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BIOPHARMACEUTICAL ANALYSIS
SUPPLEMENT TO
ADVANCES IN
Volume 34, Number s11 November 2016
www.chromatographyonline.com
A s
up
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nt to
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GC
No
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Am
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a
Ad
va
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Bio
ph
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ace
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N
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6
; ; ;
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www.chromatographyonline.com4 ADVANCES IN BIOPHARMACEUTICAL ANALYSIS NOVEMBER 2016
ArticlesIntroduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5Pat Sandra and Koen SandraAn introduction from the guest editors of this special supplement
Modern Column Technologies for the Analytical Characterization of Biopharmaceuticals in Various Liquid Chromatographic Modes. . . . . . . . . . . . . . . . . 6Szabolcs Fekete, Jean-Luc Veuthey, and Davy GuillarmeThe recent trends in column technology for reversed-phase LC, SEC, ion-exchange
chromatography, and HIC for analysis of biopharmaceuticals are critically discussed.
Monoclonal Antibodies and Biosimilars—A Selection of Analytical Tools for Characterization and Comparability Assessment . . . . . . . . . . . . . . . . . . . . . . . . . 14Koen Sandra, Isabel Vandenheede, Emmie Dumont, and Pat SandraThis article presents a selection of state-of-the-art analytical tools for mAb
characterization and comparability assessment.
Higher Order Mass Spectrometry Techniques Applied to Biopharmaceuticals. . . . . . . . . . . . . . 22Christian G. HuberAn outline of the basic principles of MS techniques used to investigate higher order
structural features of biopharmaceuticals, as well as some insights into applications
relevant to the pharmaceutical industry.
Advances in Liquid Chromatography–Tandem Mass Spectrometry (LC–MS/MS)-Based Quantitation of Biopharmaceuticals in Biological Samples . . . . . . . . . . . . . . 28Nico C. van de MerbelThe technical requirements for a successful LC–MS/MS method for the
quantitation of biopharmaceuticals are presented and the advantages and
disadvantages compared to ligand-binding assays are evaluated.
Analyzing Host Cell Proteins Using Off-Line Two-Dimensional Liquid Chromatography–Mass Spectrometry . . . . . . . . . . . . 35Koen Sandra, Alexia Ortiz, and Pat SandraThe use of off-line 2D-LC–MS for the characterization of HCPs and their monitoring
during downstream processing
November 2016
Cover images courtesy of vitstudio/shutterstock.com
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A supplement to LCGC North America
BIOPHARMACEUTICAL
ANALYSIS
BIOPHARMACEUTICAL
ANALYSIS
ADVANCES INADVANCES IN ®
NOVEMBER 2016 ADVANCES IN BIOPHARMACEUTICAL ANALYSIS 5www.chromatographyonline.com
Advances in Biopharmaceutical Analysis
An introduction from the guest editors, Pat Sandra and Koen Sandra of the Research Institute for Chromatography,
focusing on recent developments in biopharmaceutical analysis.
When we were asked to edit a follow-up to
the LCGC Europe May 2013 supplement
“Advances in Pharmaceutical Analysis,” we
immediately wanted to highlight the chal-
lenges in biopharmaceutical analysis. Indeed,
within the pharmaceutical industry and also
within our own research activities related
to pharmaceutical analysis, there has been
a remarkable shift from small to large mol-
ecules. On the market since the early 1980s,
protein biopharmaceuticals have seen an
enormous growth in the last decade. It is
even expected that within the current decade,
more than 50% of new drug approvals will
be biological in nature. A dominant role is
thereby played by monoclonal antibodies
(mAbs), of which a substantial number have
reached blockbuster status. The top 10 best-
selling pharmaceuticals are currently heavily
populated by mAbs.
Protein biopharmaceuticals are large and
heterogeneous and their in-depth analysis
during development and also during their
lifetime requires the best of both chroma-
tography and mass spectrometry (MS). In
editing this special issue, we have therefore
selected authorities in the field to illustrate
the state of the art in biopharmaceutical
analysis.
The first contribution, authored by
Szabolcs Fekete, Jean-Luc Veuthey, and Davy
Guillarme, provides an overview of the dif-
ferent liquid chromatography (LC) column
formats recently introduced in the market
for reversed-phase, size-exclusion (SEC),
ion-exchange, and hydrophobic interaction
(HIC) chromatographic analyses of thera-
peutic proteins, mAbs, and antibody–drug
conjugates (ADCs).
In the May 2013 supplement we described
the features of liquid chromatography cou-
pled to mass spectrometry (LC–MS) in the
characterization of protein biopharmaceu-
ticals. With the patents of the first genera-
tion protein biopharmaceuticals expired and
blockbuster mAbs appearing on the market,
activities in biosimilars have exploded in
recent years. More than 15 biosimilars have
already been approved in Europe, and a ver-
sion of filgrastim was launched in the United
States as the first biosimilar toward the end
of 2015. Analytical methods to compare
originators with biosimilars are highlighted
in the second contribution from our team at
the Research Institute for Chromatography.
The antibody market has been reshaped
by various next-generation formats (biospe-
cific mAbs, antibody mixtures, nanobodies,
brain penetrant mAbs, glyco-engineered
formats), and in recent years the ADCs bren-
tuximab vedotin and trastuzumab emtan-
sine have been approved by the European
Medicines Agency (EMA) and the Food
and Drug Administration (FDA). In ADCs,
a cytotoxin is coupled to an antibody that
specifically targets a certain tumor marker.
As such, highly toxic drugs can be delivered
in a targeted fashion to tumor cells without
affecting healthy cells. Compared to naked
mAbs, the conjugation of cytotoxic drugs
further adds to the complexity. The power
of MS to unravel this complexity is illus-
trated in the paper authored by Alain Beck
and by Sarah Cianferani (available online
at: www.chromatographyonline.com/har-
nessing-benefits-mass-spectrometry-depth-
antibody-drug-conjugates-analytical-char-
acterization-0).
The previous two contributions clearly
illustrate the importance of MS in the eluci-
dation of the primary structure of therapeu-
tic proteins. Higher order elements, on the
other hand, can be derived from special MS
technologies such as native MS, ion mobil-
ity MS, hydrogen–deuterium exchange MS,
and chemical cross-linking MS. In the third
contribution, Christian Huber describes the
basic principles of these techniques and illus-
trates their features for the characterization of
higher order structures of some protein bio-
pharmaceuticals.
Traditionally, ligand-binding assays
(LBAs) are applied to study the pharma-
cokinetic behavior of protein biopharma-
ceuticals in biological fluids. LBAs are
characterized by a high throughput and
sensitivity, but may suffer from long devel-
opment times and potential interferences
from other proteins present in the matrix.
In addition, the generation of drug-specific
antibody tools is a time-consuming process.
Liquid chromatography coupled to tandem
mass spectrometry (LC–MS/MS) methods
are used more and more as alternatives to
LBAs, often offering improved figures-of-
merit while at the same time being generi-
cally applicable. Some of the technicalities
and advantages and disadvantages of LC–
MS/MS compared to LBAs for monitor-
ing biopharmaceuticals in biological fluids
are addressed in the fourth contribution by
Nico C. van de Merbel.
The presence of residual host cell proteins
(HCPs) is a potential safety risk in any bio-
pharmaceutical product. Despite enormous
purification efforts, these HCPs may be left
behind from the expression hosts. HCPs are
normally dosed during downstream process-
ing and in the final biopharmaceutical prod-
uct by enzyme-linked immunosorbent assays
(ELISA). As mentioned in the previous paper,
LBAs are more and more complemented
or even replaced by LC–MS/MS and this
is illustrated in the last contribution by our
group. The use of off-line two-dimensional
LC–MS/MS in the characterization of HCPs
is described and the added value of using
multidimensional chromatography is clearly
demonstrated.
We hope that the contributions in this
supplement are of interest and even a source
of inspiration to the numerous analysts in the
biopharmaceutical industry. It was a pleasure
for us to edit and review the contributions
of outstanding (preselected) colleagues. We
would like to thank all of them for their
excellent work.
Pat Sandra Koen Sandra
6 ADVANCES IN BIOPHARMACEUTICAL ANALYSIS NOVEMBER 2016 www.chromatographyonline.com
Szabolcs Fekete, Jean-Luc Veuthey, and Davy Guillarme
Modern Column Technologies for the Analytical Characterization of Biopharmaceuticals in Various Liquid Chromatographic Modes
The recent trends in column technology for reversed-phase liquid
chromatography (LC), size-exclusion chromatography (SEC), ion-exchange
chromatography, and hydrophobic interaction chromatography (HIC) for
analysis of biopharmaceuticals at the protein level is critically discussed.
Therapeutic proteins are large and het-
erogeneous molecules subjected to a
variety of enzymatic and chemical
modifications during expression, purifica-
tion, and long-term storage. These changes
include several possible modifications, such
as oxidation, deamidation, glycosylation,
aggregation, misfolding, or adsorption,
leading to a potential loss of therapeutic
efficacy or unwanted immune reactions.
Regulatory bodies require a detailed char-
acterization (for example, verifying primary
structure and appropriate post-translational
modifications, secondary and tertiary
structure), lot-to-lot and batch-to-batch
comparisons, stability studies, impurity
profiling, glycoprofiling, determination of
related proteins and excipients as well as
determination of protein aggregates. For
this purpose, a single analytical technique
is generally not sufficient, and a variety of
orthogonal methods are required to fully
describe such a complex sample.
Today, one of the most widely used ana-
lytical techniques for therapeutic protein
characterization is liquid chromatography
(LC). This is probably a direct result of
the remarkable developments of the past
few years, which have enabled a new level
of chromatographic performance. These
developments include ultrahigh-pressure
liquid chromatography (UHPLC), col-
umns packed with wide-pore superficially
porous particles (SPPs), and organic mono-
lith columns, which have allowed a dra-
matic increase in separation efficiency, even
with large intact biomolecules.
This article reviews the possibilities
and trends of current state-of-the-art LC
column technology applied for different
modes of chromatography for the charac-
terization of therapeutic proteins.
Hydrophobic Interaction
Chromatography
Hydrophobic interaction chromatography
(HIC) has been historically used for protein
purification; more recently, the two main
application fields have been in the determi-
nation of the drug-to-antibody ratio (DAR)
of antibody–drug conjugates (ADCs) and
in monitoring post-translational modifica-
tions of monoclonal antibodies (mAbs).
In HIC, proteins are retained and sepa-
rated on the basis of their hydrophobicity as
a result of the van der Waals forces between
the hydrophobic ligands of the stationary
phase and the nonpolar regions of proteins
(1). The binding of proteins to a hydropho-
bic surface is affected by a number of fac-
tors including the type of ligand, the ligand
density on the solid support, the backbone
material of the stationary phase, the hydro-
phobic nature of the protein, and the type
of salt added to the mobile phase. Dur-
ing the separation, a negative salt gradient
(typically from 2–3 M to 0 M) is applied
under aqueous mobile phase at around pH
6.8–7.0. The structural damage to the bio-
molecules is therefore minimal and its bio-
logical activity is maintained (2).
Analytical-scale HIC columns are based
either on silica or polymer particles. Both
porous and nonporous particles are avail-
Ph
oto
cred
it: J
org
Gre
uel
/Get
ty I
mag
es
In the sample chromatograms shown here, 5 μm non-porous SP-F column was used for the separation of human IgG mAb charge variants. Even higher resolution was achieved using 3 μm particles.
Phone: +1.610.266.8650Email: [email protected]: www.ymcamerica.comStore: store.ymcamerica.com
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YMC Products for BioseparationsYMC-BioPro SP-F for mAb Charge Variant Analysis
YMC-Pack Diol-SEC is a size exclusion medium utilizing a silica gel base. Diol-120, 200, and 300 are available in 2, 3, and 5 μm particle diameters, and are suitable for separation or molecular weight determination of proteins with molecular weights of 10,000 to several hundred thousand.
Diol-60 is the most suitable for separation of peptides or oligosaccharides whose molecular weights are 10,000 or less.
YMC-Triart is a hybrid reversed phase material, and YMC Meteoric Core is a core-shell material.
Both are well-suited to the rigors of bioseparations – they perform well in aqueous phases, respond well to gradient changes, and offer high resolution and long column life.
YMC-Triart is offered in capillary columns, UHPLC columns, and standard HPLC columns. Meteoric Core is offered in standard HPLC columns. Both are ideal choices for LC-MS.
Original separation using 5μm.
Switch to 3μm resolves additional peak.
YMC Products for Bioseparations
YMC-Pack Diol Size Exclusion (SEC) Columns for Separation of mAb
YMC-Triart and Meteoric Core for Peptide Mapping and Reversed Phase
YMC-Pack Diol, 2μm, 300Å, 150x4.6mmSmaller Particles and Smaller Columns Enable Lower Flowrates, Higher Throughput
5μm
2μmSample: 1mg/mL dilution of 25mg/mL AvastinMobile Phase: 100mM NaPO4 with 200mM NaCl at pH=7.0
Sample PreparationMonoclonal antibody samples were denatured, reduced (BME), and desalted prior to trypsin digestion.
Operating ParametersMobile Phase A: 0.1% TFA in HPLC WaterMobile Phase B: 0.1% TFA in AcetonitrileColumn Temp: 40°C Flowrate: 0.6mL/minInj. Volume: 10uL Detection: 215nm
GradientStep Time Flow %A %B
1 0 0.6 98 22 0.67 0.6 98 23 14 0.6 55 454 16 0.6 0 1005 16.67 0.6 98 26 20 0.6 98 2
Porous
Non-Porous
Particle Sizes:
3, 5, 6, 10, 30, 75 μm
Particle Sizes:
3, 5, 6, 10, 30, 75 μm
8 ADVANCES IN BIOPHARMACEUTICAL ANALYSIS NOVEMBER 2016 www.chromatographyonline.com
Table I: Recent state-of-the-art and some widely used “reference” columns applied for the separation of therapeutic proteins in HIC, SEC, ion-exchange, and reversed-phase LC modes
Column Name ChemistryParticle size/Macropore Size (μm)
Max Temperature
(°C)pH Range
Max Pressure
(bar)
HIC Columns
TSKgel (Tosoh)
Butyl-NPR C4 (non porous) 2.5
50 2–12
200
Ether-5PW Ether (porous) 1050
Phenyl-5PW Phenyl (porous) 10
Protein-Pak Hi Res HIC (Waters) C4 2.5 60 2–12 200
Thermo
MAbPac HIC-Butyl C4 5 60 2–12 300
MAbPac HIC-20 Alkylamide 5 60 2–9 400
ProPac HIC-10 Amide/ethyl 5 60 2.5–7.5 300
Ion-Exchange Columns
Proswift (Thermo)(monolith)
SAX-1SStrong anion exchange
(quaternary amine)
Information not
available
70
2–12 70
WAX-1SWeak anion exchange
(tertiary amine)60
WCX-1SWeak cation exchange
(carboxylic acid)60
SCX-1SStrong cation exchange
(sulfonic acid)60
TSKgel (Tosoh)
SCXStrong cation exchange
(sulfonic acid)5
45
2–14
50
SuperQ-5PWStrong cation exchange
(trimethylamino)10 2–12
SP-STATStrong cation exchange
(sulfopropyl)7, 10 3–10
Q-STATStrong anion exchange
(quaternary ammonium)7, 10 3–10
Bio Mab (Agilent)Weak cation exchange
(carboxylate)
1.7 3 5 10
80 2–12
270 410 550 680
Antibodix (Supelco, Sepax)Weak cation exchange
(carboxylate)
1.7 3 5 10
80 2–12
270 410 550 680
Protein-Pak Hi Res IEX (Waters)
SPStrong cation exchange
(sulfopropyl)7
60 3–10
100
CM Weak cation exchange
(carboxymethyl)7 100
QStrong anion exchange
(quaternary ammonium)5 150
MAbPac SCX-10 (Thermo)Strong cation exchange
(sulphonic acid)
3 5 10
60 2–12480 480 200
Bio-Pro (YMC)
QAQA-F
Strong anion exchange (quaternary ammonium)
5 60 2–12
30 120
SPSP-F
Strong cation exchange (sulfopropyl)
30 120
SEC Columns
Thermo Silica-based 3 60 2.5–7.5 200
YMC-Pack Diol-SEC Diol modified silica-based 5 40 5–7.5 200
Acclaim SEC-300 (Thermo)Hydrophilic polymethacrylate
resin5 60 2–12 1200
TSKgel SW aggregate (Tosoh) Diol 3 30 2.5–7.5 120
TSKgel SW mAb (Tosoh) Diol 4 30 2.5–7.5 120
SRT-SEC (Sepax) Surface-coated silica-based 5Information not
available2–8.5
Information not available
Zenix-SEC (Sepax) Surface-coated silica-based 3 ~250 2–8.5 80
NOVEMBER 2016 ADVANCES IN BIOPHARMACEUTICAL ANALYSIS 9www.chromatographyonline.com
able. Highly cross-linked nonporous
poly(styrene–divinylbenzene) (PS–DVB)
and polymethacrylate-based particles are
frequently used in protein separations as a
result of their advantageous mass transfer
properties (the main contribution to the
band broadening of large biomolecules,
namely trans-particle mass transfer resis-
tance is negligible). Table I summarizes
the most widely used and the latest HIC
columns applied for mAb and ADC sepa-
rations.
