ISSN 1860-6768 · BJIOAM 7 (12) 1421–1532 (2012) · Vol.

7 · Dezember 2012

Systems & Synthetic Biology ·

Nanobiotech · Medicine

12/2012 Cover illustration

Biopharmaceuticals
Purification
Protein engineering
Antibodies

Special issue Biopharmaceuticals

Drug discovery and production are highly interlinked, because the main product
attributes need to be defined early on. This Special issue edited by Alois Jung-
bauer (University of Natural Resources and Life Sciences, Vienna, Austria) and
Klaus Graumann (Sandoz GmbH, Kundl, Austria) covers several areas relevant
to the discovery, development and manufacturing of biopharmaceuticals.
Topics include antibodies, protein engineering and purification.
Images: Antibodies © Elena Pankova – Fotolia.com; Biopharmaceutical Produc-
www.biotechnology-journal.com
tion © Sandoz.

Biotechnology Journal – list of articles published in the December 2012 issue.

Review Research Article
Antigen generation and display in therapeutic antibody Periodic counter-current chromatography – design and
drug discovery – a neglected but critical player operational considerations for integrated and continuous
Hilmar Ebersbach and Sabine Geisse purification of proteins
http://dx.doi.org/10.1002/biot.201200066 Rahul Godawat, Kevin Brower, Sujit Jain, Konstantin
Konstantinov, Frank Riske and Veena Warikoo
Mini-Review http://dx.doi.org/10.1002/biot.201200068
Current strategies in antibody engineering: Fc engineering
and pH-dependent antigen binding, bispecific antibodies Technical Report
and antibody drug conjugates Endotoxin removal and prevention for pre-clinical biologics
Karen J. Vincent and Mauro Zurini production
http://dx.doi.org/10.1002/biot.201200250 Anne Serdakowski London, Brendan Kerins,
William R. Tschantz and Kasey Mackay
Review http://dx.doi.org/10.1002/biot.201200220
Structure-function studies with G protein-coupled receptors
as a paradigm for improving drug discovery and development Technical Report
of therapeutics A high-throughput assay for enzymatic polyester hydrolysis
Patrick M. McNeely, Andrea N. Naranjo and activity by fluorimetric detection
Anne S. Robinson Ren Wei, Thorsten Oeser, Susan Billig and Wolfgang
http://dx.doi.org/10.1002/biot.201200076 Zimmermann
http://dx.doi.org/10.1002/biot.201200119
Review
The sweet tooth of biopharmaceuticals: Importance Perspective
of recombinant protein glycosylation analysis How can measurement, monitoring, modeling and control
Nico Lingg, Peiqing Zhang, Zhiwei Song and advance cell culture in industrial biotechnology?
Muriel Bardor Manuel J. T. Carrondo, Paula M. Alves, Nuno Carinhas,
http://dx.doi.org/10.1002/biot.201200078 Jarka Glassey, Friedemann Hesse, Otto-Wilhelm Merten,
Martina Micheletti, Thomas Noll, Rui Oliveira, Udo Reichl,
Review Arne Staby, Ana P. Teixeira, Henry Weichert and
Minimizing immunogenicity of biopharmaceuticals Carl-Fredrik Mandenius
by controlling critical quality attributes of proteins http://dx.doi.org/10.1002/biot.201200226
Miranda M.C. van Beers and Muriel Bardor
http://dx.doi.org/10.1002/biot.201200065

Review
Multimodal chromatography: An efficient tool in downstream
processing of proteins
Kristian Kallberg, Hans-Olof Johansson and Leif Bulow
http://dx.doi.org/10.1002/biot.201200074

© 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.biotechnology-journal.com

Biotechnol. J. 2012, 7, 1433–1443 DOI 10.1002/biot.201200066 www.biotechnology-journal.com

Review

Antigen generation and display in therapeutic antibody drug

discovery – a neglected but critical player

Hilmar Ebersbach1 and Sabine Geisse2

1 NBC/NT, Novartis Institutes for BioMedical Research, Basel, Switzerland
2 NBC/PPA, Novartis Institutes for BioMedical Research, Basel, Switzerland

Disease intervention by targeting a critical pathway molecule through a blocking antibody or

Received 08 JUN 2012
interference by therapeutic proteins is currently en vogue. Generation of blocking antibodies or Revised 26 AUG 2012
therapeutic proteins inevitably requires the production of recombinant proteins or cell-based Accepted 25 SEP 2012
immunogens. Thus, one could call the antigen molecule the neglected player in antibody drug dis-
covery. The variety of methods available for making recombinant proteins or recombinant cell lines
that present the target on the cell surface is extensive. These need to be addressed in conjunction
with biochemical and biophysical quality criteria and the experimental application intended. Fun-
damentally, successful production and isolation of monoclonal antibodies requires optimized
antigen preparation and presentation to the immune host. This review summarizes the most im-
portant aspects of antigen generation and display, enabling logical decision making to give rise to
potent high-affinity antibodies.

