Production platforms for biotherapeutic glycoproteins 147
Biotechnology and Genetic Engineering Reviews - Vol. 28, 147-176 (2012)
*To whom correspondence may be addressed (email@example.com)
Abbreviations: ADA, anti-drug antibody; alpha-Gal, Galα1-3Galb1-(3)4GlcNAc; bp, base pair; BHK, baby
hamster kidney; BSA, bovine serum albumin; CHO, Chinese hamster ovary; CMAH, CMP-N-acetylneu-
raminic acid hydroxylase; CMP, Cytidine monophosphate; DMB, fluorphore 1,2-diamino-4,5-methylene-
dioxybenzene; ELISA, Enzyme-linked immunosorbent assay; EPO, Erythropoietin; Fc, Fragment, crystal-
lizable of Ig; FDA, Food and Drug Administration; Gal, galactose; GlcNAc, N-acetylglucosamine; hEScs,
human embryonic stem cells; HD, Hanganutztiu-Deicher; HPLC, High-performance liquid chromatography;
Ig, immunoglobulin; KO, knock-out; Mab, monoclonal antibody; Man, mannose; MEFs, mouse embryonic
fibroblasts; Neu5Ac, N-acetylneuraminic acid; Neu5Gc, N-glycolylneuraminic acid; PEG, polyethylene
glycol; Sia, sialic acid; and, TBA, thiobarbituric acid.
Production platforms for biotherapeutic
glycoproteins. Occurrence, impact, and
challenges of non-human sialylation
DARIUS GHADERI1, MAI ZHANG1, NANCY HURTADO-ZIOLA1, AND AJIT
1Sialix, Inc. 1396 Poinsettia Ave. Vista, CA 92081-8504 USA; 2Glycobiology
Research and Training Center, Departments of Medicine, and Cellular &
Molecular Medicine, University of California, San Diego, La Jolla, CA, 92093,
One of the fastest growing fields in the pharmaceutical industry is the market
for therapeutic glycoproteins. Today, these molecules play a major role in the
treatment of various diseases, and include several protein classes, i.e., clotting
factors, hormones, cytokines, antisera, enzymes, enzyme inhibitors, Ig-Fc-Fusion
proteins, and monoclonal antibodies. Optimal glycosylation is critical for therapeutic
glycoproteins, as glycans can influence their yield, immunogenicity and efficacy,
which impact the costs and success of such treatments. While several mammalian
cell expression systems currently used can produce therapeutic glycoproteins that are
mostly decorated with human-like glycans, they can differ from human glycans by
presenting two structures at the terminal and therefore most exposed position. First,
natural human N-glycans are lacking the terminal Galα1-3Gal (alpha-Gal) modification;
148 D. GhaDeri et al.
and second, they do not contain the non-human sialic acid N-glycolylneuraminic
acid (Neu5Gc). All humans spontaneously express antibodies against both of these
glycan structures, risking increased immunogenicity of biotherapeutics carrying
such non-human glycan epitopes. However, in striking contrast to the alpha-Gal
epitope, exogenous Neu5Gc can be metabolically incorporated into human cells and
presented on expressed glycoproteins in several possible epitopes. Recent work has
demonstrated that this non-human sialic acid is found in widely varying amounts on
biotherapeutic glycoproteins approved for treatment of various medical conditions.
Neu5Gc on glycans of these medical agents likely originates from the production
process involving the non-human mammalian cell lines and/or the addition of animal-
derived tissue culture supplements. Further studies are needed to fully understand the
impact of Neu5Gc in biotherapeutic agents. Similar concerns apply to human cells
prepared for allo- or auto-transplantation, that have been grown in animal-derived
tissue culture supplements.
Therapeutic proteins play an increasingly important role in the pharmaceutical
industry, achieving annual total sales of ~$48 billion in 2009 (Aggarwal, 2010). Unlike
in the past, therapeutic proteins are now given to patients with a whole variety of
disease conditions, sometimes in high milligram quantities per dose. They represent
an integrated part of treatment for various cancer types, autoimmune diseases, and
replacement therapies such as enzyme and hormone substitutes. Among the biggest
blockbusters in the biopharmaceutical industry are therapeutic proteins like Insulin
(Lantus®; Sanofi Aventis), Erythropoietin (EPO, Epogen®; Amgen), and the chimeric
IgG1• monoclonal antibody Infliximab (Remicade®; Centocor Ortho Biotech Inc.)
with annual sale volumes of $2.6, $3.2, and $3.2 billion each in 2009, respectively
(Aggarwal, 2010). Unlike insulin (which is not a glycoprotein), EPO, Infliximab and
the vast majority of therapeutic proteins require posttranslational modification with
N-glycans and less frequently, O-glycans.