These materials can now withstand pres-
sure drops of up to 100–400 bar. Columns
are typically packed with 10-, 7-, 5-, 3-, and
even 2.5-μm particles. Column diameters
between 2 mm and 8 mm are available, but
4.6-mm i.d. columns are the most widely
used in current HIC applications. It is
worth mentioning that there is a need for
150 mm × 2.1 mm column formats, which
are often applied for the analysis of proteins
in modern chromatographic practice.
HIC allows both the characterization
of the distribution of drug-linked species
and the determination of average DAR of
ADCs (3). Conjugation of the drug-linker
to the antibody increases the hydrophobic-
ity; therefore, HIC appears as a suitable
tool to separate the different DAR species.
A good example of the HIC profile of a
native IgG1 ADC is shown in Figure 1 (3).
Recently off-line mass spectrometric
(MS) detection was applied for the char-
acterization of brentuximab-vedotin. Each
individual HIC peak was collected, buffer
exchanged, and analyzed by native MS
(4). HIC was also successfully applied for
monitoring various post-translational mod-
ifications, including proteolytic fragments,
domain misfolding, tryptophan oxidation,
and aspartic acid isomerization in therapeu-
tic mAbs (5).
Ion-Exchange Chromatography
Ion-exchange chromatography is widely
used for the characterization of therapeutic
proteins and can be considered as a
reference marker and powerful technique
for the qualitative and quantitative
evaluation of charge heterogeneity.
Table I: Continued
Column Name ChemistryParticle Size/Macropore Size (μm)
Max Temperature
(°C)pH Range
Max Pressure
(bar)
SEC Columns (continued)
Bio SEC (Agilent) surface-coated silica-based3 Information not
available2–8.5 240
5
Acquity UPLC BEH SEC (Waters) diol modified hybrid-based1.7
60 2–8 6002.5
Reversed-Phase LC Columns
ProSwift (Thermo)(Monolith)
RP-1S
Phenyl
1 70 1–14 200
RP-2H 2.2 70 1–14 200
RP-3U 5.1 70 1–14 200
RP-10RInformation not available
80 1–10 300
Acquity BEH 300 (Waters) C18, C4 1.7 80 1–12 1000
Zorbax (Agilent)
300SB RRHD C18, C8 1.8 80 1–8 1200
Poroshell SB300 C18, C8, C35 (0.25-μm thickness)
90 1–8 600
Poroshell 300Ex-tend
C185 (0.25-μm thickness)
60 2–11 600
AdvanceBio RP-mAb C8, C4, diphenyl3.5 (0.25-μm
thickness)90 1–8 600
Aeris (Phenomenex)
Widepore C18, C8, C43.6 (0.2-μm thickness)
90 (C18,C8), 60 (C4)
1.5–9 600
Peptide C18
3.6 (0.5-μm thickness)
2.6 (0.35 μm thickness)
1.7 (0.22-μm thickness)
90 1.5–9600
1000
Halo (Advanced Materials Technology)
Peptide C18, CN
2.7 (0.5-μm thickness)
4.6 (0.6 μm thickness)
100 1–9 600
Protein C18, C83.4 (0.2-μm thickness)
90 1–9 600
Flare Widepore (Diamond Analytics) C183.6 (0.1-μm thickness)
100 1–13 400
10 ADVANCES IN BIOPHARMACEUTICAL ANALYSIS NOVEMBER 2016 www.chromatographyonline.com
Among the different ion-exchange
modes, cation-exchange chromatography
is the most widely used for protein
characterization (6).
Two modes of elution are often applied
for protein characterization, namely the
salt-gradient and the pH-gradient. In
salt-gradient mode, solutes are eluted in
order of increasing binding charge, which
correlates more or less with the isoelectric
point (pI) and equilibrium constant. In
this case, the mobile-phase pH is kept
constant, while the ionic strength is
continuously increased. In pH-gradient
mode, the ionic strength is kept constant
and the pH is varied during the gradient
program. This mode of elution is often
referred to as chromatofocusing.
Regarding the stationary phase, there are
two main aspects that need to be considered
for successful ion-exchange separation: the
strength of interaction and associated reten-
tion (strong or weak ion exchanger), and
the achievable peak widths (efficiency) (7).
Both cation and anion exchangers can be
classified as either weak or strong exchang-
ers. Weak cation exchangers are composed
of a weak acid that gradually loses its
charge as the pH decreases (for example,
carboxymethyl groups), while strong cat-
ion exchangers are composed of a strong
acid that is able to sustain its charge over
a wide pH range (for example, sulfopropyl
groups). On the other hand, strong anion
exchangers contain quaternary amine func-
tional groups, while weak anion exchanger
possesses diethylaminoethane (DEAE)
groups. As a rule of thumb, it is preferable
to begin the method development with a
strong exchanger, to ensure that a broad pH
range can be worked on. Strong exchangers
are also useful if the maximum resolution
occurs at an extreme pH. However, silica-
based ion exchangers can only be operated
in a restricted pH range. In contrast, poly-
meric ion exchangers can be used over a
wide pH range.
Commercially available ion-exchange
columns are based on silica or polymer
particles but organic–polymeric monoliths
are also available. Both porous and
nonporous particles are available but
for large molecules, which possess low
diffusivity, nonporous materials are clearly
preferred. Highly cross-linked nonporous
PS–DVB materials are most frequently
used in protein separations because of their
high pH stability (2 ≤ pH ≤ 12). These
materials can now withstand a pressure
drop of up to 500–600 bar in some cases.
Columns packed with 10-, 5-, or 3-μm
nonporous particles are often used, but
sub-2-μm materials are also available to
perform UHPLC separations (see Table
I). Suitable peak capacity can be attained
with large biomolecules on those columns
within a reasonable analysis time (for
example, 15–20 min). However, some
limitations can be expected in terms of
loading capacity and retention when
applying nonporous materials.
A recent study systematically compared
the latest state-of-the-art cation exchanger
columns applied for the characterization of
therapeutic mAbs in pH- and salt-gradient
modes (8).
Figure 2 shows an example of the
separation of four intact antibody charge
variants using a 100 mm × 4.6 mm, 5-μm
strong cation polymeric exchanger column
packed with nonporous particles and a
20-min long gradient (7)
Size-Exclusion Chromatography
Size-exclusion chromatography (SEC) is a
powerful technique for the qualitative and
quantitative evaluation of protein aggre-
gates. The main advantage of SEC is the
mild mobile phase conditions that permit
the characterization of proteins with mini-
mal impact on the conformational struc-
ture and local environment.
200
150
100
50
1210
DAR 4
DAR 6
Time (min)
Sig
nal
DAR 8
DAR 2
DAR 0
8640
Figure 1: HIC separation of an ADC for the determination of DAR. Adapted and re-produced with permission from reference 3, ©American Chemical Society.
Sig
nal
2015
Time (min)
1050
1 2 3 4
Figure 2: Ion-exchange separation of four intact mAbs (natalizumab [1], cetuximab [2], adalimumab [3], and denosumab [4]). Adapted and reproduced with permission from reference 7, ©Elsevier.
NOVEMBER 2016 ADVANCES IN BIOPHARMACEUTICAL ANALYSIS 11www.chromatographyonline.com
SEC separates biomolecules according
to their hydrodynamic radius. The sta-
tionary phase consists of spherical porous
particles with a carefully controlled pore
size, through which the biomolecules
diffuse based on their molecular size
difference using an aqueous buffer as
the mobile phase. Basically, SEC is an
entropically controlled separation pro-
cess in which molecules are separated
on the basis of molecular size differences
(filtering) rather than by their chemical
properties (8). Therefore, retention fac-
tor (thermodynamic) in SEC is different
from other chromatographic modes. Here,
the thermodynamic retention factor is the
fraction of the intraparticle pore volume
that is accessible to the analyte (9).
Since no retention occurs in SEC, large
pore volumes (high porosity) are required
to ensure appropriate resolution. Gener-
ally, this large pore volume is provided by
long-and wide-bore columns. In routine
SEC applications, a 30-cm column length
with internal diameters (i.d.) of 7.8, 8.0, or
10 mm is generally employed. These SEC
columns are referred to as standard-bore col-
umns. Now, several vendors offer narrow-
bore columns with 4.6-mm i.d. and 15-cm
length that are packed with very efficient,
small particles of ~3 μm. Similar separation
power can be attained using these columns
as with 5-μm particles in 30-cm standard-
bore columns, but the analysis time can be
reduced by a factor of 3 to 4 (10).
There are mainly two types of SEC
packing materials: silica, with or without
surface modification, and cross-linked
polymeric packings, which possess non-
polar (hydrophobic), hydrophilic, or ionic
character (8). The most common silica
packing consists of chemically bonded
1,2-propanediol functional groups that
provide a hydrophilic surface. This sta-
tionary phase blocks or reacts with many
of the acidic silanol groups allowing the
surface to be neutralized. Bare silica is
also a suitable packing material for non-
aqueous polar or nonpolar organic mobile
phases; however, it is not recommended
with aqueous mobile phases because of
the presence of active silanol sites. The lat-
est type of silica-related packing is an eth-
ylene-bridged hybrid inorganic-organic
(BEH) material that is currently avail-
able at particle sizes of 1.7 μm—the first
sub-2-μm SEC packing—and 2.5 μm
(11). Compared to regular silica packings,
BEH particles have improved chemical
stability as well as reduced silanol activity.
The 1.7-μm BEH material can be oper-
ated at up to 600 bar.
There have been a number of different
hydrophilic cross-linked packings devel-
oped for the SEC of biopolymers. Most of
these packings are proprietary hydroxyl-
ated derivatives of cross-linked polymeth-
acrylates (10). Unusual polymeric pack-
ings for aqueous SEC include sulfonated
cross-linked polystyrene, polydivinylben-
zene derivatized with glucose or anion
exchange groups, a polyamide polymer,
and high-performance, crossed-linked
agarose (8).
Today, columns for aqueous and non-
aqueous SEC applications with pore sizes
of 125 to 900 Å are commercially avail-
able (12). Very fast separations of peptides,
myoglobin, and insulin aggregates have
been demonstrated with 1.7-μm SEC
columns (13). These columns were also
applied for the characterization of recom-
binant mAbs (11).
Applying 1.7- and 2.5-μm particles
in SEC has opened up a new level of
separation performance, but it should be
kept in mind that on very fine particles,
the separation quality is improved at the
cost of pressure (and frictional heating
temperature gradients). Therefore, there
is a risk of creating on-column aggregates
when analyzing sensitive proteins under
high-pressure (>200 bar) conditions (11).
Reversed-Phase
Liquid Chromatography
In reversed-phase liquid chromatography
(LC), the solute retention is predominantly
mediated through hydrophobic interac-
tions between the nonpolar amino acid
residues of the proteins and the bonded
n-alkyl ligands of the stationary phase.
Compared to the HIC mode, the reversed-
phase LC mobile phase typically consists
of water, acetonitrile or methanol, and
0.1–0.2% trifluoroacetic acid or formic
acid. The separation mechanism is based
on a combination of solvophobic and elec-
trostatic interactions, the latter being gov-
erned by the interaction of trifluoroacetic
acid with basic side chains of a few amino
acids (that is, arginine, lysine, and histi-
dine) and the N-terminus as well as ionic
interactions between the positive charges
at the surface of the protein and the nega-
tively charged residual silanols (14). The
efficiency of reversed-phase LC is always
superior to other chromatographic modes,
and its superior robustness makes it well
suited for use in a routine environment (15).
Current reversed-phase LC stationary
phases used for proteins analysis can be
classified as silica-based particulate mate-
rials and organic monoliths. The pore
size of particulate phases is an important
factor that must be considered. For the
analysis of peptides and small proteins, a
pore size of 100–200 Å may be acceptable.
However, porous materials with pore sizes
of greater than 200 Å are mandatory for
the separation of larger proteins or mAbs
fragments because the solute molecular
64 5
Time (min)
31 20
Sig
nal
L H
HC
2xLC2xHC
Reduction
+
Figure 3: Reversed-phase LC analysis of reduced IgG1 mAb. Unpublished results from the authors’ laboratory.
12 ADVANCES IN BIOPHARMACEUTICAL ANALYSIS NOVEMBER 2016 www.chromatographyonline.com
diameter must be approximately one-
tenth the size of the pore diameter to
avoid the restricted diffusion of the solute
and to allow the total surface area of the
sorbent material to be accessible. An aver-
age pore size of 250–300 Å is often men-
tioned as the reference value for protein
separations, but recently it was shown that
400 Å particles completely eliminated
restricted diffusion effects for molecules
up to about 500 kDa.
The two main trends today in reversed-
phase LC analysis of therapeutic proteins
are the use of fully porous small par-
ticles (FPPs) (sub-2-μm) and superficially
porous particles (SPPs), which possess
particle sizes between 3 and 4 μm.
Columns packed with FPPs have
constraints in separation speed and
efficiency because of limitations in the
stationary phase mass transfer, which
results from the relatively long diffusion
times required for proteins to cross the
porous structure. Therefore, Horváth
first applied the concept of SPPs in the
late 1960s (16,17). They were initially
intended for the analysis of macromol-
ecules such as peptides and proteins. SPPs
are made of a solid, nonporous silica core
surrounded by a porous shell layer. They
have similar properties to the fully porous
materials conventionally used in high per-
formance liquid chromatography (HPLC).
The rationale behind this concept was to
improve column efficiency by shortening
the diffusion path that molecules must
travel, in addition to improving their mass
transfer kinetics.
It was recently shown that columns
packed with wide-pore 3.6-μm and 3.4-
μm SPPs showed significant gain in anal-
ysis time and peak capacity compared to
FPPs for intact protein analysis (18,19).
These wide-pore SPPs are now available
with C4, C8, and C18 chemistries and
can be operated up to 600 bar. Figure 3
shows an example of fast separation of
heavy-chain (Hc) and light-chain (Lc)
variants of an IgG1 mAb performed on a
wide-pore C4 SPP column.
In another study, efficiency and analy-
sis times of 1.7-μm SPPs and FPPs were
compared for peptides and moderate size
intact proteins (20). This study suggests a
two-fold increase in terms of achievable
peak capacity and analysis time for large
proteins when using SPPs compared to
FPPs of the same size. For the separation
of peptides and moderate size proteins, a
160 Å SP packing was also introduced
(21,22). Recently, 1.3- and 1.6-μm SPPs
were also applied for peptide mapping of
mAb samples (23,24). By combining long
columns (200–300 mm) with extended
analysis time, peak capacity around 1000
can be reached with 1.3-μm SPPs for
0.5–2 kDa peptides.
An alternative, carbon-nanodiamond-
based C18 superficially porous mate-
rial was recently introduced (25). The
core of this material is a carbonized
poly(divinylbenzene) particle with a
diameter of approximately 3.4-μm.
Poly(allylamine)-nanodiamond het-
ero-layers are deposited onto the surface of
the carbonized core by a modified layer-
by-layer method. The resulting core–shell
is synthesized to a shell thickness of ca. 0.1
μm and a finished particle size of 3.6 μm.
This superficially porous carbon-based
material was successfully applied for real-
life protein separations.
Another interesting alternative to SPPs
was proposed by Hayes and colleagues
(26). The so-called sphere-on-sphere
(SOS) approach provides a simple and
fast one-pot synthesis in which the thick-
ness, porosity, and chemical substituents
of the shell can be controlled by using
the appropriate reagents and conditions
(27). SOS particles have been shown to
be microporous with a pore diameter of
less than 2 nm. However, while the sur-
face of the material might not exhibit
significant porosity, when packed into a
HPLC column the spaces between surface
nanospheres provide superficial macropo-
rosity. It has been proposed that for large
molecules, larger pores as well as reduc-
tion of the shell thickness can be advan-
tageous, because of the shorter diffusion
distance and greater access to the surface
area of the material (28). SOS particles
were demonstrated to have similar chro-
matographic performance compared to
commercial SPP materials (26). Figure
4 shows the separation of reduced ADC
(brentuximab-vedotin) fragments on a
column packed with SOS particles.