Keywords: Antigen preparation · Expression systems · Immunization · Phage display · Quality parameters

1 Introduction preparation and its presentation because the underlying

Recently, considerable progress has been made in gener-
ating full antibodies, fragments thereof, or other function-
ally blocking molecules based on various scaffolds as bio-
pharmaceuticals, and the different routes to achieve this
have been extensively discussed in the public domain.
However, none of the reviews [1–5] recently compiled
mention the generation of antigen material in sufficient
quantity and quality; a crucial aspect that is clearly neg-
lected.
Fundamental to the successful isolation of a mono-
clonal antibody (mAb) or binding molecule is antigen
Correspondence: Dr. Hilmar Ebersbach, NBC/NT, Novartis Institutes for
BioMedical Research, Novartis Campus, WSJ-506.313A, CH-4002 Basel,
Switzerland
E-mail: Hilmar.Ebersbach@novartis.com
Abbreviations: CHO, Chinese hamster ovary; FACS, fluorescence-activated
cell sorting; GPCR, G-protein coupled receptor; HEK293, human embryon-
ic kidney cell line; LAL, limulus amoebocyte lysate; mAb, monoclonal anti-
body; SEC, size-exclusion chromatography; MALS, multiangle light scatter-
ing
main principle of immunization is the presentation of the
antigen to the immune host in a format that elicits the
strongest and, at the same time, most specific immune
response. Therefore, the quality, integrity, and folding
state of an antigen protein are essential parameters for
successful antibody generation and its presentation
should mimic the situation encountered in vivo as close-
ly as possible.
Yet, the nature and inherent characteristics of target
molecules selected for detection or intervention by anti-
bodies or related molecules vary considerably, and like-
wise, the formats and production modes can vary as well.
In this review, several approaches to antigen generation
are discussed and summarized with particular attention
paid to their application and product quality.
2 Soluble antigen generation by chemical
synthesis or recombinant expression
The vast majority of antigens currently under investiga-
tion comprise soluble protein entities, such as full-length
proteins (such as cytokines), sub-domains of soluble pro-

