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Part IV
Process Engineering, Gene Therapy
and Vaccines
Chapter 6
Metabolic andProcess Engineering
toControl Glycan Structures
forBiopharmaceuticals Produced
inCultured Mammalian Cells
RanyaPranomphon, VijayTejwani, HussainDahodwala,
MontaropYamabhai, andSusanT.Sharfstein
Abstract Biopharmaceuticals (also known as biologics) play an increasing role in
the treatment of a wide range of diseases, particularly cancer, autoimmune diseases,
and infectious diseases. In 2017 and 2018, 11 of the 15 best-selling drugs worldwide were biopharmaceuticals, generally produced in cultured mammalian cells.
The vast majority of biopharmaceuticals are glycoproteins, in which the attached
glycan moieties play important and often critical roles in controlling activity, clearance, and immunogenicity. In addition to glycoproteins, carbohydrates, particularly
glycosaminoglycans (GAGs) such as heparin, the most widely used anticoagulant
drug in the world, are critically important biopharmaceutical products.
Many blockbuster biopharmaceuticals such as adalimumab (Humira), trastuzumab (Herceptin), and bevacizumab (Avastin) have recently come off patent,
providing an opportunity for production of biosimilar versions by companies
other than the innovator. In addition, there is increasing interest in producing
R. Pranomphon
College of Nanotechnology, Science and Engineering, University at Albany, State University
of NewYork, Albany, NY, USA
School of Biotechnology, Institute of Agricultural Technology, Suranaree University
of Technology, Nakhon Rachasima, Thailand
V. Tejwani · S. T. Sharfstein (*)
College of Nantechnology, Science and Engineering, Albany, NY, USA
e-mail: ssharfstein@albany.edu
H. Dahodwala
Institute for Bioscience and Biotechnology Research, University of Maryland,
Rockville, MD, USA
M. Yamabhai
College of Nanotechnology, Science and Engineering, State University of New York,
Albany, NY, USA
K. Gadamasetti, S. A. Kolodziej (eds.), Bioprocessing, Bioengineering
and Process Chemistry in the Biopharmaceutical Industry,
https://doi.org/10.1007/978-3-031-62007-2_6
135© The Author(s), under exclusive license to Springer Nature Switzerland AG 2024
136
R. Pranomphon etal.
glycosaminoglycans from cultured mammalian cells, rather than the current purication from animal tissues with the attendant risks of contamination by adventitious
agents and adulteration due to isolation under non-cGMP conditions. To successfully produce biosimilars and bioengineered GAGs, control of glycan composition
and structures is critical. This control is challenging as glycan synthesis is a nontemplated process, which is controlled by a complex collection of factors including
the glycoprotein being synthesized or the core protein to which the GAG is attached,
production host, enzyme activities, and bioprocess conditions.
In this chapter, we review some basics of glycoprotein and GAG biosynthesis
with a particular focus on our current understanding of how glycan structures are
controlled invivo. We then review studies in which glycan structures for recombinant proteins and glycosaminoglycans have been optimized by cell line metabolic
engineering and bioprocess manipulations. In particular, CRISPR has permitted
exquisite editing of host cells, allowing tailored production of glycan structures,
facilitating the production of biosimilars and bioengineered GAGs as well as setting
the stage for “biobetters” in which improved functionality is obtained by glycoengineering. However, complicated new products including bispecic antibodies, intricate Fc-fusion proteins and molecules not yet envisioned may necessitate new hosts
and further advances in glycoengineering.
Keywords Glycosylation · Glycoengineering · Bioprocessing · Chinese hamster
ovary cells · Monoclonal antibodies · Biosimilars
6.1 Introduction
Biological therapeutics, including monoclonal antibodies (mAbs), vaccines, hormones, and other proteins, play an increasing role in the global pharmaceutical
market, providing novel treatments for a wide range of conditions and indications.
Since 2002 there have been more than 300 biopharmaceutical approvals by the FDA
(U.S.Food and Drug Administration), and this number is continuously growing.
The vast majority of these biologics are produced in mammalian cell culture with
Chinese hamster ovary (CHO) cell lines serving as the preferred host, but other cell
lines including human embryonic kidney cells (HEK293), human retinal cells
(PERC.6), baby hamster kidney (BHK), murine myeloma (NS0) cells, and murine
(SP2/0) hybridoma cells have also been employed [1, 2]. Mammalian cells are utilized due to their ability to produce the appropriate posttranslational modications
including proper folding, glycosylation, phosphorylation, and disulde bond formation, resulting in high-quality proteins [3, 4].
Protein glycosylation is the most common posttranslational modication in
eukaryotes, formed by the attachment of glycans onto proteins [5]. Glycosylation
occurs in the endoplasmic reticulum (ER) and/or the Golgi apparatus including
6 Metabolic and Process Engineering to Control Glycan Structures…
137
N- and O-linked glycosylation, where glycan chains are added to the amide nitrogen of the asparagine residue and hydroxyl group of the serine/threonine residues,
respectively [6–8]. Glycosylation is a complex, nontemplated process, resulting
in heterogeneity in the glycan prole (microheterogeneity) and in glycan siteoccupancy (macroheterogeneity) [9, 10]. Many factors including cell type and
cell engineering, media-feed compositions, and cell culture conditions such as
temperature, pH, and dissolved oxygen and ammonia concentrations can affect
glycan patterns [1, 11, 12]. Glycosylation patterns on therapeutic glycoproteins
signicantly affect biological and physiochemical properties including efcacy,
immunogenicity, invivo half-life, protein conformation, stability, and solubility
[13–15]. Therefore, glycosylation is a critical quality attribute (CQA) of therapeutic proteins [16, 17].