As an alternative to particle-based sta-
tionary phase formats for the LC separa-
tions of proteins, organic polymer-based
monoliths offer some advantages, includ-
ing high permeability and rapid mass
transfer (29). Polymeric monolithic sta-
tionary phases have shown great poten-
tial for the reversed-phase LC separations
of large biomolecules, including intact
proteins, oligonucleotides, and peptides.
With this material, the mass transfer is
mainly driven by convection, rather than
diffusion, because of the absence of mes-
opores (30). The fact that the solvent is
forced to pass through the macropores of
the polymer because of pressure leads to
faster convective mass transfer compared
to the slow diffusion process into the stag-
nant pore liquid that is present in porous
beads packed columns. As a result of their
open channel structure, monoliths gener-
ally possess a high permeability, allowing
the application of elevated flow rates at
moderate back pressure. It was previously
demonstrated that polymeric stationary
Sig
nal
128 10Time (min)
62 40
H1
H0
L1L0
H2H3
Figure 4: Reversed phase LC separation of reduced ADC (brentuximab-vedotin) frag-ments. Unpublished results from the authors’ laboratory.
NOVEMBER 2016 ADVANCES IN BIOPHARMACEUTICAL ANALYSIS 13www.chromatographyonline.com
phases led to superior performance over
silica-based materials in the reversed
phase analysis of very large proteins (MW
> 50 kDa) (31).
Conclusion
There is always a need to use several chro-
matographic methods to draw reliable con-
clusions regarding the quality of biophar-
maceuticals. Ion-exchange chromatography,
SEC, and HIC are historical techniques
and are still used in any laboratory deal-
ing with the analytical characterization of
mAbs or ADCs. These techniques were
known to offer poor resolving power, which
is why the stationary phases employed in
ion-exchange chromatography, SEC, and
HIC have strongly evolved over the last few
years, in terms of chemistries, dimensions,
and chemical stability.
The most important improvements for
protein analysis were brought to reversed-
phase LC materials. In the past, this tech-
nique was rarely used for biopharmaceuti-
cal characterization. However, because
this is the only chromatographic approach
directly compatible with MS, providers
have improved and developed their exist-
ing materials. The performance that can
be achieved today with columns packed
with wide pore sub-2-μm fully porous or
sub-4-μm superficially porous are highly
competitive, and even if the selectivity of
reversed-phase LC is still limited for sepa-
rating charge or size variants of proteins,
this is (at least partially) compensated by
the high kinetic performance generated by
modern reversed-phase LC columns.
Acknowledgments
The authors acknowledge Alain Beck
(Pierre Fabre, Saint-Julien Genevois, France)
for providing mAb and ADC samples, and
Stephanie Schuster (Advanced Materials
Technology) and Tony Edge and Richard
Hayes (Thermo Fisher Scientific) for pro-
viding stationary phases.
Davy Guillarme wishes to thank the
Swiss National Science Foundation for
support through a fellowship to Szabolcs
Fekete (31003A_159494).
References
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Pharm. Biomed. Anal. 111, 169–176 (2015).
(8) H.G. Barth and G.D. Saunders, LCGC North
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Chrom. Rel. Techn. 35, 2923–2950 (2012).
(10) S. Fekete, A. Beck, J.L. Veuthey, and D. Guil-
larme, J. Pharm. Biomed. Anal. 101, 161–173
(2014).
(11) S. Fekete, K. Ganzler, and D. Guillarme, J.
Pharm. Biomed. Anal. 78–79, 141–149 (2013).
(12) E. Gazal, “Can Size Exclusion Chromatography
(SEC) Be Done on Sub-3-μm Particles?,”
presented at the 17th annual meeting of the
Israel Analytical Chemistry Society, Tel Aviv,
Israel, 2014.
(13) S.M. Koza, P. Hong, and K.J. Fountain,
“Advantages of Ultra Performance Liquid
Chromatography Using 125 Å Pore Size, Sub-
2-μm Particles for the Analysis of Peptides
and Small Proteins,” poster presented at
Medimmune, Rockville, Maryland, 2012.
(14) S. Fekete, J.L. Veuthey, and D. Guillarme, J.
Pharm. Biomed. Anal. 69, 9–27 (2012).
(15) K. Sandra, I. Vandenheede, and P. Sandra, J.
Chromatogr. A 1335, 81–103 (2014).
(16) C. Horvath, B.A. Preiss, and S.R. Lipsky, Anal.
Chem. 39, 1422–1428 (1967).
(17) C. Horvath and S.R. Lipsky, J. Chromatogr.
Sci. 7, 109–116 (1969).
(18) S. Fekete, R. Berky, J. Fekete, J.L. Veuthey,
and D. Guillarme, J. Chromatogr. A 1236,
177–188 (2012).
(19) S.A. Schuster, B.M. Wagner, B.E. Boyes, and
J.J. Kirkland, J. Chromatogr. A 1315, 118–126
(2013).
(20) S. Fekete, K. Ganzler, and J. Fekete, J. Pharm.
Biomed. Anal. 54, 482–490 (2011).
(21) F. Gritti and G. Guiochon, J. Chromatogr. A
1218, 907–921 (2011).
(22) S.A. Schuster, B.M. Wagner, B.E. Boyes, and
J.J. Kirkland, J. Chromatogr. Sci. 48, 566–571
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(23) S. Fekete and D. Guillarme, J. Chromatogr. A
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(24) B. Bobály, D. Guillarme, and S. Fekete, J. Sep.
Sci. 37, 189–197 (2014).
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Pharm. Biomed. Anal. 104, 130–136 (2015).
(26) R. Hayes, P. Myers, T. Edge, H. Zhang, Ana-
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Myers, and H. Zhankg, J. Chromatogr. A
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Irgum, Chem. Mater. 8, 744–750 (1996).
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Anal. Chem. 68, 315–321 (1996).
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Guillarme, Anal. Bioanal. Chem. 405, 3137–
3151 (2013).
Szabolcs Fekete holds a PhD degree
in analytical chemistry from the Techni-
cal University of Budapest, Hungary. He
worked at the Chemical Works of Gedeon
Richter Plc at the analytical R&D depart-
ment for 10 years. Since 2011, he has
worked at the University of Geneva in
Switzerland. He has contributed 70 jour-
nal articles and authored book chapters.
His main interests include liquid chroma-
tography, column technology, pharmaceu-
tical, and protein analysis.
Jean-Luc Veuthey is professor at
the School of Pharmaceutical Sciences,
University of Geneva, Switzerland. He has
also acted as President of the School of
Pharmaceutical Sciences, Vice-Dean of the
Faculty of Sciences, and finally Vice-Rector
of the University of Geneva. His research
domains include development of separa-
tion techniques in pharmaceutical sciences,
and, more precisely, the study of the
impact of sample preparation procedures
in the analytical process; fundamental
studies in liquid and supercritical chroma-
tography; separation techniques coupled
with mass spectrometry; and analysis of
drugs and drugs of abuse in different
matrices. He has published more than 300
articles in peer-reviewed journals.
Davy Guillarme holds a PhD degree
in analytical chemistry from the University
of Lyon, France. He is senior lecturer at
the University of Geneva in Switzerland.
He has authored 140 journal articles relat-
ed to pharmaceutical analysis. His exper-
tise includes HPLC, UHPLC, HILIC, LC–MS,
SFC, and analysis of proteins and mAbs.
He is an editorial advisory board member
of several journals including Journal of
Chromatography A, Journal of Separation
Science, and LCGC North America. ◾
14 ADVANCES IN BIOPHARMACEUTICAL ANALYSIS NOVEMBER 2016 www.chromatographyonline.com
Koen Sandra, Isabel Vandenheede, Emmie Dumont, and Pat Sandra
Monoclonal Antibodies and Biosimilars—A Selection of Analytical Tools for Characterization and Comparability Assessment
Monoclonal antibodies (mAbs) have emerged as important therapeutics
for the treatment of life-threatening diseases including cancer and
autoimmune diseases. With the top-selling mAbs evolving out of patent
there has been a growing interest in the development of biosimilars.
In demonstrating comparability to the originator product, biosimilar
developers are confronted with an enormous analytical challenge. This
article presents a selection of state-of-the-art analytical tools for mAb
characterization and comparability assessment.
It was Paul Ehrlich who, around 1900,
reported on “magic bullets” to cure a
wide range of diseases, thereby indi-
rectly referring to antibodies (1,2). The
development of the hybridoma tech-
nology by Köhler and Milstein, which
allowed the production of monoclonal
antibodies (mAbs), bridged the gap
between concept and clinical reality (3).
Since the approval of the first therapeu-
tic murine mAb in 1986, advances in
antibody engineering have allowed the
production of chimeric (mouse–human),
humanized, and human monoclonal
antibodies, thereby substantially improv-
ing safety and efficacy and paving the
way for the full exploitation of mAbs for
therapeutics purposes (4,5). More than
40 mAbs are now marketed in the United
States and Europe for the treatment of a
variety of diseases including cancer and
autoimmune diseases (6,7).
In 2013, 18 mAbs displayed block-
buster status and six of these prod-
ucts had sales greater than $6 billion
(Humira, Remicade, Enbrel, Rituxan,
Avastin, and Herceptin). MAbs are cur-
rently considered to be the fastest grow-
ing class of therapeutics with sales grow-
ing from $39 billion in 2008 to almost
$75 billion in 2013, a 90% increase.
Sales of other recombinant protein bio-
pharmaceuticals have only increased
by 26% in the same time period, while
small-molecule drugs are stagnating
(6,7). The successes of their predeces-
sors have triggered the development of
various next-generation mAb formats
such as bispecific mAbs, antibody–drug
conjugates (ADC), antibody mixtures,
antibody fragments (nanobodies, Fab),
Fc fusion proteins, and brain penetrant
mAbs next to glyco-engineered formats
(4,5,8). With several hundreds of prod-
ucts in preclinical development, the
future looks very bright.
The knowledge that the top-selling
mAbs are, or will become, open to the
market in the coming years has resulted
in an explosion of biosimilar activi-
ties. Last year witnessed the European
approval of the first two monoclonal
antibody biosimilars (Remsima and
Inflectra), which both contain the same
active substance, inf liximab (9). Remi-
cade, inf liximab’s blockbuster origina-
tor, reached global sales of $8.9 billion
in 2013. It is clear that the biosimilar
market holds great potential, but it is
simultaneously confronted with major
hurdles. In contrast to generic versions
of small molecules, exact copies of
recombinant mAbs cannot be produced
because of differences in the cell clon-
ing and the manufacturing processes
used. Even originator companies expe-
rience lot-to-lot variability. As a con-
sequence, regulatory agencies evaluate
Ph
oto
cred
it: J
un
os/G
etty
Im
ages
NOVEMBER 2016 ADVANCES IN BIOPHARMACEUTICAL ANALYSIS 15www.chromatographyonline.com
biosimilars based on their level of simi-
larity to, rather than the exact replica-
tion of, the originator. In demonstrating
similarity, an enormous weight is placed
on analytics, and both the biosimilar
and originator need to be characterized
and compared in great detail. In con-
trast to small-molecule drugs, mAbs are
large and heterogeneous (as a result of
the biosynthetic process and subsequent
manufacturing and storage), making
their analysis very challenging (10–13).
This article reports on selected state-of-
the-art chromatographic and mass spec-
trometric tools for detailed mAb charac-
terization and comparability assessment.
Protein A Chromatography
for Clone Selection
Protein A from Staphylococcus aureus has
a very strong affinity for the Fc domain
of IgG, allowing its capture from com-
plex matrices such as cell culture super-
natants. Affinity chromatography mak-
ing use of Protein A is the gold standard
in therapeutic mAb purification and
typically represents the first chromato-
graphic step in downstream processing.
Protein A chromatography finds applica-
tions beyond this large-scale purification.
At the analytical scale it is being used
early on in the development of mAbs
for the high-throughput determination
of mAb titer and yield directly from
cell culture supernatants and to purify
microgram amounts of material for fur-
ther measurements by techniques such as
mass spectrometry (MS) and chromatog-
raphy (14–16).
Figure 1 shows an overlay of the
protein A chromatograms of 12 trastu-
zumab-producing Chinese hamster
ovarian (CHO) clones, generated in the
framework of a Herceptin biosimilar
development program. Herceptin (sci-
entific INN name trastuzumab) is being
used in the treatment of HER2 positive
breast cancer. It is open to the European
market and evolves out of patent in the
United States in 2019 (17). Given its
market potential (global sales of $6.5
billion in 2013), dozens of companies
are actively developing a Herceptin
biosimilar. The unbound CHO mate-
rial is eluted in the f low-through while
the mAb is captured and only released
after lowering the pH. From these chro-
matograms, a distinction can already
be made between low and high mAb
producing clones. Absolute mAb con-
centrations can be determined by link-
ing the peak areas to an external cali-
bration curve constructed by diluting
Herceptin originators. Obtained mAb
titers are visualized in the bar plot in
Figure 1. From the findings, clear deci-
sions can be made for further biosimilar
development, that is, high trastuzumab
producing clones can be selected and
taken further in development.
Next to the mAb titer, the second
important criterion in clone selection is
based on the structural aspects. In the case
of biosimilar development, the structure
should be highly similar to the originator
product, within the originator batch-to-
batch variations. Figure 2 shows the ion
mobility (IM) quadrupole time-of-flight
(QTOF) MS measurements of inter-chain
reduced Herceptin and protein A–puri-
fied trastuzumab from a high titer CHO
84876
1106
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<�C18
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<�ES-Cyano
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<�OH5
©2016 Sigma-Aldrich Co. LLC. All rights reserved. Sigma-Aldrich and Supelco are trademarks
of Sigma-Aldrich Co. LLC, registered in the US and other countries. Ascentis is a registered
trademark of Sigma-Aldrich Co. LLC. Solutions within is a trademark of Sigma-Aldrich Co. LLC.
Sigma-Aldrich Corp. is a subsidiary of Merck KGaA, Darmstadt, Germany.
16 ADVANCES IN BIOPHARMACEUTICAL ANALYSIS NOVEMBER 2016 www.chromatographyonline.com
clone. Samples were introduced into the
MS system via a reversed-phase on-line
desalting cartridge and light (Lc) and
heavy chain (Hc) were resolved in the IM
drift cell. Two Lc forms with identical
m/z and molecular weight (MW) but with
a different drift time, hence conformation,
are highlighted. Deconvoluted spectra
reveal that clone derived trastuzumab and
originator display the same Lc and Hc
MW values. In addition, the same N-gly-
cans, which are of the complex type, are
observed on the Hc of the originator and
clone derived mAb. These are considered
the most important attributes of biosimi-
larity according to US and European
regulatory authorities (primary sequence
should be identical and glycosylation
should be preserved). While glycosylation
is similar from a qualitative perspective,
quantitative differences are observed. Our
experience in clone selection studies has
found that it is not always the case that
MW values are identical between origi-
nators and mAbs derived from high-titer
clones or subclones. In these situations,
mAbs are typically not taken further in
development.
Reversed-Phase LC–MS Analysis
of Intact, Reduced, Papain, and
IdeZ Cleaved mAb
When a mAb is taken further in devel-
opment, a detailed characterization
and comparability assessment has to be
performed. Structural characteristics
such as amino acid sequence and com-
position, molecular weight and struc-
tural integrity, N- and O-glycosylation,
N- and C-terminal processing, S-S
bridges, deamidation (asparagine, glu-
tamine), aspartate isomerization, and
oxidation (methionine, tryptophan)
need to be assessed. In that respect,
reversed-phase liquid chromatography
(LC) is extremely powerful. Figure 3
shows highly efficient reversed-phase
LC–ultraviolet (UV) chromatograms
obtained on intact, inter-chain reduced,
papain-digested, and nonreduced and
reduced IdeZ-cleaved Herceptin. All
of these chromatograms are generated
using exactly the same chromatographic
conditions making use of widepore sub-
2-μm C8 particles, elevated column
temperatures (80 °C), and trif luoroace-
tic acid as ion-pairing reagent in a water–
acetonitrile mobile phase system. Under
these conditions, many of the challenges
encountered in performing reversed-
phase LC of proteins (peak tailing, peak
broadening, and adsorption) are tackled
(18–19). Moreover, these conditions are
compatible with MS, which allows an
in-depth characterization and compara-
bility assessment of mAbs.