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teins, or full extracellular domains of cell-surface recep-
tors (such as G-protein coupled receptors, GPCRs). Tech-
niques such as chemical peptide synthesis as well as the
entire repertoire of recombinant protein expression tech-
nologies in prokaryotic and eukaryotic hosts can be
applied to generate these molecules and are summarized
in the following sections. Two considerations should
govern the decision for one expression technology over
another: (i) the nature of the molecule to be expressed
and (ii) its biochemical and biophysical characteristics.
The yield should be a homogenous, properly folded and
displayed antigen such that the critical epitopes are ac-
cessible.
2.1 Synthetic peptide antigens
Synthetic peptides are often used as immunogens, in that
they represent surrogates for protein domains or even
complete proteins. This approach is applied in hybridoma
generation, for example, because it is fast, straightfor-
ward, and comes into play when the required amount of
soluble, functionally active antigen is difficult to produce
by recombinant expression technologies. Moreover, for
cell-surface receptors, representing a class of highly
interesting targets for antibody intervention, peptide
antigen mimics have been successfully used in immu-
nization or phage display [6, 7]. Yet, the selection of suit-
able peptides derived from cell-surface receptors provides
challenges. Apart from immunogenic attributes, they also
need to have the appropriate conformational properties to
enable selection of mAbs capable of recognizing the
native antigen displayed on the cell surface, in particular,
if the retrieval of functionally blocking antibodies is
envisaged. Detailed information on all aspects that should
be considered when working with peptide antigens was
nicely summarized in a recent review by Trier et al. [8].
2.2 Recombinant antigen expression in bacteria
Recombinant protein expression in Escherichia coli
strains is undoubtedly the fastest and most cost-effective
approach to protein generation, but frequently results in
the formation of inclusion bodies. Here, the protein of in-
terest accumulates in high concentration and purity, but
is insoluble and non-functional. If the antigen exceeds
small to medium size, features multiple disulfide bonds, or
needs to undergo other secondary modifications essential
for function, the choice of a bacterial expression system
may thus be suboptimal [9, 10]. Periplasmic expression by
co-expression of folding chaperones has shown some
benefits in this respect; specific release from only the
periplasm is achieved by osmotic shock or leakage [11,
12].
Moreover, remarkable progress has been made in the
successful refolding of complex proteins from inclusion
bodies. The refolding process comprises several steps
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from inclusion body isolation and solubilization to refold-
ing techniques, such as chromatographic methods, mem-
brane controlled denaturant removal, or direct dilution in
conjunction with optimization of redox conditions in the
case of disulfide-bonded proteins.
Improved refolding strategies, the use of enhancers
such as low-molecular-weight additives, and the deter-
mination of optimum refolding parameters by coupling
dynamic and static light scattering methodologies has
driven recent progress and achievements, as document-
ed impressively in multiple reviews [13–18].
Likewise, the solubility of the antigen protein can be
enhanced by fine-tuning of expression parameters, rang-
ing from plasmid construct design, selection of E. coli host
strain, to modulation of process parameters [19].
For purification of soluble recombinant proteins
expressed in E. coli, classical methods based on the
physico-chemical properties of the molecule (such as size,
charge, hydrophobicity, solubility, or binding affinity) are
applied. Certain techniques, such as hydrophobic inter-
action, ion exchange or size exclusion chromatography,
enable individual proteins to be purified from a crude
source. The biological affinity of a protein interacting with
a specific partner, such as enzyme–substrate, ligand–
receptor, and antibody–antigen interactions, can be
exploited for affinity chromatography. Alternatively, artifi-
cial N- or C-terminal extensions to the gene of interest can
be introduced into the molecule to serve as affinity purifi-
cation tags. Comprehensive reviews of affinity tags in the
context of bacterial expression were recently compiled by
Young et al. [20] and Walls and Loughran [21].
As an example, polyhistidine tags are very common,
easy to use, and usually result in relatively pure protein
samples [22, 23]. “Pseudo-affinity” resins contain bound
metal ions, either nickel or cobalt, to which the polyhisti-
dine tag binds with micromolar affinity. High concentra-
tions of imidazole, EDTA or buffers with low pH in the
sample elution fraction can be exchanged against a phys-
iological buffer by dialysis or application of size exclusion
chromatography as a subsequent purification step.
2.3 Recombinant expression in eukaryotic systems
E. coli-derived antigens, lacking the natural sugar
residues attached by glycosylation, may give rise to anti-
bodies directed against exposed epitopes that are not
accessible in the mammalian system and hence are of lit-
tle value [24]. In contrast, yeast expression systems using,
for example, the strain Saccharomyces cerevisiae as a
host may yield hyper-glycosylated proteins, which in turn
could lead to masking of immunogenic epitopes [25],
unless a yeast strain modified by metabolic engineering
towards a human glycosylation pattern is employed [26,
27].
Infecting insect cell lines, such as Spodoptera frugi-
perda (Sf) or Trichoplusia ni (T.ni) derivatives, with re-
© 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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combinant baculoviruses is very effective for the produc-
tion of recombinant proteins of an intracellular, cell-sur-
face-bound, or secreted nature [28–30]. Insect cells, in
comparison to mammalian cells, are easy to handle and
cultivate and require less complex and thus less expen-
sive culture media and cell culture equipment. Moreover,
the efficient infectious process and short run time facili-
tates the generation of multiprotein complexes, either
from a mixture of single recombinant baculoviruses by co-
infection, or from the so-called MultiBac system [31].
Insect cells are capable of modifying proteins by phos-
phorylation, fatty acid acylation, N-terminal acetylation,
carboxymethylation, isoprenylation, and N- and O-glyco-
sylation. Yet, an insect-specific N-glycosylation pattern of
the high-mannose type has led to efforts in pathway