In addition to glycoprotein therapeutics, there is increased interest in the recombinant or bioengineered production of carbohydrate drugs, particularly glycosaminoglycans (GAGs) such as heparin, the most widely used anticoagulant drug
worldwide [18]. GAGs are synthesized and attached to a core protein, and the structure of the GAG is controlled by an extensive biosynthetic pathway consisting of
more than 20 enzymes. Consequently, GAGs are highly heterogeneous, and their
properties are strongly inuenced by the cell line and culture conditions used for
production. The CQAs for GAGs include their sulfation pattern and for heparin, the
anticoagulant activity.
CQAs are dened as physical, chemical, biological, or microbiological properties or characteristics that must be monitored during manufacturing processes and
controlled within an appropriate limit, range, or distribution to ensure the desired
product quality and hence, the safety and efcacy of the drug products [19–21]. Due
to the natural glycan heterogeneity, there is not “one correct glycan,” though there
are certainly undesirable glycans including immunogenic forms described in Sects.
6.4 and 6.5. Moreover, the glycosylation patterns, such as degree of branching,
sialylation and fucosylation have signicant effects on biological activity as
described in Sect. 6.6.3. Consequently, glycoengineering and process engineering
may be performed to achieve a specic biological goal for the therapeutic protein.
Most critical is the need to maintain consistency throughout the product lifetime,
which may include a variety of process changes and manufacturing at many different sites.
In addition to the need to maintain quality attributes in the production of novel
therapeutics, the end of patent protection for many biotherapeutics (e.g., adalimumab (Humira), trastuzumab (Herceptin), and bevacizumab (Avastin)) provides an
opportunity for the production of biosimilars. Biosimilars are off-patent biologic
products that are highly similar but not identical to the originator biologic product
(Reference Biologic) in terms of structure, function, biological activity, immunogenicity, efcacy, and safety due to differences in manufacturing processes [22, 23].
However, in biosimilar production, it is vitally important to approximate the CQAs
of the innovator molecule to ensure efcacy. Signicant effort is often employed
during the process development of biosimilars to match the reference product
including host and cell line selection as well as optimization of bioprocess
138
conditions [24]. Furthermore, CQA identication is a key step in the quality by
design (QbD) approach to product development, a systematic approach that begins
with predened objectives and emphasizes product and process understanding and
process control, based on sound science and quality risk management. QbD has
been implemented for developing and manufacturing biologics, including biosimilars [25–27].
To understand how these steps are being implemented, this chapter provides an
overview of protein glycosylation, strategies for glycoengineering cell lines to control glycan patterns, and a discussion of the effects of process conditions on the
glycosylation prole of therapeutic protein products.
R. Pranomphon etal.
6.2 Biology ofGlycosylation
The addition of oligosaccharides onto a protein is a complex metabolic pathway,
characterized by the en-bloc transfer of polysaccharide chains, as well as the stepwise addition and removal of individual monosaccharides. The number of pathways
traversed is dependent on reaction site accessibility, the enzymatic substrate specicities, as well as spatial localization of the various enzymes and nucleotide-sugar
substrates that are necessary for the reactions to proceed in a particular order
[28, 29].
6.2.1 N-Linked Glycosylation
N-linked glycosylation initiates primarily in the ER of mammalian cells. The entire
pathway in mammalian cells involves a highly complex and interconnected reaction
network, catalyzed by glycosidases and glycosyltransferases contained within different compartments of the ER and Golgi apparatus, as depicted in the schematic in
Fig.6.1. The biosynthesis of mammalian N-glycans initiates at the cytoplasmic face
of the ER membrane with the transfer of a phosphorylated N-acetylglucosamine
(GlcNAc-P) from uridine diphosphate (UDP-GlcNAc) to the dolichol phosphate
(Dol-P) lipid carrier to generate dolichol pyrophosphate N-acetylglucosamine (DolP- P-GlcNAc) [30]. Fourteen sugars are then sequentially added to Dol-P-P-GlcNAc
to form an oligosaccharide precursor (Glc3Man9GlcNAc2) [10]. Next, oligosaccharyltransferase identies Asn-X-Ser/Thr sequons in a nascent polypeptide and
proceeds with an en-bloc transfer of Glc3Man9GlcNAc2 to the side chain amide of
asparagine, releasing Dol-P-P in the process [31]. The glucose (Glc) residues on the
precursor are sequentially trimmed by ER α-glucosidase I and II to form a monoglucosylated glycan, which is a key intermediate in the calnexin/calreticulin-associated
glycoprotein folding control cycle, an ER protein-quality assurance mechanism [32].
6 Metabolic and Process Engineering to Control Glycan Structures…
139
Fig. 6.1 An overview of glycosylation pathway in most eukaryotic cell systems. (Adapted from
[33] with permission from Springer Science+Business Media and from [1] with permission from
Oxford University Press)
Once correctly folded, the precursor is trimmed by ER α-mannosidase I to yield
Man8GlcNAc2-protein before exiting the ER. After translocation into the cisGolgi, the Man8GlcNAc2 glycoform is further trimmed by Golgi α-mannosidases I
to give Man5GlcNAc2, a key intermediate along the pathway to form hybrid and