Figure 4 shows the reversed-phase
LC–UV–MS analysis of IdeZ-cleaved
and TCEP-reduced Herceptin originator
and biosimilar. IdeZ or immunoglobu-
lin-degrading enzyme from Streptococ-
cus equi ssp zooepidemicus is a highly
specific protease similar to IdeS that
cleaves mAbs at a single site below the
hinge region, yielding F(ab′)2 and Fc/2
fragments (20,21). Following reduction,
the F(ab′)2 fragment is converted into
the Lc and Fd′. From the simultaneously
0,8
0,7
0,6
0,5
0,4
0,3
0,2
0,1
0,03
Co
nce
ntr
ati
on
(m
g/m
L)
6 8 9 10 14 24
Clone
Time (min)
mA
U
21.510.50
700
600
500
400
300
200
100
25 26 27 28 32
0
mAb
Figure 1: Overlaid UV 280 nm protein A chromatograms of 12 trastuzumab-produc-ing CHO clones with graphical representation of the mAb titer.
mAb structure
mAbs are tetrameric immunoglobulin G (IgG) molecules with a MW of 150 kDa
composed of two light (Lc – 25 kDa) and two heavy (Hc – 50 kDa) polypep-
tide chains connected through inter-chain disulphide bridges. Twelve intra-chain
disulphide bridges, four within each Hc and two within each Lc, furthermore
guarantee its structural integrity. Six different globular domains, that is, one vari-
able (VL) and one constant domain (CL) for the Lc and one variable (VH) and
three constant domains (CH1, CH2, CH3) for the Hc, are recognized. The struc-
ture can also be divided in the antigen-binding fragment (Fab), composed of VL,
CL, VH, and CH1 and the crystallizable fragment (Fc) composed of CH2 and
CH3. Antigen-binding is mediated by the Fab fragment while the Fc fragment is
responsible for the effector function, that is, antibody-dependent cell-mediated
cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). All mAbs
are glycoproteins with two conserved N-glycosylation sites in the Fc region that
can be occupied with complex and high mannose type N-glycans. These glycan
structures are known to play a role, amongst others, in the effector function.
NOVEMBER 2016 ADVANCES IN BIOPHARMACEUTICAL ANALYSIS 17www.chromatographyonline.com
acquired MS data it can be deduced
that peaks b, b′, d, d′, g, and g′, corre-
sponding to, respectively, Fc/2, Lc and
Fd′, are identical in both the origina-
tor and biosimilar. The measured MW
values obtained are well below 0.005%
of the theoretical MW values, which is
expected when using high-resolution
and accurate mass instrumentation.
Upon examining the spectra of the Fc/2
fragment, the biosimilar appears to be
enriched in the N-glycan G0F while a
more even distribution between G0F
and G1F is observed in the originator.
This is also ref lected in the chromato-
graphic peak shape. The broader peak
b′ indicates a partial separation of the
G0F and G1F species, with the former
eluting slightly later. Several other differ-
entiating peaks are observed in the sepa-
ration of the biosimilar, that is, peaks a,
c, e, and f. Compared to peak b, peak a
displays a 128 Da mass increase, which
can be explained by the presence of a
C-terminal lysine. To provide some more
background on this particular event, the
Hc is cloned with a lysine residue at the
Reduction
Reduction
Papain
2 * Lc 2 * Hc
2 * Fab 2 * Fc
1 * F(ab)’2
F(ab)’2 Fd’
Fc/2
Lc
Fc
Fab
Hc
Lc
2 * Fc/2 2 * Lc 2 * Fd’ 2 * Fc/2
Fc/2
IdeZ
6 8 10 12 14 16 18
6 8 10 12 14 16 18
20
64 8 10 12 14 16 18 20 228 10 12 14 16 18 20 22
Time (min)
Time (min)
Time (min)
Time (min)
Time (min)
10 12 14 16 18 20 22
mA
U
mA
U
mA
Um
AU
mA
U
200
150
100
50
0
500
400
300
200
100
0
500
400
300
200
100
0
500
400
300
200
100
0
240
200
160
120
80
0
40
35
30
251000 1500
Drift Time (ms) vs. m/z
2000 2500 1290 1295 1300 13101305
Drift Time (ms) vs. m/z
1315
195025
30
35
40
32
31
34
33
36
35
38
37
1952 1954 1956 1958Drift Time (ms) vs. m/z
1960
Heavy chain
44+
12+
12+ 11+10+
9+
13+
13+
14+15+
16+17+
18+
40+38+
36+
42+
Light chain
Light chain
Light chain (12+)
Light chain (12+)
Light chain 1 (18+)
G0G0F G1F
G2F
Heavy chain (39+)
23100 23200 23300
23439.9
23440.1
0
1
2
3
x106
0
1
2
3
x106
0
1
2
3
4
x106
0
1
2
x106
Counts vs. Deconvoluted Mass (amu) Counts vs. Deconvoluted Mass (amu)
23400 23500 23600 23700 23800 23900 49800 50200
50598.4
50760.6
50922.4
50922.850452.3
50598.150760.7G0F
G0 G2F
Originator
Biosimilar
Originator
Biosimilar
G1F
50600 51000 51400 51800
Figure 3: Reversed-phase LC–UV separations of intact, dithiothreitol (DTT)-reduced, papain-digested, nonreduced IdeZ-cleaved and tris(2-carboxyethyl)phosphine (TCEP) reduced IdeZ-cleaved Herceptin. These represent extremely powerful separations for comparability assessment and for detailed characterization. Conditions are compat-ible with MS allowing identification of the observed peaks.
Figure 2: IM-QTOF-MS profile of inter-chain reduced Herceptin (top). Deconvoluted light and heavy chain spectra of a Herceptin originator and a trastuzumab-producing clone (bottom).
18 ADVANCES IN BIOPHARMACEUTICAL ANALYSIS NOVEMBER 2016 www.chromatographyonline.com
C-terminus. During protein matura-
tion, this lysine is removed by host cell
carboxypeptidases. This process is more
dominant in the host cell producing the
originator product than in the host cell
producing the biosimilar mAb. From the
MS data it can be deduced that peak c
originates from the Lc plus 1 and 2 hex-
ose units. This potentially originates
from a glycation event, which appears to
be negligible in the originator mAb. Peak
e shows a 1 Da mass increase compared
to peak d, indicating a deamidation in
the Lc. This event is apparent in both
the originator and biosimilar with an
increased occurrence in the biosimilar.
In analogy with peak c, peak f displays
162 Da spacings on Fd′, which is indica-
tive of glycation.
Reversed-Phase LC–MS
Analysis for Peptide Mapping
As previously demonstrated, protein mea-
surement is extremely powerful but does
not provide the complete picture. While
it is indicative for identity and highlights
dominant modifications, it does not pro-
Fc/2Lc Fd’
Biosimilar
Originator
b
a
b’
8 9 10 11 12 13
Response Units vs. Acquisition Time (min)
x10 2
x10 2
2
1.5
1
0.5
2.5
x10 3
x10 4
x10 4
5
x10 3
8
6
4
2
0
x10 4
8
x10 5
1.25
1
0.75
0.5
0.25
0
6
4
2
0
Fc/2 + K(G0F) a
b
b’
25365.2
Fd’ + 1 Hex
Fd’ + 2 Hex
f
g
g’
25707.6
Fd’
25384.0
Fd’25384.3
25546.1
x10 3
x10 4
x10 3
x10 5
1
0.5
0
4
2
S
S
S
S
S
N
DS
S
S
S
0
2
3
2
1
0
0
7.5
5
2.5
Lc + 2 Hex
Lc + 1 Hex
Lcd
c
d’
e
23766.8
23605.4
23443.8
Lc + deam23444.7
Lc23443.7
Fc/2 (G0F)
NHYTQKSLSLSPG
NHYTQKSLSLSPGK
25237.0
Fc/2 (G1F)25399.2
Fc/2 (G1F)
Fc/2 (G2F)
25399.1Fc/2 (G0F)
Fc/2 (G0)
25236.9
25090.7
25560.9
25254.0
4
3
2
1
0
4
3
2
1
0
2.5
2
1.5
1
0.5
024800 24900 25000 25100 25200 25300 25400
Counts vs. DeconvolutedMass (amu)
24800 25000 25200 25400 25600 25800 26000 26200 26400 2660024600
Counts vs. DeconvolutedMass (amu)
22800 23000 23200 23400 23600 23800 24000 24200 24400
Counts vs. DeconvolutedMass (amu)
25500 25600 25700 25800 25900 26000
2
1.5
1
0.5
0
0
14 15 16 17 18 19 20 21
d’g’
c
d
ef
g
Figure 4: Reversed-phase LC–UV–MS analysis of IdeZ-cleaved and TCEP-reduced Herceptin originator and biosimilar and decon-voluted MS spectra associated with the annotated peaks.
T45 + G2F
Originator
Originator
Biosimilar
Biosimilar
Originator
Biosimilar
Originator
Biosimilar
T45 + G0F
T45 + G1Fb
T45 + G1FaT45 + G0
T45 + G0F
T45 + G1F T45 + G0
T62
T62
T3
T3
13.1%T3 deam
10.5%T62+K
0
20 40 60 80
100
120
0
20
40
60
80
5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33
10.6 10.8 11 11.2 15.4 16.2 17 17.8 18.4 18.8 19.2
Response Units vs. Acquisition Time (min)
Figure 5: Reversed-phase LC–UV peptide map of Herceptin originator and biosimi-lar with detail in some specific regions showing post-translational modifications. T45: EEQYNSTYR, T62: SLSLSPG, T3: ASQDVNTAVAWYQQK. Peak identities were as-signed by the simultaneously acquired MS and MS/MS data.
NOVEMBER 2016 ADVANCES IN BIOPHARMACEUTICAL ANALYSIS 19www.chromatographyonline.com
CEX
SEC
HIC
Asndeamidation
mA
Um
AU
mA
U
1000
800
600
400
200
35
350
300
250
200
150
100
50
0
30
25
20
15
10
5
0
2
10 12 14 16 18 20
4 6 8 10 12 14 16
0
12.5 15 17.5 20 22.5 25 27.5 30 32.5
Buffer excipients
Dimer0.4%
Time (min)
Time (min)
Time (min)
Figure 6: CEX, SEC, and HIC separations of Herceptin. These techniques are used in characterization and release testing.
vide the actual amino acid sequence nor
does it localize the modifications. For
example, the measurement presented in
Figure 4 reveals a deamidation on the Lc
(peak e) but it cannot be traced back to a
specific asparagine or glutamine residue.
The Lc of the measured mAb contains
six asparagine and 15 glutamine resi-
dues, which are all prone to this chemical
modification. These characteristics can
further be assessed at the peptide level
following proteolytic digestion. When
digesting Herceptin with the enzyme
trypsin, which cleaves the protein next to
arginine and lysine residues, 62 identity
peptides are formed. Taking into account
post-translational modifications and
incomplete and aspecific cleavages taking
place, more than 100 peptides with vary-
ing physicochemical properties in a wide
dynamic concentration can be expected.
This is a particularly complex sample and
demands the best in terms of separation
technique. Again, reversed-phase LC
is the method of choice to resolve these
complex mixtures. Figure 5 shows the
UV peptide maps of both the origina-
tor and biosimilar. By taking advantage
of the simultaneously acquired MS data,
more than 99% of the peptide sequence
can be covered in both the originator
and biosimilar thereby confirming iden-
tity. While peptide maps are highly com-
parable, differences in post-translational
modifications can be detected (Figure
5). Obtaining a good knowledge of all
of these modifications is important since
they could be critical to the potency and
safety of a mAb. A deviating glycosylation
profile between originator and biosimilar
is already revealed at the protein level (Fig-
ure 4). At the peptide level, the different
N-glycosylated variants are nicely resolved
chromatographically and are shown to be
located on peptide EEQYNSTYR. Again,
the undergalactosylation of the biosimilar
is apparent. The peptide map also reveals
the presence of a lysine at the C-terminal
peptide of the heavy chain (SLSLSPGK)
and slightly increased deamidation in a
light chain peptide (ASQDVNTAVAWY-
QQK). This particular peptide contains
four potential deamidation sites (3 Gln
and 1 Asn). Based on MS measurement
one cannot discriminate between the
four sites. Upon performing MS/MS and
carefully interpreting the fragment ions
observed, the deamidation can be traced
back to the N (11). This deamidation in
fact corresponds to the deamidation event
observed in the reduced IdeS digest (Fig-
ure 4). At that time this event could be
linked to the Lc but could not be traced
back to a specific residue.
As discussed, mAb digests can be quite
complex and their analysis demands the
best in terms of separating power. If one-
dimensional (1D) separations are not able
to provide the separation power needed,
one can opt for two-dimensional (2D)
LC. Compared to 1D-LC, 2D-LC and
especially comprehensive LC (LC×LC)
will drastically increase resolution. We
have recently described the analysis of
Herceptin originator and biosimilar
digests on the combination reversed-
phase LC×reversed-phase LC (22). It is
important to point out that orthogonality
in reversed-phase LC×reversed-phase LC
peptide mapping is only obtained when
operating the two reversed-phase LC col-
umns at different pH values. This is a
direct result of the zwitterionic nature of
peptides, which gives rise to major selec-
tivity differences at pH extremes. These
reversed-phase LC×reversed-phase LC
20 ADVANCES IN BIOPHARMACEUTICAL ANALYSIS NOVEMBER 2016 www.chromatographyonline.com
peptide maps provide a wealth of infor-
mation and allow both identity and
purity to be assessed. This makes it an
attractive technology for the comparison
of different production batches and to
compare innovator biopharmaceuticals
with biosimilars.
Native Chromatographic Tools:
Size-Exclusion Chromatography,
Cation-Exchange Chromatogra-
phy, and Hydrophobic Interac-
tion Chromatography
In contrast to reversed-phase LC,
size-exclusion chromatography (SEC),
ion-exchange chromatography, and
hydrophobic interaction chromatog-
raphy (HIC) are nondenaturing tech-
niques that provide complementary
information to the afore-mentioned
chromatographic mode (Figure 6).
These techniques are used early on in
mAb characterization and comparability
assessment and are subsequently applied
in routine testing. A major advantage
of these chromatographic modes com-
pared to reversed-phase LC is that they
preserve the structure, and so minor
variants can be collected and subjected
to complementary techniques such
as potency determination. SEC, ion-
exchange chromatography, and HIC are
not directly compatible with MS because
of the presence of nonvolatile salts in
the mobile phases. The identification
of peaks requires their collection and
subsequent desalting or dilution before
MS measurement. Desalting of the col-
lected fractions can be performed in an
automated manner using a setup such as
a small reversed-phase cartridge hyphen-
ated to an MS system.
Ion-exchange chromatography is an
excellent tool to highlight charged vari-
ants that might arise from modifications
such as deamidation, lysine trunca-
tion, or N-terminal cyclization (23,24).
Since most therapeutic mAbs have a
higher proportion of basic residues,
cation-exchange chromatography is the
most commonly used technique. The
cation-exchange separation of Herceptin
(Figure 6) highlights the asparagine
deamidation discussed earlier. A deami-
dation renders a protein more acidic,
which explains this earlier elution.
SEC is the chromatographic mode
with the lowest efficiency or resolution
of the afore-mentioned techniques, but
it is extremely powerful when determin-
ing aggregation and fragmentation. It is
recognized that aggregates may stimulate
immune responses and it is therefore very
important to measure this critical quality
attribute. Aggregation can typically not
be assessed using the other chromato-
graphic modes discussed. Figure 6 pres-
ents the SEC analysis of Herceptin and
illustrates that dimers can be measured
accurately at levels as low as 0.4%.
In recent years, HIC has been revisited
mainly from the perspective of ADC’s
governing a separation based on the
number of conjugated drugs allowing
the drug-to-antibody ratio (DAR) to be
determined. In the separation of naked
mAbs it is useful to highlight heteroge-
neities originating from oxidation, aspar-
tate isomerization, deamidation, succin-
imide formation, C-terminal lysine, and
clipping. The HIC analysis of Herceptin
gives rise to a single chromatographic
peak (Figure 6).
Hydrophilic Interaction
Liquid Chromatography
for Glycan Profiling
As demonstrated, glycosylation can be
(a) OriginatorG0F
G0F
G1Fa
G1FaMan5G0
F-G
lcN
Ac
G1Fb
G1Fb
G2FG0
Biosimilar
LU14
12
10
8
6
4
2
0
LU
14
16
10 12.5 15 17.5 20 22.5 25 27.5
(b) Biosimilar
Biosimilar: 4x
Biosimilar: 8x
Biosimilar: 16x
Biosimilar: 24x
LU
10
10
5
0
LU
8
4
0
LU
8
4
0
LU8
4
0
LU8
4
0
12.5 15 17.5 20 22.5 25 27.5
10 12.5 15 17.5 20 22.5 25 27.5
10 12.5 15 17.5 20 22.5 25 27.5
10 12.5 15 17.5 20 22.5 25 27.5
10 12.5 15 17.5 20 22.5 25 27.5
Time (min)
Time (min)
Time (min)
Time (min)
Time (min)
Time (min)
Time (min)
10 12.5 15 17.5 20 22.5 25 27.5
12
10
8
6
4
2
0
Figure 7: (a) Overlaid HILIC-FLD chromatograms of the 2-AB labeled N-glycans en-zymatically released from Herceptin originator and Protein A purified biosimilar. (b) N-glycan profiles of the biosimilar obtained by growing the CHO clone at dif-ferent galactose, uridine, and manganese chloride concentrations. Separations were performed on superficially porous HILIC particles.