engineering of insect cells to exhibit a human-like glyco-
sylation pattern [32, 33].
Rapidly growing interest in the development of bio-
pharmaceuticals is paralleled by a steep rise in recombi-
nant protein production using mammalian cells as hosts,
not only for manufacturing purposes, but also to supply
the discovery process with high-quality proteins, such as
antigens. For the rapid generation of material on a scale
of 1–100 mg of purified protein, transient expression in
the human embryonic kidney cell line (HEK293) and Chi-
nese hamster ovary (CHO) cells has therefore become the
method of choice in recent years [34–36]. In many in-
stances, these host cell lines have been engineered by
stable integration of viral elements derived from
Epstein–Barr virus or simian virus 40 (SV40), which, in
conjunction with the corresponding interacting virus-de-
rived elements incorporated into the expression plasmid,
give rise to high-level expression. For both host cell lines,
detailed protocols for suspension cultivation of cells under
serum-free conditions and transfection by lipoplexes and
polycations on a small to large scale have been worked out
and described [37–40].
The availability of disposable bioreactors, such as the
WAVE™ system [41], along with inexpensive transfection
reagents, such as polyethylenimine (PEI) [42], has enabled
transient protein production up to a scale of >100 L at a
reasonable price, giving rise to protein yields in the multi-
milligram to gram range [43, 44]. The impact of this might
not be obvious, since only small amounts of antigen are
required to initiate the antibody generation process, but
advanced antibody drug candidates in preclinical or clin-
ical phases still rely on antigen supply in increasing
amounts at the highest quality for manufacturing of the
product [45]. Furthermore, the expression process and
scale can be flexibly tailored to expression levels and
requested amounts of an individual protein.
Applying techniques paralleling those used for pro-
teins derived from bacteria is possible, provided that the
antigen is secreted from cells and the protein is subse-
quently captured from the culture medium of insect or
mammalian cells. Tagging of the protein with various cap-
© 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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turing tags is often the method of choice to allow for its re-
trieval from the rather complex composition of cell culture
media. A variety of small to large tags, such as HA-tag,
FLAG-tag, his6-tag, and Fc-fusions, have been described.
Small tags usually remain attached to the recombinant
protein and binders raised against these entities during
the primary antibody selection process require a subse-
quent counter-selection step for de-selection. Large tags
can also be removed by an additional cleavage and purifi-
cation step, if a protease cleavage site is introduced into
the cDNA construct (e.g. PreScission, enterokinase,
thrombin) [21, 46, 47].
In some instances, a capturing tag on the antigen may
impair correct folding and/or biological activity. In these
cases, a tagless protein can be produced and retrieved by
affinity chromatography, providing that a suitable anti-
body against the target is available in sufficient quantity.
To conclude this section on soluble expression of anti-
gens, there is increasing interest to tackle complex multi-
domain antigens in different indications. Each domain is
capable of possessing a separate independent function
and three-dimensional structure and could harbor func-
tional epitopes. Both isolated individual expression of sin-
gle domain entities and expression of the full-length
extracellular domain can be exploited for antigen genera-
tion. However, the determination of the protein domain
boundaries as an extension or deletion of a single amino
acid is critical and may result in soluble expression in rea-
sonable yields. Unfortunately, the respective entries in rel-
evant databases may be incorrect and ill-described at
times [48].
2.4 Cell-surface displayed antigens
2.4.1 Naturally presented antigens
Instead of defined polypeptides, naturally occurring
whole cells can be used for immunization and in vitro
display; an approach especially useful for the identifica-
tion and characterization of novel cell-surface biomark-
ers. For example, normal or cancerous human cells are
potent immunogens for the generation of mAbs, result-
ing in a strong immune response in animals [49–51]. For
cell-based immunization, cells can be collected from tis-
sue culture or isolated directly from tissue samples or
blood samples, such as blood mononuclear cells. With
this approach, a prerequisite for success is the high
overexpression of the target molecule versus all other
cell-surface entities displayed. Additionally, isogenic
control cells expressing low or no target molecule are
needed for counter-selection. Overall, extensive screen-
ing must be successful in isolating highly specific bind-
ing molecules when non-recombinant technologies are
applied [52, 53].
A further specialized technique for antibody genera-
tion, which uses naturally occurring and presented anti-
gens, is in vivo phage display, for which antibody libraries
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are injected into animals. The phage enriched for binding
to the target is then recovered by isolation of cells or even
tissues of interest. This technique is often used for spe-
cific cancer cell targets [54].
2.4.2 Recombinant antigens presented on cell surface
To circumvent the inefficiency of screening for mAbs that
recognize the native cell-displayed antigens, receptor
molecules can be overexpressed recombinantly. If the in-
tended application involves cell-based screening, a tran-
sient expression approach may be less desirable due to
the heterogeneity of expression frequently encountered
in these cell pool populations. Furthermore, if repeated
cell batches are required, the inherent inter-experimental
variation observed in transient expression trials could be
problematic. Novel transfection strategies with very high
transfection rates, such as large-scale electroporation
using the MaxCyte™ device [55] combined with a proto-
col for freezing of transiently transfected cell batches [56],
are potential means to overcome these problems.
Alternatively, the generation of stable episomal cell
pools can be applied. Subsequent to transfection, cell pop-
ulations exhibiting resistance to commonly used selection
antibiotics, such as hygromycin or geneticin, are estab-
lished. To circumvent any lengthy and tedious single-cell
cloning procedure, enriched fractions of positive recombi-
nant pools can be isolated by fluorescence-based cell sort-
ing (FACS). Two to three rounds of FACS enrichment usu-
