mechanisms of action in which ADCC is not desirable, IgG4 may be a more appropriate isotype, as IgG4 mAbs do not mediate ADCC to the same degree as IgG1 (part c) although this isotype can still engage macrophage effector function via nanomolar affinity binding to Fc?RI. Nivolumab, an IgG4 mAb that blocks programmed cell death protein 1 (PD1) on T cells, is one such example. Producing radioimmunoconjugates involves linking the radioisotope to the mAb. A stable linker is most desirable (part d) to limit the leakage of the free radioactive isotope. Conversely, optimal antibody-drug conjugates (ADCs) use a cleavable linker (part e). To avoid nonspecific toxicity, it is desirable for drugs used in ADCs to be cytotoxic once inside the target cell but nontoxic when bound to the mAb in the circulation. Linkers that are pH-sensitive or enzymatically cleaved are now a standard component of ADCs. Chimeric antigen receptor (CAR) T cells get their specificity from mAb variable regions but are a form of gene, not protein, therapy. They are produced by inserting DNA coding for the mAb variable region fused to DNA coding for signaling peptides into T cells (part f). Some bispecific antibodies lack a functional constant region so that they do not nonspecifically crosslink activating receptors and activate T cells (part g). The lack of a constant region on such constructs results in a short half-life, thus requiring continuous infusion to achieve the desired exposure.

Source: Weiner [10]. Reproduced with permission of Nature Publishing Group.

Humanized and other chimeric versions of these antibodies now dominate the market [11] and in the past 5 years have accounted for nearly 30% of all approvals. Various antibody isotypes are now being explored to provide a wealth of functional diversity that is present through the various IgG subclasses that can be exploited to improve clinical safety and performance by increasing stability, reducing adverse events, modulating effector functions, and by the engagement of two antigens by a single antibody [8]. Several variants that have been Fc engineered for reduced effector function have entered the clinic, for example, Eculizumab, a novel engineered IgG isotype, IgG2m4, with reduced Fc functionality. IgG2m4 is engineered based on the IgG2 isotype with four key amino acid residue changes derived from IgG4 (H268Q, V309L, A330S, and P331S). This antibody was demonstrated to have an overall reduction in complement and Fc gamma receptor binding in in vitro binding analyses while maintaining the normal in vivo serum half-life in rhesus [12].

Biosimilars (biologically identical antibodies, for example) and so-called biobetters (moieties with improved properties such as pharmacodynamic (PD) and pharmacokinetic (PK) readouts, higher potency, longer half-lives, and less immunogenicity, for example) are also starting to emerge, which presents new challenges in terms of testing for the presence of liabilities such as degradative properties, changes in immunogenicity through addition of novel contaminant proteins from new manufacturing processes, and so on. New formats such as glucagon-like peptide 1GLP fused proteins, for example, Eperzan (albiglutide) [13], and PEGylated proteins such as Plegridy (e.g., peginterferon beta-1a) [14] offer improved PK or PD properties but also increased analytical challenges due to their larger masses and increased heterogeneity.

Typically, expression of non-mAb biotherapeutics has been performed in Escherichia coli or a noneukaryotic system. This has many advantages for biotherapeutics that are not reliant on PTMs for their optimal activity. Over the years, however, there has been a gradual increase in the prevalence of mammalian expression systems. Of the mammalian expression systems, the Chinese hamster ovary (CHO) cell-based model (reviewed by Krawitz and Sandoval in Ref. [11]) remains the most employed expression system with a smaller percentage of therapeutics manufactured in other mammalian cell lines such as the murine myeloma line, NSO, and baby hamster kidney cells [15, 16]. Nonmammalian eukaryotic expression systems such as yeast [17] are also utilized, each again presenting their own challenges with regard to the correct PTM of the protein, occasionally adding to adverse properties [18, 19].

More recently transgenic animal production systems (e.g., expression of recombinant products in the animals' milk [19, 20], rabbits, and goats) have been explored as a means of biopharmaceutical production although to date there are many challenges associated with this type of biotherapeutic production with few benefits. Throughout this book the challenges of characterizing both the biotherapeutic moiety itself and the contaminant proteins such as CHO-derived proteins are discussed.

1.3 Types of Analyses Performed

Throughout this book a variety of analytical procedures are described. Many of them have been implemented for characterizing biotherapeutic molecules for as long as these moieties have existed. Others have evolved as the need arises. One such example of developing such sets of tools to answer a newly arisen problem is for the de novo sequencing of antibodies [21, 22]. Occasionally antibodies are discovered that are of great interest for preclinical testing, for which the cDNA or any genetic information is not available. In these scenarios, researchers have to sequence...