NOVEMBER 2016 ADVANCES IN BIOPHARMACEUTICAL ANALYSIS 21www.chromatographyonline.com
revealed at both the protein and peptide
level. A detailed insight into the sugars,
however, can only be obtained follow-
ing their removal from the protein–pep-
tide backbone. This is preferably done
enzymatically using the deglycosidase
PNGase F. The liberated sugars are sub-
sequently labeled via reductive amina-
tion to improve their chromatographic
separation and detectability (f luores-
cence or mass spectrometric detection).
The f luorescence trace is typically used
for quantitative purposes while the MS
trace is used for qualitative purposes.
Figure 7a displays the analysis of 2-ami-
nobenzamide (2-AB) labelled Herceptin
originator and biosimilar N-glycans
using hydrophilic interaction chroma-
tography (HILIC) with f luorescence
detection (FLD). In this particular case,
a column packed with superficially
porous HILIC particles compatible
with 600 bar high performance liquid
chromatography (HPLC) instrumenta-
tion was used. This measurement pro-
vides information on the glycans and
allows structural isomers, that is, G1Fa
and G1Fb which differ in the position-
ing of the galactose residue either on
the α1-3 or α1-6 branch of the complex
type glycan, to be resolved.
The same type of complex N-glycans
are observed on both the originator and
biosimilar but quantitative differences
are revealed with an overexpression of
G0F species on the biosimilar, which is
in accordance with the measurements
performed at protein and peptide level.
Since glycosylation is a critical quality
attribute, this undergalactosylation does
not make the product similar enough to
be considered by regulatory authorities
as a Herceptin biosimilar.
The biosimilar-producing CHO
cell culture medium was subsequently
tuned by feeding uridine (U), galac-
tose (G), and manganese chloride (M)
at different concentrations (25). These
are the substrates and activator of the
galactosyltransferase responsible for
donating galactose residues to G0F and
G1F acceptors. Figure 7b shows the
N-glycan profiles obtained by growing
the biosimilar producing CHO clone at
different U, G, and M concentrations.
It is observed that the ratio G1F–G0F
increases with increasing concentration
of U, G, and M. From these results it
can be concluded that conditions can
be found that allow the glycosylation of
the biosimilar to fit within the origina-
tor specifications.
Conclusion
In the development of biosimilars, a com-
prehensive comparability exercise involv-
ing the originator product is required
to demonstrate similarity in terms of
physicochemical characteristics, efficacy,
and safety. In that respect, an enormous
weight is placed on analytics and the
analytical package for a biosimilar mAb
submission is considerably larger than
that of a stand-alone mAb. Structural
differences define the amount of pre-
clinical and clinical studies required. A
wide range of analytical tools providing
complimentary information is available
to guide biosimilar development.
Acknowledgments
The authors acknowledge Maureen
Joseph (Agilent Technologies, Wilming-
ton, Delaware), David Wong (Agilent
Technologies, Santa Clara, California)
and Lindsay Mesure (Promega, Leiden,
The Netherlands).
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Analyst at the Research Institute for Chro-
matography (RIC).
Emmie Dumont is an LC–MS Specialist
at the Research Institute for Chromatog-
raphy (RIC).
Pat Sandra is Chairman at the Re-
search Institute for Chromatography (RIC)
and Emeritus Professor at Ghent Univer-
sity in Ghent, Belgium. ◾
22 ADVANCES IN BIOPHARMACEUTICAL ANALYSIS NOVEMBER 2016 www.chromatographyonline.com
Christian G. Huber
Higher Order Mass Spectrometry Techniques Applied to Biopharmaceuticals
The recent trends in mass spectrometric techniques—including native
mass spectrometry (MS), ion mobility spectrometry (IMS), hydrogen–
deuterium exchange MS (HDX MS), and chemical cross-linking MS
(CXMS)—used to elucidate higher-order structures of protein complexes
and the practical implementations of these methods are discussed.
Since its invention in the early
20th century (1), mass spec-
trometry (MS) has been used
to discover new chemical elements
and their isotopes (2), explore mar-
tian soil for organic matter (3), and
study biological processes by profiling
proteomes or metabolomes (4,5). Bio-
polymers, and especially proteins, are
the subject of intensive investigation.
They are characterized by different
levels of structural organization, rang-
ing from the primary structure repre-
sented by the amino acid sequence,
over secondary structural elements
such as α-helices and β-sheets, and
the three-dimensional (3D) orienta-
tion of the polypeptide chain, and
f inally to the assembly of subunits
into protein complexes.
Analysis of protein structure using
MS was first possible in the mid-1980s,
when the soft ionization techniques of
electrospray ionization (ESI) (6) and
matrix-assisted laser desorption–ion-
ization (MALDI) (7) were introduced.
Implementation of MS for protein
analysis initia lly focused on large-
scale identification (8) and the deter-
mination or confirmation of primary
structure (9), whereas newer technol-
ogies have laid the ground work for
the study of tertiary and even higher
order structures of protein molecules
and complexes.
The analysis of biopharmaceuticals
(therapeutic proteins developed for
disease treatment) requires analytical
techniques that are able to elucidate
the various structural levels to ensure
their eff icacy and safety in patients.
Several MS techniques are indispens-
able in the toolbox of physicochemi-
cal characterization methods available
for the analysis of therapeutic proteins
(10). Some of the methods used in
the elucidation of higher order struc-
tural elements of proteins—including
native MS, ion mobility MS, hydro-
gen–deuterium exchange MS, and
chemical cross-lining MS—are dis-
cussed in this article.
Native Mass Spectrometry
Experiments using electrospray ion-
ization mass spectrometry (ESI–MS)
for the analysis of intact proteins were
first performed by J.B. Fenn’s group
(6). The study used strongly dena-
turing conditions created by using
50–90% organic solvent contain-
ing acetic acid or trif luoroacetic acid
and was beneficial for the detection
of multiple-charge protein ions with
a quadrupole mass spectrometer with
an upper nominal mass limit of m/z
1500. However, it was soon discovered
that the charge-state distribution of
electrosprayed proteins was signif i-
cantly inf luenced by the protein struc-
Ph
oto
cre
dit
: D
ou
g A
rman
d/G
etty
Im
ages
NOVEMBER 2016 ADVANCES IN BIOPHARMACEUTICAL ANALYSIS 23www.chromatographyonline.com
ture prevalent under nondenaturing or
denaturing conditions (11). This dis-
covery then led to the implementation
of native MS.
Native MS aims to maintain the
3D structure of proteins or protein
complexes as much as possible during
an experiment (12) by using condi-
tions that ref lect the protein’s native
environment. The overall charge of a
protein ion is limited by the number
of ionizable functionalities that are
accessible on the surface for charg-
ing, predominantly through proton-
ation or deprotonation, leading to the
observation of low-charged species in
mass spectra. Weakly bound, nonco-
valent complexes—including proteins
interacting with inhibitors, cofactors,
metal ions, carbohydrates, or pep-
tides—can be preserved during the
electrospray process facilitating the
study of the structure, stoichiometry,
and association constant of such bio-
molecular complexes (13). The reduc-
tion of charge requires the use of mass
spectrometers with an extended mass
range such as time-of-f light (TOF)
(14), or more recently orbital ion trap
(15) mass analyzers to detect the low-
charged protein species.
Trastuzumab (INN; trade name
Herceptin) is a monoclonal antibody
that interferes with the human epider-
mal growth factor receptor 2 (HER2)
and is used to treat HER2-positive
breast cancers. Figures 1a and 1c
illustrate the difference between mass
spectra of trastuzumab when analyzed
under denaturing (Figure 1a) versus
nondenaturing conditions (Figure 1c).
Under denaturing conditions (Figure
1a), charge states from 33+ to 60+ are
detected in an m/z range of 2200–4500,
whereas nondenaturing conditions
(Figure 1c) yield charge states of 22+
to 28+ at m/z 5200–6700. Deconvolu-
tion of both mass spectra gives equiva-
lent masses for the uncharged species
with masses in the range of 147–148
kDa and also reveals several different
protein species that represent the dif-
ferent glycoforms of the monoclonal
antibody. Such analysis can therefore
readily reveal the glycosylation pat-
tern of the protein, a highly important
quality parameter of recombinant bio-
pharmaceuticals.
Native MS has been shown to be a
highly efficient tool for determining
binding stoichiometry of a monoclonal
antibody with its antigen. Humanized
murine monoclonal antibody (hzmAb),
directed against the junctional adhe-
sion molecule A (JAM-A) to have anti-
proliferative and antitumoral prop-
erties, was titrated with its antigen
and then analyzed using native MS
to reveal noncovalent complex stoi-
chiometries (17). Three species were
detected when equimolar amounts of
antibody and its target antigen were
incubated including free antibody, 1:1,
and 1:2 antibody–antigen complex.
bc
a
1000
20
00
40
00
60
00
80
00
40
00
2000 3000 4000
3000 4000 5000 6000 7000 8000
m/z
m/z
5 10
Drift Time (millisec)
15 20 25
5 10
Drift Time (millisec)
15 20 25 30
d
e
(a) (b)
(c) (d)
45+
42+
23+27+
25+
ESI -sourceIon
guide Quadrupole TWIMS cell
Trap Transfer
Pusher Detector
Reflectron
Figure 1: MS and IMS analysis of intact trastuzumab. (a) and (c): Intact MS analy-sis of trastuzumab. ESI-TOF mass spectra of trastuzumab in denaturing (a) or na-tive (c) conditions. The inserts shows an extended view of the 44+ (a) and 25+ (c) charge states with resolution of the different glycoforms: (a) 147 917.1 ± 1.1 Da (G0/G0F), (b) 148 061.7 ± 0.8 Da (G0F/G0F), (c) 148 222.4 ± 0.9 Da (G0F/G1F), (d) 148 383.8 ± 0.8 Da (G1F/G1F), and (e) 148 544.3 ± 1.0 Da (G1F/G2F). (b) and (d): IMS analysis of trastuzumab. Ion mobility mobilograms of trastuzumab in de-naturing (b) or native (d) conditions. IMS data obtained in native conditions (d) reveal small amounts of dimeric mAb. Adapted and reproduced with permission from reference 16, ©American Chemical Society.
Figure 2: Schematic diagram of a quadrupole-traveling wave ion mobility–time of flight instrument.
24 ADVANCES IN BIOPHARMACEUTICAL ANALYSIS NOVEMBER 2016 www.chromatographyonline.com
Two molar equivalents of antigen led
to an almost quantitative formation of
the 1:2 complex, while eightfold molar
excess yielded a 1:4 complex with a
small portion of 1:3 complex.
Ion Mobility Spectrometry
Developed in the 1960s, ion mobil-
ity spectrometry (IMS) enables the
generation of size- and conformation-
dependent information that is not pos-
sible using MS alone. When coupled
to MS this technique has the potential
to separate isomers, isobars, and con-
formers; significantly reduce chemical
background; and detect aggregates of
biopharmaceuticals. IMS separation
of ions is possible using differing sepa-
ration powers, analyte detection, and
hyphenation to mass spectrometry
(18), including drift-time ion mobil-
ity spectrometry (DTIMS); aspiration
ion mobility spectrometry (AIMS);
field-asymmetric waveform ion mobil-
ity spectrometry (FAIMS); and trav-
eling-wave ion mobility spectrometry
(TWIMS). DTIMS and TWIMS are
the two principles most often used in
commercial instruments. In a DTIMS
device ions are moved through a uni-
form, linear-field drift tube filled with
a so-called “buffer gas” through a small,
uniform electric f ield. The moving
ions are attenuated by collisions with
the buffer gas depending on their over-
all charge and collision cross-section
(18). Ions with multiple charges and a
small cross-sectional area move faster
through the drift cell than low-charge
ions with large collisional cross-sec-
tions. TWIMS is a type of IMS that
utilizes traveling waves created by a
series of ring-shaped electrodes that
split the structure of the drift cell into
a series of segments (Figure 2). Instead
of a uniform linear field, a high field
is applied to one segment of the cell
that is subsequently swept through the
cell in the direction of ion migration.
Consequently, movement and sepa-
ration of ions in the mobility cell is
accomplished by means of pulses of an
electric field passing through.
An example for the appl icat ion
of T W I M S t o t he a n a l y s i s of
monoclonal antibodies is illustrated
in Figures 1b and 1d. IMS analysis
of t ra stuzumab under denaturing
condit ions revea ls a la rge number
of highly charged species clustering
at dri f t t imes between 10 and 15
ms, while native conditions clearly
dist inguish between the dif ferent,
low-charged species in a drift time
range of 7–25 ms. This contrast in
drift behavior is advantageous for the
analysis of more complex mixtures
of biopharmaceutica ls, particularly
when looking at sequence variants
o r o t h e r p o s t - t r a n s l a t i o n a l
modif ications such as ox idation or
pyroglutamate formation.
A n o t h e r p r a c t i c a l e x a m p l e
o f I M S c h a r a c t e r i z a t i o n o f
biopharmaceut ica l s ( les s directed
towards higher order elucidation) is
outlined in Figure 3. Here, a reduced
mouse monoclonal antibody (IgG1,
κ) sample comprising of both heavy
and l ight cha ins was int roduced
into an ESI-quadrupole-IMS-TOF
system (19). The two-dimensiona l
(2D) ion mobilogram-mass spectrum
depicted in Figure 3a clearly shows
that light and heavy chains can be
readily separated as different species
without any other upfront separation
technique. Multiple charged species
related to the light and heavy chains
were d i f f e r ent i a t ed u s i ng m a s s
spectra extracted from the encircled
areas in Figure 3a without mutually
interfering signals. Extracted mass
spectra (Figures 3b and 3d) were
deconvoluted using a ma x imum
entropy algorithm, yielding spectra
Light Chain
Heavy Chain
2000
1500
1000
700
10026+
(b)
(a)
(d)
(e)(c)
28+ 23+
20+
17+
52+
45+
41+
36+
800 1200 1600 2000 2400 28000
%
100
024050 24150 24250 24350 24450 49600 49800 50000 50200 50500
massmass
50249
49779
49924
50086
** *
*
** *
* *
2422624177
24199
%
100
0
%
100
800 1200 1600 2000 2400 2800
m/zm/z
0
%
3.2 6.4
Drift Time (millisec)
m/z
9.6 12.8
Figure 3: On-line LC–MS analysis of a completely reduced IgG1 antibody using ion mobility-TOF mode. (a) Two-dimensional plot of ion drift time versus m/z for the reduced IgG1 obtained using the ion mobility separation (7.5 V pulse). (b) Combined raw mass spectrum of the light chain. (c) Deconvoluted mass spec-trum of the light chain. (d) Combined raw mass spectrum of the heavy chain. (e) Deconvoluted mass spectrum of the heavy chain. Adapted and reproduced with permission from reference 19, ©John Wiley and Sons.
NOVEMBER 2016 ADVANCES IN BIOPHARMACEUTICAL ANALYSIS 25www.chromatographyonline.com
of uncharged species, as i l lustrated
in Figures 3c and 3e. As expected,
the light chain is detected as a single
species, while the spectrum of the
heavy chain revea ls at lea st three
glycoforms, characterized by different
ga lactose content in the at tached
N-glycan (162 Da mass difference).
This example nicely demonstrates the
benefits of an additional dimension
of separation, although an upfront
sepa rat ion method such a s high
performance liquid chromatography
(HPLC) or capillary electrophoresis
(CE) may be necessary—especia l ly
for the quantitative analysis of trace
amounts of impurities that is often
indispensable for quality control.
Hydrogen–Deuterium
Exchange Mass Spectrometry
The native state of proteins is gener-
ally characterized by a tightly folded,
compact structure that exposes a
well-def ined surface to its environ-
ment. Denaturation under conditions
such as high temperature, extreme pH,
adsorption to surfaces, or dissolution
in organic solvent results in the protein
unfolding and forming a significantly
different surface. Similarly, interac-
tions of a protein with other molecules
such as small drugs, nucleic acids, or
other proteins can lead to a significant
change in surface properties.