ally give rise to nicely positive, albeit not fully homoge-
nous, cell populations. A caveat to this approach is the
necessity to monitor the expression stability of the
enriched pool populations over time. Moreover, the avail-
ability of a detection antibody capable of recognizing a na-
tive epitope and thus suitable for immunofluorescence
staining and cell sorting may pose a constraint, in partic-
ular, for novel targets. In cases where no detection anti-
body is available against the target protein, if the attach-
ment of a small peptide detection tag, such as the his6 tag,
at the N terminus of the receptor is not desired, or the N
and C termini of the receptor molecule reside intracellu-
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larly, expression of a second detector molecule, such as
green fluorescent protein, or an unrelated short receptor
fragment, either by direct fusion to the molecule of inter-
est or by co-expression, has been tried successfully
[57–59].
Commonly used cell lines for recombinant receptor
expression comprise HEK293 and CHO derivatives, but to
maximize the specific antibody response in the mouse
upon whole-cell immunization, human target antigens
can also be cloned into and overexpressed on the surface
of mouse cell lines derived from a strain syngenic to the
immunization host strain, for example, BALB/c mice. The
stably transfected cells are used for immunizing the syn-
genic mouse strain and subsequent screening of hybrido-
ma clones derived from cell fusions, while the non-trans-
fected cell line serves as the negative control (Fig. 1). In
contrast to human cells, recombinant murine cells will
typically exhibit a significantly reduced background
response when using the syngenic inbred mouse strain
because the cells are genetically identical to the mouse
strain apart from the transfected and expressed target
molecule. In addition, it is also possible to use cell-based
immunization to generate mAbs against the secreted
forms of antigens by tethering the secreted proteins, such
as serum proteins and cytokines, to the cell surface as
antigens for immunization.
A condensed overview for the selection of recombi-
nant expression hosts in relation to the antigen to be pro-
duced is given in Fig. 2. To conclude, awareness at the
beginning of a discovery project should be raised that
usually more than one antigen needs to be prepared per
project. Depending on the sequence homology of the tar-
get between human, rodents, and monkeys, antigens of
all species may be required for cross-reactivity assess-
ment. Notably, even if a protocol for successful expression
of a human antigen has been worked out, this may not
translate into similarly successful production of the relat-
ed, but not sequence-identical cynomolgus or mouse pro-
tein. Furthermore, paralogue antigens for counter-screen-
ing and selection should also be included in planning.
Figure 1. Transfected cells as antigens
for immunization and subsequent mAb
screening. A human target antigen is
overexpressed on the surface of a mouse
cell line derived from the BALB/c strain.
The stable transfected cells are used as
an antigen for immunizing BALB/c mice
and for the differential screening of
hybridoma supernatants of the transfect-
ed versus non-transfected cells by flow
cytometry. Expression profiles of an anti-
gen on transfected and control cells are
shown.
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2.4.3 Complex membrane proteins as antigens
Isolation of soluble membrane proteins, such as most
GPCRs, following recombinant expression in either mam-
malian or bacterial systems remains a challenging task.
The identification of functional binding molecules,
regardless of whether they are agonistic or antagonistic,
requires expression and purification of GPCRs in a func-
tional state. The amount of recombinant membrane-
inserted receptors produced depends on the optimal com-
bination of a particular GPCR and the production host,
which needs to be determined experimentally. In addition
to classical membrane preparation from overexpressing
cells, receptors can be extracted from membranes by the
use of detergents. The choice of an appropriate detergent
for solubilization and purification is crucial for retaining
the functionality of the receptor. Many GPCRs become
unstable upon detergent extraction from lipid membranes
and their quality should be controlled as described below,
for example, by ligand binding assays [60–62].
Alternatively, Lebon et al. [63] described a method to
engineer a GPCR gene by point mutagenesis to obtain
sufficient amounts of soluble and thermostable trans-
membrane protein for crystallization trials and biochemi-
cal analysis. In fact, thermostabilized transmembrane
proteins obtained by protein engineering are a valuable
antigen source and might be the method of choice if oth-
er trials for antigen preparation are unsuccessful.
Another option for antigen presentation as an
immunogen (especially suited for membrane proteins) is
the generation of virus-like particles, as shown for cell re-
ceptors and ion channels. A number of different ap-
proaches exist, however, they all follow the same princi-
ple of co-overexpression of the antigen with a virus coat
protein for encapsulation of the antigen into so-called
virus-like particles. The main benefit of this approach over
merely transfected cells is a higher expression or concen-
tration of the antigen combined with a better defined sur-
face, that is, reduced presentation of irrelevant proteins
© 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Figure 2. Which approach to choose for which
type of antigen? In an abbreviated format, an
overview of the different choices for expression
hosts, depending on the type of protein and its
sub-cellular localization, is shown. Dashed
arrows indicate potentially less preferable
choices. BEVS, baculovirus expression vector
system; ORF, open reading frame.
from the parental cell line. It is also very easy to produce
“null” particles in an identical parental cell line not ex-
pressing the target antigen for counter-selection purpos-
es. Exosomes, which are microparticles naturally shed by
a variety of cell types, can also be engineered towards
presentation of antigens [64–66].
Finally, cell-free expression offers an interesting alter-
native method to produce proteins that lead to cell toxic-
ity in other systems. Potential translation directly into
membrane mimetics is beneficial for membrane proteins.
Further stabilization of the expressed proteins can be
achieved by almost unlimited supplementation of cofac-
tors, ligands, or chaperones to the translation reaction
mixture. Recent developments have resulted in well-
established systems of prokaryotic and eukaryotic origins
[67–69].
2.4.4 DNA Immunization