Glycosylation is a very critical modification of therapeutic proteins, known to
significantly modulate yield, bioactivity, solubility, stability against proteolysis,
immunogenicity, and clearance rate from circulation (Arnold et al., 2007; Durocher and
Butler, 2009; Higgins, 2009). This review begins with an overview about established and
potentially new production platforms for such recombinant biotherapeutic glycoproteins,
highlighting critical differences in the glycosylation patterns among available expression
systems. Depending on the source, the glycosylation pattern of the recombinant protein
product varies greatly: starting with bacterial systems that do not glycosylate, followed
by yeast, plants and insect cell systems generating immunogenic glycan types that are
absent in humans, to mammalian systems with human-like complex glycans (Figure
1). Significant progress has been made over the past decade to overcome the current
limitations of non-mammalian expression systems by glycoengineering approaches to
achieve expression of human-like glycosylation patterns (Durocher and Butler, 2009;
Jacobs and Callewaert, 2009). Thus, non-mammalian expression systems might become
more prevalent for therapeutic glycoprotein production in the future. But currently,
production of therapeutic glycoproteins is dominated by mammalian production
platforms with their natural ability to express human-like glycosylation.
Production platforms for biotherapeutic glycoproteins 149
Figure 1. Non-human glycan structures in available glycoprotein expression systems. (Modified from
Cummings and Doering, 2009). Eukaryotic cells share the ability to modify proteins by N-glycosylation.
Shown are common examples of N-glycan structures occurring in different conceivable production platforms
for biotherapeutic glycoproteins. Among eukaryotic cells, the first steps of N-glycosylation occur in the
endoplasmatic reticulum (ER). The glycosylation machinery of the ER is highly conserved between all
species and results in the biosynthesis of the common Man3GlcNAc2 core structure. All further modifications
of the N-glycan core take place in the Golgi apparatus whereupon the glycosylation repertoire varies greatly
among species. For example, yeast mainly expresses high-mannose glycan structures harboring up to 100
mannose residues in different linkages. By contrast, most N-glycans found on insect cell proteins belong
to the paucimannosidic type which represents the core structure, but further modifications by additional
mannose, fucose, and galactose residues have been found to a lesser extent. Higher plants even synthesize a
significant portion of complex type glycans with two antennae. However, non-human highly immunogenic
xylose residues occur with a high frequency. By contrast, animals mainly express multiantennary complex
type N-glycans and carry sialic acids at outermost positions of glycan chains. But humans have lost
the ability to synthesize two of the major mammalian glycan epitopes, Gala1-3Gal (alpha-Gal) and
N-glycolylneuraminic acid (Neu5Gc). Normal humans have antibodies directed against these structures.
However, two critical differences have been identified between humans and most
other mammals: humans have lost the ability to biosynthesize both the terminal
Gal•1-3Galb1-(3)4GlcNAc (alpha-Gal) epitope, and a major mammalian sialic acid,
N-glycolylneuraminic acid (Neu5Gc), structures that are widely present on non-human
mammalian cells (Padler-Karavani and Varki, 2011). These non-human glycan epitopes
can potentially affect immunogenicity and/or efficacy of therapeutic glycoproteins in
a disadvantageous manner for the patient, since all humans tested have circulating
antibodies against them (Padler-Karavani and Varki, 2011). In contrast to the alpha-Gal
epitope, Neu5Gc can even be taken up by human cells and metabolically incorporated
into cell surface glycoconjugates, which occurs despite the presence of a circulating
polyclonal anti-Neu5Gc antibody response (Tangvoranuntakul et al., 2003; Nguyen
et al., 2005; Padler-Karavani et al., 2008; Padler-Karavani et al., 2011). This makes
Neu5Gc the first example of a “xeno-auto-antigen” (Padler-Karavani and Varki, 2011;
Varki et al., 2011).