Denaturing and complex formation
can also have a profound inf luence on
the exchangeability of protons at the
surface of a protein molecule—the
acidic protons of the carboxyl groups
or acidic side chains of aspartate and
glutamate are normally the most eas-
ily and rapidly exchanged while the
amide protons of the protein back-
bone are much less prone to exchange.
Exchange can be monitored by dissolu-
tion of a protein in heavy water (D2O),
which leads to a hydrogen exchange by
deuterium in a few minutes to hours.
In proteins, the exchange rates for
the different hydrogen atoms strongly
depend on accessibility and therefore
on protein conformation or associa-
tion into higher order structures. The
substitution of exchangeable hydrogen
atoms with deuterium atoms forms the
basis of hydrogen–deuterium exchange
mass spectrometry (HDX-MS) (20).
A schematic workf low of HDX-MS
is depicted in Figure 4. In brief, a
protein with exchangeable hydrogens
is dissolved for different periods of
time at ambient or elevated incuba-
tion temperature (20–40 °C) in deu-
terated water (buffered to pH around
7). Depending on exchangeability,
hydrogen atoms are replaced by deu-
terium atoms during the incubation
time, before the exchange is quenched
upon acidif ication and cooling to
0 °C. Proteins are then digested under
quenching conditions, and the result-
ing peptides are separated by low-tem-
perature HPLC, and finally analyzed
by tandem mass spectrometry (MS/
MS) upon fragmentation either by col-
lision-induced dissociation (CID) or
electron-transfer dissociation (ETD).
Characteristic mass shifts in the frag-
ment ions are indicative for the pres-
ence and positions of deuterium atoms.
Analysis of the kinetics of deuterium
uptake yields information about the
accessibility of hydrogens at different
positions in the protein, which allows
valuable insights into the 3D structure
of proteins or protein complexes.
HDX-MS has been successfully used
to compare 3D structures of biophar-
maceuticals, which is essential to dem-
onstrate manufacturing consistency to
regulatory agencies or provide a proof
of structural equivalence between an
originator biopharmaceutica l and
its biosimilar. The advantage of this
approach is that it probes the whole
molecule instead of just certain sub-
structures. The results of an interroga-
tion of the 3D structure of interferon-
β-1a, a 20-kDa cytokine used to treat
multiple sclerosis (traded under the
names Avonex (Biogen), Rebif (Merck
Serono or Pfizer), or CinnoVex (Cin-
naGen) as a biosimilar, are shown in
Figure 5 (22). Hydrogen exchange
rates determined for f ive different
peptic peptides effectively show the
impact of different manufacturing
conditions as well as post-translational
modif ications—modif ication with
poly(ethylene glycol) (PEG); or oxi-
dation at C17, M1, M36, M62, and
M117—on protein structure.
No s i g n i f i c a n t a l t e r a t i on i n
h y d r o g e n – d e u t e r iu m e x c h a n g e
prof i le was observed, even though
p r o d u c t i o n i n v o l v e d d i f f e r e n t
batches, using different cell media,
and was subjected to N-termina l
modification with PEG. A significant
i mpa c t w a s howe ve r f ou nd f o r
methionine or cysteine ox idat ion,
and because the ox idized peptides
i n c o r p o r a t e d m o r e d e u t e r i u m
compared to the reference analogues,
it could be concluded that oxidized
i nte r f e ron-β-1a i s more so lvent
exposed and less hydrogen bonded.
H HH H
Z7
t1
H/D exchange
Quench
(pH 2.5, 0oC )
(pepsin, pH 2.5, 0oC )
Cooled
LC-MS
Time
co
nte
nt
Time
(protein)
Gas-phase
Solution-phase
cleavage
cleavage
(peptide)
Gas-phase
cleavage
D2O
t2
t3
t4
C7
C6
C5
C4
C3
C2
C1
R1
N
O
NN
NN
NN
NOH
OOOO
O O O
2
R3
R5
R7
R8
R6
R4
R2
Z6
Z5
Z4
Z3
Z2
Z1
H
H
H
H
H
HH
H
HH
H
H
H
H
HH
H
H
H
H
HH
H H
HH
H H
H
H
HH H
H
H
D
DD
D
DD
D
D
DDD
D
D
D D
D
D
H
HH
H
H
H
H H
H
H
HH H
H
H
D
DD
D
DD
D
D
DDD
D
D
co
nte
nt
D
D
D
H
HH
H
H
H
HH
H
H
HH H
H
H
D
DD D
D D
D
D
DDD
D
D
D
H
HH
H
H
H
Figure 4: Principle of hydrogen/deuterium exchange mass spectrometry. Adapted and reproduced with permission from reference 21, ©American Chemical Society.
26 ADVANCES IN BIOPHARMACEUTICAL ANALYSIS NOVEMBER 2016 www.chromatographyonline.com
Although this example convinc-
ingly demonstrated the applicability of
HDX-MS for revealing structural dif-
ferences in homogenous biopharma-
ceuticals, the authors also pointed out
that it is not capable, at the moment,
to detect conformational differences
in coexisting, low-level (<10%) com-
ponents of the population (22).
Chemical Cross-Linking
Mass Spectrometry
Three-dimensional structures of pro-
teins can be determined with atomic
resolution by using high-resolution
methods such as X-ray crystallography
or nuclear magnetic resonance (NMR)
spectroscopy, but the high amount
of sample required for these meth-
ods (typically in the milligram range)
makes them impractical for biological
studies. In comparison, low-resolution
structural data generated by chemical
cross-linking MS (CXMS) uses much
less sample amounts (in the nanogram
to picogram range) to generate highly
valuable data (23). Low-resolution
structural information is obtained by
chemically cross-linking functional
groups in a protein by means of a
bifunctional cross-linker, which gives
information about the distance of the
cross-linked functional groups in a
protein molecule or a protein complex.
The most common functional groups
available for cross-linking in proteins
are the lysine amino groups. Sulfydryl
groups of cysteines are another possi-
bility, but they can become involved
in the 3D structure of a protein par-
ticularly when created by reduction of
disulfide-bridges in the native protein.
Although formaldehyde is the oldest
cross-linking reagent, the most com-
monly utilized reagents are based on
bifunctional N-hydroxy-succinimide
esters, which readily react with free
amino groups (and in a side reaction
also with hydroxyl groups of tyrosine)
to create a stable amide or imide bond
upon release of N-hydroxysuccinimide.
Depending on the length of the cross-
linking spacer, different distances of
amino acids can be probed, ranging
from (almost) zero for formaldehyde to
6.4 Å for disulfosuccinimidyl tartrate,
11.4 Å for bis(sulfosuccinimidyl)suber-
ate (10 atoms), and 16.1 Å for ethylene
glycol bis(sulfosuccinimidyl succinate
(14 atoms) (24).
Figure 6 shows an outline of a cross-
linking experiment. After the forma-
tion of intramolecular or intermolecu-
lar cross-links, the protein or protein
complex is proteolytically digested
and the resulting peptides are ana-
lyzed via HPLC–MS/MS. Because the
crosslink remains unaffected by the
proteolysis, cross-linked amino acids
are revealed through the correspond-
ing cross-linked peptides. To more
easily identify cross-linked products,
isotope-labeled cross-linking reagents
with 50% heavy isotope exchange can
be used. Thus, cross-linked peptides
are recognized by 1:1 doublets of mass
signals for the light and heavy ver-
sions, which is usually achieved with
the help of computer-based searching
algorithms (25). The distance infor-
mation obtained from the cross-link-
ing experiment is then used to build
and verify structural models for pro-
teins or protein complexes.
Chemical cross-linking can also
be utilized to directly analyze stabi-
lized protein complexes. For example,
disuccinimidyl suberate, as well as
1,1′-(suberoyldioxyl)bisazabenzotri-
azole) were used as cross-linkers to
stabilize the complex between the
bovine prion protein and a specif ic
1. (8 - 15)FLQRSSNF
76543210
0 1 10 100 1000
5
4
3
2
1
00 1 10 100 1000
12
10
8
6
4
2
00 1 10 100 1000
12
10
8
6
4
2
00 1 10 100 1000
5678
43210
0 1 10 100 1000
76543210
0 1 10 100 1000
5
4
3
2
1
00 1 10 100 1000
12
10
8
6
4
2
00 1 10 100 1000
12
10
8
6
4
2
00 1 10 100 1000
5678
43210
0 1 10 100 1000
76543210
0 1 10 100 1000
5
4
3
2
1
00 1 10 100 1000
12
10
8
6
4
2
00 1 10 100 1000
12
10
8
6
4
2
00 1 10 100 1000
5678
43210
0 1 10 100 1000
76543210
0 1 10 100 1000
5
4
3
2
1
00 1 10 100 1000
12
10
8
6
4
2
00 1 10 100 1000
12
10
8
6
4
2
00 1 10 100 1000
5678
43210
0 1 10 100 1000
Rela
tive a
bu
nd
an
ce (
Da)
Rela
tive a
bu
nd
an
ce (
Da)
Rela
tive a
bu
nd
an
ce (
Da)
Rela
tive a
bu
nd
an
ce (
Da)
2. (62 - 67)MLQNIF
3. (88 - 101)LANVYHQINHLKTV
4. (121 - 134)HLKRYYGRILHYLK
5. (154 - 162)FYFINRLTG
(a)Differentbatches
(c)Different
media
(b)Pegylated
(d)Oxidized
1. (8- 15)
Time (min) Time (min) Time (min) Time (min) Time (min)
3. (88- 101)
5. (154- 162)2. (62- 67)
4. (121- 134)
Figure 5: Deuterium incorporation graphs generated for five interferon-β-1a (IFN) peptic peptides from four different HDX MS comparability experiments. In each graph, the reference IFN data are the black lines with closed triangles, while the experimental IFN data, to which it is being compared, is the red line with open circles. Row a: Comparison of two different large scale IFN batches prepared over eight years apart; row b: Comparison of IFN versus N-terminally PEGylated IFN; row c: Comparison of IFN produced using different cell culture media and growth conditions; row d: Comparison of IFN versus oxidized IFN (oxi-dation of Met and Cys residues C17, M1, M36, M62, and M117 was 100%). Adapt-ed and reproduced with permission from reference 22, ©John Wiley and Sons.
NOVEMBER 2016 ADVANCES IN BIOPHARMACEUTICAL ANALYSIS 27www.chromatographyonline.com
antibody against it, the antibody
3E7 (26). Direct analysis of the reac-
tion products by matrix-assisted laser
desorption–ionization MS revealed
both the free prion protein and the
free antibody together with 1:1 and
1:2 antibody–prion protein complexes.
Conclusions
In conclusion, MS, tradit iona l ly
regarded as one of the most important
analytical methods for the determina-
tion of the primary structure of pro-
teins, is increasingly contributing to
the elucidation of diverse fundamental
aspects of the tertiary and even quater-
nary structure of proteins and protein
complexes. In spite of providing less
spatial resolution, the major strength
of MS-based investigations compared
to NMR spectroscopy or X-ray crys-
tallography lies within the compara-
tively low amounts of sample required
for successful analysis, typically a few
picograms to nanograms. Such stud-
ies are, however, only feasible with
substantial support through elabo-
rate computational algorithms and
workf lows, which requires significant
involvement of bioinformatics into
data evaluation.
Acknowledgments
The financial support by the Austrian
Federal Ministry of Economy, Family,
and Youth, the National Foundation
of Research, Technology, and Devel-
opment, and by a Start-up Grant of
the State of Salzburg is gratefully
acknowledged.
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(11) J.A. Loo, H.R. Udseth, and R.D. Smith,
Biomed. Environ. Mass Spectrom. 17, 411–
414 (1988).
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186 (2000).
(14) M.C . Fitzgerald, I. Chernushevich, K .
G. Standing, C. P. Whitman, and S. B.
Kent, Proc. Natl. Acad. Sci. U.S.A . 93,
6851–6856 (1996).
(15) S. Rosati, R .J. Rose, N.J. Thompson,
E . van Duijn, E . Damoc, E . Denisov,
A. Makarov, and A. J. R. Heck, Angew.
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(16) A. Beck, S. Sanglier-Cianferani, and A.
Van Dorsselaer, Anal. Chem. 84, 4637–
4646 (2012).
(17) C . Atmanene, E . Wagner-Rousset, M.
Malissard, B. Chol, A. Robert, N. Cor-
vaia, A. Van Dorsselaer, A. Beck, and
S. Sanglier-Cianferani, Anal. Chem. 81,
6364–6373 (2009).
(18) A.B. Kanu, P. Dwivedi, M. Tam, L. Matz,
and H.H. Hill, J. Mass Spectrom. 43,
1–22 (2008).
(19) P. Olivova, W. Chen, A.B. Chakraborty,
and J.C. Gebler, Rapid Commun. Mass
Spectrom. 22, 29–40 (2008).
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Soc. 115, 6317–6321 (1993).
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(27) h t t p : //d a l t o n l a b . i q m .u n i c a m p . b r /
research.html.
Christian Huber was educated as
an analytical chemist from 1985 to 1993
at the University of Innsbruck, Austria.
Following a lecturing qualification at
the University of Innsbruck in 1997, he
held the chair of analytical chemistry
position at Saarland University in
Germany from 2002 to 2008. In 2008,
he was made a professor of chemistry
for biosciences at the Department of
Molecular Biology of the University of
Salzburg, Austria. His research interests
include bioanalytical chemistry and
proteome and metabolome analysis, as
well as in-depth characterization of
therapeutic proteins. ◾
Proteolysis
LC-MS/MS
Cross-linking
Map of cross-links
Intra and inter-chainSelection of structural models cross-links
Set of distance restraints
Protein complexProtein 1
Protein 2
Protein 3
Figure 6: Schematic of a workflow for chemical cross-linking of protein complexes followed by digestion and analysis by HPLC–MS/MS. Reproduced with permission from reference 27.
28 ADVANCES IN BIOPHARMACEUTICAL ANALYSIS NOVEMBER 2016 www.chromatographyonline.com
Nico C. van de Merbel
Advances in Liquid Chromatography–Tandem Mass Spectrometry (LC–MS/MS)-Based Quantitation of Biopharmaceuticals in Biological Samples
Liquid chromatography coupled to tandem mass spectrometry
(LC–MS/MS) has recently become a more popular alternative to
traditional ligand-binding assays for the quantitative determination
of biopharmaceuticals. LC–MS/MS offers several advantages such
as improved accuracy and precision, better selectivity, and generic
applicability without the need for raising analyte-directed antibodies.
Here we discuss the technical requirements for a successful LC–MS/MS
method for the quantitation of biopharmaceuticals and evaluate the
advantages and disadvantages compared to ligand-binding assays.
The development of protein-based
pharmaceuticals, or biopharma-
ceuticals, is by far the fastest
growing part of the pharmaceutical
industry today. With more than 1500
biopharmaceuticals in clinical devel-
opment and more companies shifting
their research and development (R&D)
efforts towards this sophisticated and
relatively profitable class of drugs, the
pharmaceutical landscape has changed
beyond recognition compared to 20 or
even 10 years ago. As a result, the field
of bioanalysis that supports drug devel-
opment by measuring the concentra-
tions of drugs or relevant endogenous
molecules in biological samples has also
seen many changes. The quantitative
determination of biopharmaceuticals
has traditionally been the domain of
ligand-binding assays, such as enzyme-
linked immunosorbent assay (ELISA).
However, in the past few years there
has been a clear increase in the applica-
tion of alternative analytical platforms,
in particular liquid chromatography
coupled to tandem mass spectrometry
(LC–MS/MS), which has been the
workhorse for small-molecule bioanaly-
sis for more than 20 years (1–5).
Over the past decade, there have
been many advances in the LC–MS/
MS-based quantitation of biopharma-
ceuticals, both from an analytical and
a conceptual point of view. In this
article, an overview is given of the
many aspects of this field of analytical
research by reference to a selection of
recent applications.
Protein Digestion
MS/MS remains the detection tech-
nique of choice for the quantitative
determination of biopharmaceuticals
because of its sensitivity and wide-
spread availability in the pharmaceu-
tical and related industries. However,
the use of LC–MS/MS to quantify
biopharmaceuticals is more complex
than for small molecules because it
is not directly compatible with mol-
ecules with a mass above around
5000 Da. The ions of larger analytes
are distributed over many dif fer-
ent charge states and usually do not
readily fragment, which considerably
reduces sensitivity.
Therefore, a typical step in the
analysis is the (enzymatic) digestion
of a biopharmaceutical into a mixture
of smaller peptides, followed by the
analysis of the digest and quantitation
of one or more so-called signature pep-
tides as a measure for the intact pro-
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tein. Digestion is usually performed using the enzyme tryp-
sin, which cleaves the amino acids chain in proteins after a
lysine or arginine. Trypsin is popular because it is readily
available at a reasonable price and can cleave proteins into
peptides of a size (500–2000 Da) that is well suited for MS/
MS detection.