For the sake of completeness, an approach that does not
rely on a preformulated proteinaceous antigen directly
should be mentioned. An additional strategy for dealing
with difficult to express antigens is to use a DNA immu-
nization approach. This method exploits the flexibility
and ease of working with plasmid DNA, allowing the gen-
eration of many different DNA constructs encoding pep-
tides, protein domains, or full-length proteins. DNA can
also be delivered by several different means, including
injection (hypodermic needle), a gene gun using DNA-
coated gold beads, pneumatic injection, and lipid formu-
lations [70]. It is thought that the resulting antigenic pep-
tides are presented by antigen presenting cells, such as
dendritic cells, and elicit both a strong humoral and cellu-
lar immune response [71, 72].
The main limitation of this approach is the resulting
lack of available protein antigen for subsequent screening
purposes. Thus, combination with the approaches
described above using cells, which endogenously express
the protein of interest, or even more recent techniques
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employing next-generation sequencing have to be con-
sidered [73, 74]. Sequencing of the complete antibody
repertoire versus the sequencing results obtained from a
naïve animal may lead to the identification of highly spe-
cific binders albeit at low frequency.
3 Chemical modification of antigens
for immunization and screening
The generation of mAbs to peptide antigens by classical
immunization usually requires coupling of the peptide to
a carrier protein, for example, keyhole limpet hemocyanin
or BSA. Likewise, in vitro display systems require the use
of carrier proteins due to the small size of peptide anti-
gens. The coating of the plastic surface of microtiter
plates with unconjugated peptides renders them masked
and inaccessible during blocking of free binding sites of
the plastic surface with BSA or milk.
Protein antigens can also be biotinylated either site-
specifically at the C or N terminus by attachment of the
so-called Avi-tag to the cDNA [75] or through endoge-
nously present lysines by random biotinylation. Con-
versely, chemically derived peptides can be conjugated
during synthesis. This allows the addition of antigen to
displayed binding molecules and, following a standard
panning cycle, facilitates capture of the full binding com-
plex via streptavidin or NeutrAvidin. This serves as an
alternative strategy for antigen presentation apart from
direct coating onto plastic surfaces [76, 77].
For immunization peptide or protein antigens are usu-
ally formulated with an adjuvant. The use of an adjuvant,
such as Freund’s adjuvant, not only lowers the amount of
antigen required, but also prolongs the antigen stimula-
tion of the immune host. Even though in recent years
there has been a growing number of new adjuvant
reagents available, Freund’s adjuvant has remained the
first choice for working with antigens that have poor im-
munogenicity in the immune host [78–80]. Freund’s com-
plete adjuvant contains heat-inactivated mycobacterial
elements, which enhance the immune response by
directly stimulating the activity of antigen-presenting
cells. However, it also elicits a local inflammatory reaction
that may cause a necrotic appearance at the site of injec-
tion in the animal. To minimize or eliminate this side
effect of Freund’s adjuvant, it is necessary to utilize a min-
imal dose of the antigen and complete Freund’s adjuvant
mixture in the first injection; subsequent injections are
then performed using incomplete Freund’s adjuvant. The
(partially) denaturating effect of Freund’s adjuvant gives
rise to a tendency to isolate antibodies that recognize lin-
ear exposed epitopes, even if properly folded protein
preparations are used for immunizations [81–84].
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4 Quality-control criteria and methodologies
applied
Careful monitoring of protein quality with respect to iden-
tity, integrity, and functionality is essential to ensure the
use of antigens, which mimic natural presentation and
provide the highest chance for isolation of binding mole-
cules with the desired specificity. The analytical methods
applied are highly diverse and cover a broad range with
regard to technology as well as equipment. The most
important parameters to be assessed are summarized
below.
First, an exact determination of protein concentration
is a crucial parameter for affinity determination of binding
molecules towards purified antigen. Apart from classical
methods, such as Bradford and Lowry, Nanodrop offers a
fast analysis technique with minimal sample consump-
tion by measurement of the absorbance at 280 nm with a
known extinction coefficient. Alternatively, HPLC pro-
vides data on protein quantity and purity, but clearly has
limitations for glycosylated proteins. Another convincing
method, which combines analysis of quantity and purity,
is LabChip (Caliper Life Sciences, Zurich, Switzerland).
Here SDS-PAGE-like size separation is electronically eval-
uated to obtain concentrations of individual protein
bands based on an internal reference standard.
Likewise, confirmation of the protein identity should
be a routinely performed analysis and can be achieved by
different methods. Most commonly used and powerful is
HPLC coupled to mass spectrometry (LC-MS), which sep-
arates proteins on a reverse-phase column following
analysis of protein mass by TOF electrospray ionization
(TOF-MS). Evaluation of individual results can confirm
the identity, purity, and integrity of the protein, but also
allow identification of post-translational modifications,
such as phosphorylation, deamidation, and glycosylation
[85, 86]. More sophisticated and laborious methods to
confirm the protein identity comprise mass spectrometry
after proteolytic cleavage (peptide mapping), N-terminal
sequencing or tandem MS [87–89].
In addition, classical SDS-PAGE analysis under reduc-
ing and non-reducing conditions gives an indication of
protein purity and amount of contaminants by host cell
proteins, but also oxidation state and degradation of the
respective protein.
Importantly, quantified concentrations of purified
protein may differ dramatically from effective functional
protein concentration due to multimerization and aggre-
gation because these non-native folding states are
unlikely to contribute to the specific interaction with the
binding partner. Analytical size-exclusion chromatogra-
phy (SEC) or different light-scattering methods (multian-
gle light scattering (SEC-MALS) or dynamic light scat-
tering (DLS)) assist in quality control regarding aggrega-
tion [90].
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Table 1. Overview on quality parameters, methods, and their readout