150 D. GhaDeri et al.
Notably, pre-clinical trials in small animals to test for efficacy, potential side effects,
and immunogenicity of a putative new drug cannot study consequences of the presence
of immunoreactive Neu5Gc because organisms such as mice, rats, or rabbits are
characterized by the natural occurrence of Neu5Gc. Thus, alternative modified animal
models need to be developed to analyze Neu5Gc-dependent immunoreactivity. One
available system is the Cmah knockout mouse, which is Neu5Gc-deficient (Hedlund
et al., 2007) and can be induced to express anti-Neu5Gc antibodies at human-like
levels (Padler-Karavani et al., 2011; Taylor et al., 2010). Recent work confirmed that
immunoreactive Neu5Gc is not only found in biotherapeutic glycoproteins approved
for treatment of various medical conditions, but also that its presence can reduce
half-life and increase immunogenicity in the Cmah knockout mouse model (Ghaderi
et al., 2010). In the following review, we will focus on the occurrence and impact of
non-human sialylation patterns on recombinant biotherapeutic glycoproteins derived
from mammalian cells. Table 1 summarizes current FDA-approved biotherapeutic
glycoproteins and rates their chance of carrying critical immunoreactive Neu5Gc
depending on the expression host (Table 1). Besides recombinant expression of
biotherapeutic glycoproteins in mammalian cells, the issue of Neu5Gc-contamination
also applies for recombinant glycoproteins derived from transgenic animals and
animal plasma, as well as for human stem cells if maintained on non-human feeder
cell layers and/or cultured in the presence of animal sera or animal-derived cell culture
supplements. Finally, this review will discuss the currently available, highly sensitive
methods to detect Neu5Gc-contamination and outline promising, cost-efficient
approaches to reduce and/or eliminate Neu5Gc-contamination from recombinant
Glycosylation properties of available production platforms for biotherapeutic
BACTERIAL EXPRESSION SYSTEMS. These have limitations in glycoprotein
production due to the complete lack of the enzymatic machinery and compartmentalization
required for mammalian-type glycosylation. However, several non-glycosylated
biotherapeutic proteins are currently produced in prokaryotes including monoclonal
antibodies, hormones, enzymes, and cytokines. Moreover, several FDA-approved
biotherapeutics produced in bacteria are chemically modified with covalently linked
polyethylene glycol (PEG) chains, such as Cimzia® Certolizumab pegol by UCB and
Lucentis® Ranibizumab by Genentech Inc (DeFrees et al., 2006). This has proved
to be successful in improving therapeutic capability, by prolonging drug half-life,
enhancing stability, and reducing immunogenicity caused by the lack of glycosylation.
Also, approaches involving the engineering and functional transfer of the required
glycosylation machinery into prokaryotes to allow synthesis of mammalian-like
N-glycans in bacterial systems has made significant progress over recent years (Wacker
et al., 2002; Schwarz et al., 2010; Nothaft and Szymanski, 2010). However, additional
improvements are needed to establish a cost-efficient, reliable system.
YEAST EXPRESSION SYSTEMS. This expression system is attractive as yeasts
may be cultured in chemically defined media, have the ability to multiply rapidly to
high densities, have well characterized glycosylation machineries, and the ability
Production platforms for biotherapeutic glycoproteins 151
Table 1. Overview of currently FDA-approved therapeutic glycoproteins derived
from different mammalian sources and the likelihood for Neu5Gc contamination.
AgentMarketing Company Source Chance of
Genentech Inc., Hoffmann-La Roche
Genentech Inc., Hoffmann-La Roche
Simponi® Golimumab Centocor Ortho Biotech Inc., Merck &
Xolair® OmalizumabGenentech Inc., Novartis Pharmaceut.
Corp. Tanox Inc.
Yervoy® Ipilimumab BMS
Soliris® EculizumabAlexion Pharmaceuticals, Inc
Benlysta® Belimumab Human Genome Sciences Inc.
Synagis® PalivizumabAbbott Labs, MedImmune Inc.
Tysabri® Natalizumab Élan Pharmaceut., Biogen Idec.
Erbitux® CetuximabImClone Systems, BMS
Ilaris® CanakinumabNovartis Pharmaceuticals
Remicade® Infliximab Centocor Ortho Biotech Inc.
Reopro® AbciximabCentocor Ortho Biotech Inc.,
Eli Lilly & Co.
Simulect® Basiliximab Novartis Pharmaceuticals Corp.
Genentech Inc., Hoffmann-La Roche Ltd CHO++
Abbott Laboratories CHO++
Genentech Inc, Biogen Idec
Centocor Ortho Biotech Inc. CHO++
++Biogen Idec., Bayer Schering Pharma
Centocor Ortho Biotech Inc.Murine
+++ Wyeth Pharmaceuticals
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