Protein digestion enormously increases the complexity of a
biological sample. Matrices such as plasma contain proteins
at a total concentration of around 80 mg/mL and, when no
further cleanup of the sample is performed, each of these
proteins is cleaved into a series of peptides that are all of a
similar size and have more or less comparable physicochemi-
cal and analytical properties. Therefore, it is often challeng-
ing to detect low concentrations of a signature peptide in
a digest, because of the presence of so many endogenous
peptides, which all consist of combinations of the same 20
amino acids and often occur at much higher levels than the
signature peptide itself.
Despite the selective nature of MS/MS detection, chro-
matograms of digested biological samples often contain
many background peaks originating from endogenous pep-
tides that show a response at the mass transition of the sig-
nature peptide. Figure 1 shows this effect for a fixed con-
centration of digested salmon calcitonin in the presence of
increasing amounts of digested plasma (6). The selectivity of
the method is clearly affected by the presence of endogenous
background peptides. As a result, method sensitivity is also
heavily impacted—in this case the achievable lower limit of
quantitation (LLOQ ) increases 100-fold, from 0.2 ng/mL
(60 pM) in the absence of matrix peptides to 20 ng/mL (6
nM) in the presence of 50% of digested plasma.
A review of current literature (1,4) shows that a typical
LLOQ for a biopharmaceutical in plasma or serum, only
treated by digestion, is in the high nanograms-per-milliliter
to low micrograms-per-milliliter range (corresponding to
low nanomolar levels for many proteins). Figure 2 shows an
example chromatogram for a signature peptide of recombi-
nant human α-glucosidase at its LLOQ of 0.5 μg/mL (5 nM)
in human plasma (7).
Signature Peptide Selection
The possibilities for selecting a proper signature peptide
are usually rather limited. First and foremost, it is essential
that the selected signature peptide has a unique amino acid
sequence that does not naturally occur in any of the endog-
enous matrix proteins. Selection of a non-unique signature
peptide results in an overestimation of analyte concentra-
tions, because the same amino acid sequence that is released
from endogenous proteins would contribute to the overall
signal. This often disqualifies a large number of the theo-
retical signature peptides, particularly for biopharmaceuti-
cals with a high degree of similarity to endogenous proteins,
such as humanized antibodies (if these need to be quantified
in human plasma). In addition, other criteria are applied to
ensure robustness of the LC–MS/MS assay. Peptides con-
taining unstable amino acids, such as methionine and tryp-
tophan that can be oxidized, or glutamine and asparagine
that can be deamidated, are usually disregarded to avoid
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30 ADVANCES IN BIOPHARMACEUTICAL ANALYSIS NOVEMBER 2016 www.chromatographyonline.com
losses during analysis, although forced
oxidation of a signature peptide to a sta-
ble oxidized product has been success-
fully used (8). Similarly, peptides with
(variable) post-translational modifica-
tions—such as O- or N-glycosylated
amino acids—are typically excluded
because these would introduce unde-
sirable heterogeneity. In addition, pep-
tides that are too small, too large, too
polar, or too hydrophobic might cause
analytical problems because of adsorp-
tion, sub-optimal chromatographic
behavior, or limited selectivity and sen-
sitivity. In the end, there may be just a
few out of the many potential signature
peptides that can be successfully used
in practice.
Protein Extraction
An obvious way to improve selectiv-
ity and sensitivity of an LC–MS/MS
method is to remove interfering matrix
proteins before digestion, which can
be achieved by applying immunocap-
ture (IC) techniques. Magnetic beads
or other resins are coated with a pro-
tein that displays a high binding affin-
100
0
1.75 2.00 2.25 2.50
2.94
0%
1%
5%
10%
20%
50%
2.93
1.84 2.644.24
3.723.583.22
2.922.64
2.50
2.332.162.041.76
2.162.33
2.50
2.54
2.91
3.23 3.74 4.23 4.54 4.86 5.055.44
5.40
5.754.944.514.384.20
3.723.54
3.21
2.902.812.47
2.302.14
2.031.76
2.002.22
2.74
3.043.19
3.453.66 4.20 4.41
4.72
5.24
1.76 1.932.04
5.454.874.554.24
2.75 3.00 3.25 3.50 3.75 4.00 4.25 4.50 4.75 5.00 5.25 5.50 5.75 6.0
1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 4.25 4.50 4.75 5.00 5.25 5.50 5.75 6.0
1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 4.25 4.50 4.75 5.00 5.25 5.50 5.75 6.0
1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 4.25 4.50 4.75 5.00 5.25 5.50 5.75 6.0
1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 4.25 4.50 4.75 5.00 5.25 5.50 5.75 6.0
1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50
Time (min)
Time (min)
Time (min)
Time (min)
Time (min)
Time (min)
3.75 4.00 4.25 4.50 4.75 5.00 5.25 5.50 5.75 6.0
%
100
0
%
100
0
%
100
0
%
100
0
%
100
0
%
Figure 1: LC–MS/MS (m/z 561.9 to m/z 204.0) chromatograms of a signature peptide of 2 ng/mL salmon calcitonin in samples containing increasing amounts of human plasma digest. Analyte peak at 2.9 min. Adapted and reproduced with permission from reference 6, ©American Chemical Society.
NOVEMBER 2016 ADVANCES IN BIOPHARMACEUTICAL ANALYSIS 31www.chromatographyonline.com
2.94
1000
800
600
400
200
Inte
nsi
ty, cp
sIn
ten
sity
, cp
s
0
1000(b)
(a)
800
600
400
200
0
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0
0.5 1.0 1.5 2.0
Time (min)
Time (min)
2.5 3.0 3.5 4.0 4.5 5.0
1.04
1.04
2.96
3.20
3.43 4.054.12
1.441.52 1.62 2.50 2.64
3.173.40
3.49 3.69
4.014.11
Figure 2: LC–MS/MS (m/z 616.1 to m/z 1030.7) chromatogram of the signature pep-tide of recombinant human α-glucosidase in human plasma; (a) blank and (b) 0.5 μg/mL, pretreated with digestion only.
ity towards the analyte, typically an
antibody raised against the analyte or
the pharmacological target to which a
biopharmaceutical is directed. By mix-
ing the sample with a suspension of
the beads or passing it through a car-
tridge filled with the resin, the analyte
is selectively isolated from the complex
sample. This approach is particularly
popular for the quantitation of endog-
enous proteins such as biomarkers, for
which well-characterized immunologi-
cal reagents are widely available.
One example is an LC–MS/MS
method for parathyroid hormone
(PTH) in human serum (9). A sample
of 1 mL was treated by IC with poly-
styrene beads coated with murine
anti-PTH antibodies and the trapped
analyte digested with trypsin. The IC
treatment allowed the quantitation
of PTH down to 40 pg/mL (4 pM)
in serum, which shows the enormous
clean-up potential of this approach.
A completely 15N-labeled form of
PTH was added to the sample as an
internal standard at the very begin-
ning of the sample handling proce-
dure. In general, it is desirable that a
stable-isotope-labeled or other closely
related form of the protein analyte be
included in the method as an internal
standard, to correct for the variability
of the extraction procedure. This is
also one of the drawbacks of extract-
ing a biological sample before digestion,
because such a protein-based internal
standard can usually only be obtained
by biotechnological means, which may
be difficult, if not impossible (4).
The disadvantages associated with the
use of immunological reagents—such
as their potentially limited availability,
varying quality, and the interference
of matrix proteins with the extraction
efficiency—have prompted research-
ers to investigate alternative so-called
antibody-free extraction approaches
(5). An interesting technique is immo-
bilized-metal affinity chromatography
(IMAC), which is based on the interac-
tion of metal ions, such as Ni2+, with
amino acids that feature strong elec-
tron donor groups, such as histidine.
Proteins with such amino acids on their
surface will be selectively captured by
IMAC resins. As an example, the bio-
pharmaceutical recombinant human
tumor necrosis factor-related apop-
tosis-inducing ligand (rhTRAIL) has
been quantified in human and mouse
serum down to 20 ng/mL (340 pM)
by removing 95% of matrix proteins,
while recovering >70% of the analyte
with IMAC (8).
Another technique is solid-phase
extraction (SPE) with ion-exchange
materials, which separates proteins
based on their isoelectric point (pI).
Proteins with a relatively high pI bear a
net positive charge and can be trapped
on a cation-exchange resin at neutral
or slightly alkaline pH, at which many
endogenous proteins with a lower pI
will be negatively charged and thus
not be captured. The extraction of
rhTRAIL with strong-cation exchange
SPE was found to have a similar clean-
up potential to IMAC, with an analyte
recovery of 70% and a protein removal
eff iciency of 99%. As an illustra-
tion, Figure 3 shows chromatograms
obtained for 10 ng/mL (170 pM) of
rhTRAIL in human serum, which was
extracted by strong cation exchange or
IMAC, followed by trypsin digestion
and LC–MS/MS analysis of the signa-
ture peptide.
Peptide Extraction
Removal of interfering matrix com-
ponents is also possible after diges-
tion, that is, at the peptide level. This
approach has some distinct advantages.
From a practical point of view, the opti-
mization of an SPE procedure is more
straightforward because of the wide
availability of a range of materials that
are commonly used for small-molecule
extractions and because of the more
predictable extraction behavior of
smaller peptides compared to that of
intact proteins.
The accuracy and precision of extrac-
tions may be inf luenced by protein-
protein interactions in samples (such as
binding of a biopharmaceutical to its
target or to anti-drug antibodies), or
the occurrence of aggregates. If a sam-
ple is first subjected to digestion, these
interactions will no longer inf luence
the extraction because all proteins will
have been cleaved to peptides that are
much less likely to bind to one another
with a high affinity.
No less importantly, peptide extrac-
tion does not need a protein-based
internal standard; it can instead per-
32 ADVANCES IN BIOPHARMACEUTICAL ANALYSIS NOVEMBER 2016 www.chromatographyonline.com
form very well when using a stable-
isotope labeled form of the signature
peptide (4,6), which is considerably less
expensive and easier to obtain. It may,
however, be difficult to achieve suffi-
cient selectivity because the peptides in
a plasma digest are much more similar
to each other than the plasma proteins
were before digestion. Again, the high-
est selectivity and sensitivity is achieved
by applying immunocapture, which in
this case uses immobilized antibodies
raised against the signature peptide.
This approach is most widespread in the
field of biomarker analysis, where the
number of analytes is relatively limited
and assays are relevant to many research
groups around the globe. Large cleanup
efficiencies can be achieved in this way,
as was reported for the endogenous pro-
teins α1-antichymotrypsin (1453-fold
enrichment relative to matrix proteins)
and TNF-α (573-fold enrichment) (10).
IC at the peptide level is less popular
in biopharmaceutical analysis, probably
because of the general drawbacks of
antibody-based reagents with regard to
availability and batch-to-batch repro-
ducibility. A more generic approach
for peptide extraction from a digest is
to use conventional ion-exchange SPE,
but this needs to be carefully optimized
to obtain sufficient selectivity. A digest
of a protein-rich biological sample
(such as plasma) contains a multitude
of peptides, which all have carboxylic
and amine groups, and the signature
peptide can only be separated from
the excess of endogenous background
peptides if its pI value is sufficiently
different. Typically, the pH and ionic
strength of the loading, washing, and
elution steps need to be carefully opti-
mized for a selective extraction.
A biopharmaceutical nanobody was
quantified down to 10 ng/mL (360 pM)
in rabbit and human plasma by trypsin
digestion followed by SPE on a weak-
anion exchange phase (11). The signa-
ture peptide contained three carboxylic
acid groups and was strongly retained
by the positively charged SPE phase at
pH 5; many endogenous peptides with
less negative charges were not trapped
during sample loading or were removed
from the SPE material by a washing
step with 300 mM sodium chloride.
The mixed-mode SPE phase, which
also contained reversed-phase groups,
was subsequently neutralized at a high
pH and the (relatively polar) signature
peptide was eluted, while some less
polar endogenous peptides remained
bound by reversed-phase interactions.
In this way, two dimensions of selectiv-
ity (ion exchange and reversed phase)
were used to isolate the signature pep-
tide from the plasma digest. Figure 4
illustrates that many interfering peaks
were removed from the chromatogram
with this approach and that selectivity
was clearly improved. Of course, cat-
ion-exchange SPE can be applied in the
same way in case the signature peptide
100
%
0
100
%
0
2.32
1.513.90
4.27 5.07 5.25
5.71 5.84
4.59
2.843.52
4.06
4.20 4.55
5.03
5.305.47
6.195.88
(a)
(b)
Time (min)
Time (min)
Figure 4: LC–MS/MS (m/z 752.0 to m/z 773.3) chromatograms of the signature peptide of 10 ng/mL of a nanobody in human plasma (a) without or (b) with solid-phase extraction of the plasma digest. Analyte peak at 4.6 min. Adapted and reproduced with permission from reference 11, ©Future Science Ltd.
100 2.58e3
6.63
6.51
6.65
6.78
6.94
7.007.13
7.237.49
7.55
7.737.91
6.71
6.79 6.92
7.097.507.55
7.63
7.817.90
6.60 6.80 7.00 7.20 7.40 7.60 7.80
[rhTRAIL]=10 ng/mL [rhTRAIL]=10 ng/mL
SCX
0
%
100 2.02e3
6.60 6.80 7.00 7.20 7.40 7.60 7.80
Time (min)Time (min)
IMAC
0
%
Figure 3: LC–MS/MS (m/z 729.0 to m/z 942.4) chromatograms of the signature peptide of 10 ng/mL rhTRAIL in human serum and the corresponding blanks, pretreated with SCX or IMAC before digestion. Adapted and reproduced with permission from reference 5, ©Future Science Ltd.
NOVEMBER 2016 ADVANCES IN BIOPHARMACEUTICAL ANALYSIS 33www.chromatographyonline.com
100
012.50 13.00
12.70
(a)
Time (min) Time (min)
(b)
%100
013.00 14.00 15.00
15.4414.72
13.52
12.76
%Figure 5: LC–MS/MS (m/z 456.6 to m/z 852.5) chromatograms of the signature pep-tide (a) of 0.2 ng/mL of rhTRAIL spiked to dog saliva, plus corresponding blank, or (b) of endogenous human TRAIL in unspiked human saliva. Samples pretreated with IMAC before and SCX after digestion.
has multiple positive charges, and even
reversed-phase SPE might be an option
if the signature peptide is particularly
hydrophobic.
Combined Protein
and Peptide Extraction
As illustrated above, generic protein
or peptide extractions typically result
in LLOQs in the low nanograms-per-
milliliter (mid to high picomolar) range,
while IC extraction at the protein or
peptide level enables quantitation down
to mid picograms-per-milliliter (low to
mid picomolar) concentrations. If more
sensitivity is required, one option is to
combine protein and peptide extrac-
tions. Excellent selectivity and sensitiv-
ity can be reached even without anti-
body-based extraction materials, as was
shown for rhTRAIL in saliva (12). After
IMAC extraction of the protein analyte
and trypsin digestion, the digest was
further purified using SPE on a strong
cation exchange cartridge. Because of
the presence of four basic amino acids
in the signature peptide, the digest was
acidified before loading onto the SPE
phase. The peptide was then trapped
and endogenous peptides were removed
by washing with 200 mM sodium chlo-
ride. After elution at alkaline pH, the
signature peptide was quantified using
LC–MS/MS. As shown in Figure 5, a
TRAIL concentration as low as 0.2 ng/
mL (3.4 pM) could be quantified in
both dog and human saliva. In prin-
ciple, protein or peptide extractions
can be combined in many ways and as
long as the separation mechanisms are
orthogonal, improved selectivity and
sensitivity can be expected compared
to a single-extraction approach.
The ultimate combination of protein
and peptide extraction is IC of the pro-
tein analyte followed by digestion and
IC of the signature peptide. Although
this requires two specifically raised
antibodies and is by no means a generic
approach, it can result in impressive
sensitivities. The biomarker interleu-
kin-21 (IL-21) was quantified in human
serum and monkey tissues with an
LLOQ of 0.78 pg/mL (0.05 pM). This
was achieved by combining off-line
magnetic bead-based protein extraction
using an anti-IL-21 antibody with on-
line enrichment of the signature pep-
tides using immobilized anti-peptide
antibodies (13). Figure 6 shows repre-
sentative chromatograms. It is impor-
tant to realize that the obtained LLOQ
corresponds to a molar concentration
of the protein, which is five orders of
magnitude lower than that shown in
Figure 2 (digestion only). This con-
vincingly demonstrates the enormous
cleanup capability of this combination
of techniques.