Quality parameter Method Readout

Quantity Absorbance 280 nm Protein concentration [mg/mL]
Lab chip (Caliper)
HPLC
Purity HPLC Purity of protein [%], amount of contaminations/impurities
SDS-PAGE of expression host
Lab chip (Caliper)
LC-MS
Identity LC-MS Expected protein
Peptide mapping
N-terminal protein sequencing
Tandem MS
Immunoassays (western blot, ELISA)
Integrity LC-MS Intact protein, degradation by proteolytic cleavage,
SDS-PAGE folding state, and secondary structure
Circular dichroism (CD) spectroscopy
Dispersity Analytical SEC Multimerization and aggregation
SEC-MALS
DLS
Homogeneity LC-MS Post-translational modifications
(e.g. phosphorylation, deamidation, glycosylation)
Pyrogenicity LAL assay (turbidimetric or chromogenic) Endotoxin level [EU/mg]
Functionality BiaCORE Affinity [M]
Cellular assays Binding
FACS Specificity
Immunoassays (ELISA)
Ligand binding assays

Furthermore, protein oxidation, aggregation, and deg-
radation are known issues that can affect the outcome of
antibody generation severely. Therefore, there is a need to
carefully monitor, for example, by MS or functional assays,
the quality of the antigen produced. Additional post-
translational modification, such as deamidation, may like-
wise be critical to the successful isolation of antibodies
and needs to be appreciated [91, 92].
Glycosylation is another aspect that can affect antigen
functionality. As outlined in previous sections, the glyco-
sylation pattern is dependent on the host organism as well
as on cultivation conditions [93]. Thus, it can be essential
in some instances to characterize the sugar moieties of
recombinantly expressed proteins in detail to assess
product consistency as well as to understand the rela-
tionship between glycan structure and function. Several
methods have been described for detailed characteriza-
tion of the glycan profiles of proteins, ranging from HPLC
over capillary electrophoresis to MS-based methods [94].
In the context of in vivo application of antigens,
attention should be paid to the presence and, if necessary,
removal of endotoxins. Endotoxins, also called lipopoly-
saccharides, are major contaminants often found in bac-
teria-derived protein preparations. Another major source
of endotoxins are hydrolysates, which are frequently
added to cell culture media as a substitute for fetal bovine
serum. Due to their deleterious effect in animal studies, it
is clearly mandatory to remove endotoxins from immuno-
gens. This can be very demanding because, despite the
development of novel methods, there is no universal
recipe for removal. To determine the pyrogenicity of pro-
tein samples the turbidimetric LAL (limulus amoebocyte
lysate) technique and the chromogenic LAL technique
are most frequently used [95–97].
Last but not least, one of the most important and
meaningful parameters is the functionality of the antigen
preparation. The type of functional assessment is very
dependent on the nature and class of the antigen and
should provide information on overall binding, affinity, or
specificity. Interactions of soluble antigens to their bind-
ing partners are examined by immunoassays (e.g. ELISA)
if the reagents for antigen detection are available. Other-
wise, label-free assays, such as surface plasmon reso-
nance, come into play [98, 99]. Transmembrane proteins,
either as antigens or as antigen-binding partners, require
specific assays with a functional readout, such as fluores-

