Book Review: Glycosylation Engineering of Biopharmaceuticals: Methods and Protocols

Biopharmaceuticals represent the fastest growing sector of the pharmaceutical industry, mainly driven by a rapid expansion in the manufacture of recombinant protein-based drugs and monoclonal antibodies as therapeutic agents. Glycosylation is the most prominent posttranslational modification occurring in these protein drugs. Glycosylation of proteins to form glycoproteins is important for 2 reasons. Glycosylation alters the properties of proteins, changing their stability, solubility, and physical bulk. In addition, carbohydrates of glycoproteins act as recognition of signals that are central to aspects of protein targeting and for cellular recognition of proteins and other cells. Glycosylation can involve addition of a few carbohydrate residues or the formation of large branched oligosaccharide chains. Sites and types of glycosylation are determined by the presence on a protein of appropriate amino acids and sequences and by availability of enzymes and substrates to carry out the glycosylation reactions. All antibodies are glycosylated at conserved positions in their constant regions and the presence of carbohydrate can be critical for antigen clearance functions such as complement activation. The structure of the attached carbohydrate can also affect antibody activity. Antibody glycosylation can be influenced by the cell in which it is produced, the conformation of the antibody, and cell culture conditions. These variables should be considered in the design and production of antibodies with selected specificity and function.

Due to the importance of advances in glycoengineering to therapeutic monoclonal antibodies and other proteins, the publication of Glycosylation Engineering of Biopharmaceuticals, edited by Alain Beck, is notable and timely. The 355-page book is organized into 4 parts that contain 22 chapters contributed by 68 scientists from the United States, France, Belgium, and Sweden.

Glyco-Engineering of Therapeutic Proteins (Part I) is discussed in first 5 chapters of the book. Chapters 1 and 2 describe the use of Chinese Hamster Ovary (CHO) cells for producing recombinant glycoproteins. Linda Lindburg and colleagues used CHO cells adapted to serum-free conditions for generating stable transfectants for production of O-glycans on recombinant mucin-type immunoglobulin fusion proteins. The investigators discuss numerous benefits and drawbacks of different protocols that could influence the experimental results. Nassimal El Mai et al employed ST6Gall minigenes to optimize the ST6Gall sialyltransferase activity and used them to engineer ST6(+)CHO cells. These constructs have proven to equip cell clones with efficient transfer activity of 6-linked sialic acid (ie, human-like glycosylation of therapeutic glycoproteins based on 6 linked sialylation). Chapters 3 to 5 focus on the use of nonmammalian cells (yeasts and insects) for producing glycoproteins. In Chapter 3, Dongxing Zha describes protocol for using glycoengineered Pichia to produce monoclonal antibodies. The chapter covers monoclonal antibody expression in yeast including vector construction, yeast transformation, high-throughput strain selection for fermentation, antibody purification, and characterization. In Chapter 4, Christelle Arico and colleagues describe GlycodExpress technology for production of therapeutic recombinant glycoproteins with humanized and homogeneous N-glycan moieties in Saccharomyces cerevisiae. Finally, in Chapter 5, Sylvie Juliant et al provide methods for engineering the Baculovirus genome for producing galactosylated antibodies in lepidopteran cells.

To assess the structure of glycoproteins and glycoengineered biopharmaceuticals, state-of-the-art analytical methods are needed. Mass spectrometry (MS), capillary electrophoresis (CE), ultracentrifugation, and chromatography methods are discussed in Part II, Glycoanalytics (Chapters 6–15). In Chapter 6, Elsa Wagner-Rousset and coworkers depict a protocol for nano liquid chromatography (LC)–MS/MS analyses of the proteolytic digest from the heavy chain of A2CHM recombinant antibody, with graphitized carbon LC chips (43 mm length). Marie-Claire Janin-Bussat et al (Chapter 7) then describe a rapid electrospray ionization–MS method for characterization of Cetuximab glycosylation. Cetuximab is a potent chimeric mouse/human antibody approved worldwide for the treatment of colon and head and neck cancers. In Chapter 8, Arnaud Delobel et al give a contract research organizations perspective for batch release using high-performance LC (HPLC) and MS methods for determining N-glycans profiling, monosaccharides, and silalic acids for Trastuzumab (Herceptin)-humanized monoclonal antibody produced in CHO cells. Analysis of O-linked glycosylation is a major challenge during structural validation of recombinant glycoproteins. To address this important question, Diarmuid Kenny and colleagues (Chapter 9) present MS protocols for analyzing native and permethylated O-glycans from mucin-type immunoglobulin fusion proteins expressed in mammalian, insect, and yeast cells.