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Biotechnology
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cent imaging plate reader (FLIPR) technology or reporter
gene assays [100, 101]. The detailed setup of cell-based or
ligand-binding assays needs to be tailored to the individ-
ual antigen [102, 103].
The quality criteria discussed herein are by no means
exhaustive, but should give an overview of the key pa-
rameters summarized in Table 1. All described tech-
niques should be properly considered before application
in any antibody generation campaign to allow the pro-
duction of a highly valuable binding molecule.
5 Discussion
This review covers a plethora of different options for anti-
gen generation and validation. As an extract and to
bridge the descriptive contents with applications, we
summarize briefly our strategies for antigen generation.
The selection of an appropriate expression system for a
given antigen is based on the nature of the target and its
anticipated biochemical and biophysical behavior. It is
our experience that a multiparallel approach in different
systems (e.g. E. coli and mammalian cells) with multiple
constructs greatly enhances the chances of success,
especially for novel targets. The candidate proteins are
mostly tagged, including differently sized tags with var-
ied positions. This enables in-process analytics during
small-scale trial expression preceding upscaling of cul-
tures, as well as affinity-based purification as a first step.
The vast majority of proteins derived from mammalian
cells are purified by affinity-based capturing, followed by
a size-exclusion polishing step, if necessary. The purified
proteins are characterized by SDS-PAGE analysis under
non-reducing and reducing conditions, as well as by MS,
which can be preceded by a de-glycosylation step, if
needed. The aggregation and degradation rate of the final
product is assessed by analytical SEC, likewise the endo-
toxin level is determined. More elaborate techniques,
such as SEC-MALS, N-terminal sequencing, tryptic
digest followed by peptide mapping, and assessment of
the correct folding state of E. coli-derived antigens by DLS
are carried out on demand. Functionality of the protein is
always analyzed by appropriate assays developed by the
project teams.
Lastly, a brief comment on the rather large set of com-
mercially available antigen preparations: the possibility of
acquiring antigens as a starting material for target vali-
dation and assay development is certainly a huge advan-
tage in research. Unfortunately, apart from the cost issue,
the quality of the purchased material often does not live
up to expectations. Thus, in addition to the product char-
acteristics described by the vendor being carefully looked
at, a quality check by biochemical, biophysical, and/or
functional means is clearly advisable to avoid unpleasant
surprises at later stages of the project.
1440
Biotechnol. J. 2012, 7, 1433–1443
Hilmar Ebersbach entered the field of
protein engineering during his Ph.D.,
which was a joint venture project
between the University of Halle and a
small biotech company, Scil Proteins,
Halle, Germany. He continued to
broaden his experience in display tech-
nologies with a post-doctoral project in
the Plückthun lab at the University of
Zürich, Switzerland. In 2007 he joined Novartis Research, Biologics
Center, in Basel as an expert in antibody generation. In this position
he developed several novel libraries of different binding molecules for
drug discovery.
Sabine Geisse is a recognized expert in
eukaryotic cell biology with >20 years of
experience gathered during her Ph.D.
studies in Nutrition and Human Biolo-
gy at the Justus-Liebig-University in
Giessen and Philipps-University in Mar-
burg and as a post-doctoral researcher
at the University of Essen, Germany.
She joined Sandoz/Novartis in 1988
within Discovery Technologies and is a scientific expert for biomole-
cule production. In 2004, she received the Novartis Leading Scientist
award. Sabine Geisse built up and implemented an expression plat-
form for transient and stable expression technologies in eukaryotic cell
lines and developed, among others, a HEK293 expression system for
transient expression on small and large scale for the rapid production
of recombinant proteins.
We would like to acknowledge the many scientific contri-
butions from all our colleagues in the Novartis Biologics
Center, subgroups ‘Protein Production and Antibodies’
and ‘Novel Technologies’, that enabled us to compile the
content of this review. We are grateful to our colleague,
Dr. Anne London, for careful proofreading of the manu-
script.
The authors declare no conflict of interest.
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