In complement to MS, glycoprofiling can also be performed by HPLC and by electrophoresis-based methods. T. Shantha Raju (Chapter 10) presents details on assessment of Fc glycan heterogeneity of therapeutic recombinant monoclonal antibodies using normal phase (NP) HPLC. This investigator claims that NP-HPLC method resolves both natural and sialylated glycans, thus enabling the user to obtain a broad heterogeneity profile of Fc glycans in a single run. In Chapter 13, Richard Rustandi discusses hydrophobic interaction chromatography (HIC) for analyzing glycoproteins. This method illustrates the power of silica-based HIC HPLC column for separating full intact monoclonal antibodies with relatively high resolution. In addition, Richard Rustandi and coworkers also describe use of CE separation such as CE–sodium dodecyl sulfate gel technology (Chapters 11 and 12) for characterizing glycoproteins. In Chapter 14, Ludovic Landemarre and Eric Duverger examine the use of the novel lactin array method for the study of recombinant human therapeutic interleukin 7 (rhIL-7). This specific method allows for the analysis of glycan motifs, distribution of glycoforms population, and detection of potential immunogen glycans in rhIL-7 purified CHO-produced batches. The use of sedimentation velocity analytical ultracentrifugation (SV-AUC) for analysis of therapeutic monoclonal antibodies has become widespread throughout the biopharmaceutical industry. W. Blaine Stine describes the basic principles of designing, collecting, and analyzing data using SV-AUC for monoclonal antibodies in Chapter 15.

Glycoproteins complexes can be characterized by mass spectrophotometry methods which are discussed in Part III. In Chapter 16, Cedric Atmanene and colleagues describe noncovalent MS technologies including automated chip-based nanoelectrospray ionization–MS to study the formation of immune complexes involving mumAb 6F4 directed against human JAM-A, a newly identified antigenic protein overexpressed in tumor cells. In Chapter 17, Damian Houde and John Engen present a detailed protocol for conformational analysis of antibodies in hydrogen/deuterium exchange MS. This method can provide information about immunoglobulin G1 (IgG1) conformation in solution at peptide-level resolution, purification and formulation procedure, and changes in storage condition. Antibody epitope mapping (binding sites of monoclonal antibodies on target antigens) has emerged as an extremely useful tool in determining the uniqueness and mechanism of action of neutralizing monoclonal antibodies. In Chapter 18, Victor Obungu et al describe matrix-assisted laser desorption/ionization time of flight and LC/MS for epitope mapping of an anti-FasL antibody.

The last portion of the book, Part IV (Chapters 19-22), is entitled PK/PD Assays for Therapeutic Antibodies. Sensitive methods for analyzing biological samples are required to evaluate the impact of glycosylation of biopharmaceuticals. Toward this end, Matthieu Broussas and coworkers in Chapter 19 present an alternate method to the standard ⁵¹Cr release assay for antibody-dependent cellular cytotoxicity (ADCC) using nonradioactive assay based on the measurement of lactate dehydrogenase release. Human natural killer cells, purified by flow cytometry methods, were used as effector cells to trigger ADCC against target tumor cells in vitro. In Chapter 20, Lucile Broyer et al discuss a nonradioactive method for assessing complement-dependent cytotoxicity using a luminescence method based on adenosine triphosphate measurement to estimate tumor-damaged cells and a flow cytometry method for evaluating C1q/C4b binding. As a surrogate in vitro assay, in Chapter 21 Thierry Champion and Alain Beck describe a method for chemical coupling of protein A to a sensor chip, which is used for capturing purified antibodies or from crude cell media supernatants and used as the ligand. The chips are then used for a kinetic study of h-IgG–FcγR interaction using surface plasmon resonance-based biosensor technology. In the final chapter of the book (Chapter 22), Francois Becher and colleagues examine a MS protocol for quantification of monoclonal antibody in serum from a clearance study. The limit of quantification of the assay was 20 ng/mL in serum.

Overall, the book is well written and it provides the reader with current state of the scientific information through introductions backed by published references followed by detailed methods and protocols for glycoanalysis, functional assays, and pharmacokinetic/pharmacodynamic assays for glycoengineered biopharmaceuticals. The book is easy to read with excellent illustrations, figures, tables, and print quality. The chapter end notes about benefits and drawbacks of procedures used also prove to be useful in helping the reader grasp the subject matter. The book serves as an excellent educational resource for medicinal chemists, biochemists, biologists, and other biopharmaceutical scientists. I highly recommend it for those working in the field of glycosylation engineering of recombinant protein-based drugs and monoclonal antibodies.