LC–MS/MS Versus ELISA
Compared to ligand-binding assays,
LC–MS/MS has a number of analyti-
cal advantages such as a larger linear
dynamic range; (usually) higher accuracy
and precision because of the possibility
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34 ADVANCES IN BIOPHARMACEUTICAL ANALYSIS NOVEMBER 2016 www.chromatographyonline.com
to apply internal standards (4); the ability
to quantify multiple analytes simultane-
ously; and the fact that it does not neces-
sarily require immunological reagents (5).
The last point can be especially critical,
because such reagents may be problem-
atic to obtain or show a large batch-to-
batch variability, which makes compari-
son of results between laboratories or
over a longer period of time difficult,
if not impossible. The disadvantages of
LC–MS/MS include its generally higher
operational cost; more limited sample
throughput; and less favorable concen-
tration sensitivity. In addition, with the
digestion step that is generally needed
for LC–MS/MS, the three-dimensional
(3D) structure of a protein analyte is lost
and the analytical principle is therefore
not related to the complex molecular
structure of a protein, which determines
its pharmacological activity.
Now that more and more reports are
appearing that compare newly devel-
oped LC–MS/MS methods with exist-
ing ELISAs for the same protein ana-
lyte, it is becoming increasingly clear
that both techniques do not always give
superimposable concentration results
(14,15). Although in the world of small-
molecule quantitation, two different
results for the same sample would be
seen as proof that at least one of them
is incorrect, this is not necessarily true
for biopharmaceuticals. It should be
realized that, in contrast to small mol-
ecules, LC–MS/MS and ELISA only
use a small part of the protein molecule
for the actual quantitation, the signa-
ture peptide and the binding epitope,
respectively, and this may represent as
little as a few percent of the entire mol-
ecule. Furthermore, both techniques
are based on quite different biochemi-
cal principles, to which the structurally
complex and often heterogeneous bio-
pharmaceuticals may respond in dif-
ferent ways. Thus, neither LC–MS/MS
nor ELISA should be regarded as the
ultimate quantitation technique for bio-
pharmaceuticals, but rather as comple-
mentary tools for obtaining quantitative
information about this complicated but
very interesting class of compounds.
Acknowledgments
Stichting Technische Wetenschappen
(STW) and Samenwerkingsverband
Noord-Nederland (SNN) are gratefully
acknowledged for providing financial
support for part of the work described
in this paper.
References
(1) R. Bischoff, K.J. Bronsema, and N.C. van
de Merbel, Trends Anal. Chem. 48, 41–51
(2013).
(2) G. Hopfgartner, A. Lesur, and E. Varesio,
Trends Anal. Chem. 48, 52–61 (2013).
(3) I. van den Broek, W.M. Niessen, and
W.D. van Dongen, J. Chromatogr. B 929,
161–179 (2013).
(4) K.J. Bronsema, R. Bischoff, and N.C. van
de Merbel, J. Chromatogr. B 893-894,
1–14 (2012).
(5) D. Wilffert, R. Bischoff, and N.C. van de
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(6) K.J. Bronsema, R. Bischoff, and N.C. van
de Merbel, Anal. Chem. 85, 9528–9535
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(7) K.J. Bronsema, R . Bischoff, W.W.M.P.
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van de Merbel, Anal. Chem. 87, 4394–
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(8) D. Wilffert, C.R. Reis, J. Hermans, N.
Govorukhina, T. Tomar, S. de Jong, W.J.
Quax, N.C. van de Merbel, and R. Bischoff,
Anal. Chem. 85, 10754–10760 (2013).
(9) V. Kumar, D.R. Barnidge, L.S. Chen, J.M.
Twentyman, K.W. Cradic, S.K. Grebe,
and R.J. Singh, Clin. Chem. 56, 306–313
(2010).
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L.C. Feng, B.D. Piening, L. Anderson,
and A.G. Paulovich, Anal. Biochem. 362,
44–54 (2007).
(11) K.J. Bronsema, R. Bischoff, M.P. Bouche,
K. Mortier, and N.C. van de Merbel, Bio-
analysis 7, 53–64 (2015).
(12) D. Wilffert, unpublished results.
(13) J. Palandra, A. Finelli, M. Zhu, J. Mas-
ferrer, and H. Neubert, Anal. Chem. 85,
5522–5529 (2013).
(14) N.C. van de Merbel, K.J. Bronsema, and
M. Nemansky, Bioanalysis 4, 2113–2116
(2012).
(15) P. Bults, N.C. van de Merbel, and R .
Bischoff, Expert Rev. Proteomics 12, 355–
374 (2015).
Nico van de Merbel is scientific
director at the bioanalytical laboratories
of PRA Health Sciences in Assen, The
Netherlands and Lenexa, Kansas, and
honorary professor at the University of
the Groningen, The Netherlands. ◾
400
350
300
250
200
150
100
50
QLI
DIV
DQ
LK In
ten
sity
(a) (b)
04.5 5.0
Time (min) Time (min)5.5
400
350
300
250
200
150
100
50
04.5 5.0 5.5
Figure 6: LC–MS/MS chromatograms of two mass transitions (m/z 592.8 to m/z 943.5 in red and m/z 592.8 to m/z 830.5 in blue) of the signature peptide of IL-21 in human serum; (a) blank and (b) 0.78 pg/mL. Samples pretreated with immuno-capture both before and after digestion. Adapted and reproduced with permis-sion from reference 13, ©American Chemical Society.
NOVEMBER 2016 ADVANCES IN BIOPHARMACEUTICAL ANALYSIS 35www.chromatographyonline.com
Koen Sandra, Alexia Ortiz, and Pat Sandra
Analyzing Host Cell Proteins Using Off-Line Two-Dimensional Liquid Chromatography–Mass Spectrometry
Protein biopharmaceuticals are commonly produced recombinantly in
mammalian, yeast, or bacterial expression systems. In addition to the
therapeutic protein, these cells also produce endogenous host cell
proteins (HCPs) that can contaminate the biopharmaceutical product,
despite major purification efforts. Since HCPs can affect product
safety and efficacy, they need to be closely monitored. Enzyme-linked
immunosorbent assays (ELISA) are recognized as the gold standard for
measuring HCPs because of their high sensitivity and high throughput,
but mass spectrometry (MS) is gaining acceptance as an alternative
and complementary technology for HCP characterization. This article
reports on the use of off-line two-dimensional liquid chromatography–
mass spectrometry (2D-LC–MS) for the characterization of HCPs and
their monitoring during downstream processing.
In contrast to small-molecule drugs
that are commonly synthesized by
chemical means, protein biophar-
maceuticals result from recombinant
expression in nonhuman host cells.
As a result, the biotherapeutic is co-
expressed with hundreds of host cell
proteins (HCPs) with different physi-
cochemical properties present in a wide
dynamic concentration range. During
downstream processing, the levels of
HCPs are substantially reduced to a
point considered acceptable to regula-
tory authorities (typically <100 ppm–
ng HCP/mg product). These process-
related impurities are considered as
critical quality attributes because they
might induce an immune response,
cause adjuvant activity, exert a direct
biological activity (such as cytokines),
or act on the therapeutic itself (for
example, proteases) (1,2). To men-
tion some specif ic examples, during
the clinical development phase of
Omnitrope, Sandoz’s human growth
hormone biosimilar expressed in E .
coli, adverse events associated with
residual HCPs were encountered. The
European Medicines Agency (EMA)
only granted approval after additional
purification steps for HCP clearance
were incorporated (3–5). Scientists at
Biogen Idec demonstrated fragmenta-
tion of a highly purified monoclonal
antibody as a result of residual Chi-
nese hamster ovarian (CHO) cell pro-
tease activity in the drug substance,
despite an enormous purif ication
effort undertaken (protein A aff in-
ity chromatography with subsequent
orthogonal purif ication steps by cat-
ion- and anion-exchange chromatog-
raphy) (6). The authors of the study
state that it is of utmost importance
to identify residual protease activity
early in process development to allow
a revision of the purification scheme
or ultimately to knockdown the spe-
cific protease gene.
Multicomponent enzyme-linked
Ph
oto
Cre
dit
: M
on
ty R
aku
sen
/Get
ty I
mag
es
36 ADVANCES IN BIOPHARMACEUTICAL ANALYSIS NOVEMBER 2016 www.chromatographyonline.com
immunosorbent assay (ELISA) is pres-
ently the workhorse method for HCP
testing because of its high throughput,
sensitivity, and selectivity (1,2). Poly-
clonal antibodies used in the test are
typically generated by the immuniza-
tion of animals with an appropriate
preparation derived from the produc-
tion cell, minus the product-coding
gene. However, ELISA does not com-
prehensively recognize all HCP spe-
cies—that is, it cannot detect HCPs
to which no antibody was raised, it
only provides information on the total
amount of HCPs without providing
insight in individual HCPs, and, in a
multicomponent setup, it has a poor
quantitation power. In that respect,
MS nicely complements EL ISA
because it can provide both qualitative
and quantitative information on indi-
vidual HCPs. In recent years, various
papers have appeared dealing with the
mass spectrometric (MS) analysis of
HCPs (2,3,7–12). These studies typi-
cally rely on bottom-up proteomics
approaches in which peptides derived
from the protein following proteolytic
digestion are handled. A clear trend
is observed toward the use of upfront
multidimensional chromatography
to tackle the enormous complexity
and wide dynamic range (2,3,7,8,10).
Compared to one-dimensional liq-
Supernatants
Buffer exchange/desalting
Reduction/alkylation/digestion
Database search
Protein ID/Quant
High pH reversed-phase LC x low pH reversed-phase LC
QTOF MS/MS
Time (min)
0 5 10 15 20 25 30 35
mA
U
0
200
400
600
800
1000
1200
1400
Fraction 11-19
7x10
0
7x10
0
7x10
0
7x10
0
7x10
0
7x10
0
7x10
0
7x10
0
7x10
0
MS counts vs. Acquisition Time (min)
4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40
Fraction 12
Fraction 13
Fraction 14
Fraction 15
Fraction 16
Fraction 17
Fraction 18
Fraction 19
Fraction 11
Figure 1: Workflow for the character-ization of HCPs using off-line 2D-LC–MS/MS.
Figure 2: First-dimension reversed-phase LC–UV 214 nm chromatogram of a selected downstream manufacturing sample. HPLC system: Agilent Technologies 1200; Col-umn: 150 mm × 2.1 mm, 3.5-μm Waters XBridge BEH C18; mobile-phase A: 10 mM NH4HCO3 pH 10; mobile-phase B: acetonitrile; flow rate: 200 μL/min; gradient: 5–50% B in 30 min; column temperature: 25 °C; injection volume: 50 μL; fraction interval: 1.5 min (300-μL fractions).
Figure 3: Second dimension LC–MS/MS chromatograms of selected fractions (Fig-ure 2). HPLC system: Thermo Scientific Ultimate3000 RSLC nano; MS system: Agilent Technologies 6530 Q-TOF; column: 150 mm × 75 μm, 3-μm Thermo Scientific Acclaim PepMap100 C18; precolumn: 20 mm × 75 μm, 3-μm Acclaim PepMap100 C18 (Thermo Scientific); mobile-phase A: 2% acetonitrile, 0.1% formic acid; mobile-phase B: 80% acetonitrile, 0.1% formic acid; loading solvent: 2% acetonitrile, 0.1% formic acid; flow rate: 300 nL/min (nano pump), 5 μL/min (loading pump); gradient: 0–60% B in 60 min; column temperature: 35°C; injection volume: 20 μL.
NOVEMBER 2016 ADVANCES IN BIOPHARMACEUTICAL ANALYSIS 37www.chromatographyonline.com
uid chromatography (1D-LC), two-
dimensional LC (2D-LC) drastically
increases peak capacity as long as
the two dimensions are orthogonal
(13). In a 1D chromatographic setup
the separation space is dominated by
peptides derived from the therapeutic
protein, in 2D-LC the increased peak
capacity allows one to look substan-
tially beyond the therapeutic peptides
and detect HCPs at low levels. Three
recent papers using 2D-LC–MS/
MS demonstrate that HCPs can be
revealed at levels as low as 10 ppm
(2,3,7). In these cases, label-free quan-
tification was based on the three most
intense tryptic peptides making use of
single-point calibration against spiked
exogenous proteins.
An off-line 2D-LC–MS/MS setup
was used in our laboratory for the
characterization of HCPs throughout
the downstream manufacturing of a
therapeutic enzyme recombinantly
expressed in yeast. The workf low is
schematically presented in Figure 1.
Supernatant was collected at different
purification steps. Following desalting
of the supernatant, the proteins were
reduced using dithiothreitol (DTT)
and alkylated using iodoacetamide
(IAM) before overnight trypsin diges-
tion. The peptide mixture was subse-
quently subjected to 2D-LC–MS/MS.
In successfully applying 2D-LC ,
the selectivity of the two separation
mechanisms toward the peptides
must differ substantially to maximize
orthogonality and, hence, resolution.
Various orthogona l combinations
targeting different physicochemical
properties of the peptides have been
described. Bottom-up proteomics
setups initially relied on the com-
bination of strong-cation exchange
and reversed-phase LC to separate by
charge in the first dimension and by
hydrophobicity in the second dimen-
sion (13–15). In recent years, various
researchers have shifted their efforts
to the combination of reversed-phase
LC and reversed-phase LC (13,16–19).
The orthogonality in this nonobvious
combination is mainly directed by the
mobile-phase pH, in this instance,
high pH in the f irst dimension and
low pH in the second dimension,
and by the zwitterionic nature of the
peptides. In contrast to the combina-
tion of strong-cation exchange and
reversed-phase LC , where the f irst
dimension has an intrinsic low peak
capacity, the combination of reversed-
phase LC in both dimensions benefits
from the high peak capacities of the
two independent dimensions, which
results in an overall high peak capac-
ity of the 2D setup.
In the characterization of yeast
HCPs, we opted to use reversed-phase
LC in both dimensions with the first
dimension operated at pH 10 and
the second dimension at pH 2.6. An
acidic pH is preferred in the second
dimension since it maximizes MS sen-
Recombinant therapeutic enzyme
99.53
45
99.47
46
0.44
0.008
0.0130.43
0.015
7 1
211
20.000.07
5
0.054
0.023
99.77
0.0346
3
0.06
3
0.09
9
Exogenous glycosidase Metallopeptidase (HCP)
Serine carboxypeptidase 1 (HCP) Aspartyl peptidase (HCP)
Pe
rce
nta
ge
Pe
rce
nta
ge
Pe
rce
nta
ge
Pe
rce
nta
ge
Pe
rce
nta
ge
Pe
rce
nta
ge
Purification stage
1 2 3 1 2 3
Purification stage
Purification stage
1 2 3 1 2 3
Purification stage
Purification stage
1 2 3 1 2 3
Purification stage
Serine carboxy peptidase 2 (HCP)
Figure 4: Evolution of the therapeutic enzyme, the exogenous glycosidase, and some selected HCPs throughout the final stages of downstream manufacturing. The numbers on the bars represent the relative abundances and the number of unique peptides identified and quantified. Relative abundances were calculated based on the MS signal of identified pep-tides. Note: the therapeutic enzyme contains various fully occupied glycosylation sites. These glycopeptides are not identi-fied by the MS/MS search engine and therefore not taken into account in the calculation of relative abundances.
38 ADVANCES IN BIOPHARMACEUTICAL ANALYSIS NOVEMBER 2016 www.chromatographyonline.com
sitivity for peptides. Figure 2 shows
the f irst dimension ultraviolet (UV)
214-nm chromatogram of a selected
downstream manufacturing sample.
A reversed-phase LC column with an
internal diameter of 2.1 mm was used,
which allowed substantial amounts of
sample to be loaded, in this particu-
lar case the amount corresponding to
115 μg of protein. The peptides were
nicely spread throughout the aceto-
nitrile gradient and 22 fractions were
collected and further processed after
drying and reconstitution in 50 μL of
low-pH mobile-phase A (2% acetoni-
trile and 0.1% formic acid).
The second dimension consisted of
a reversed-phase LC capillary column
with an internal diameter of 75 μm,
which was directly coupled through a
nanospray interface to high resolution
quadrupole time-of-f light (QTOF)-
MS operated in the data-dependent
acquisition (DDA) mode. The LC–
MS/MS traces of some selected frac-
tions are shown in Figure 3 illustrat-
ing good orthogonality between first
and second dimension separations.
The MS system was programed so
that an MS survey measurement pre-
ceded three dependent MS/MS acqui-
sitions. Precursors selected twice for
collision-induced dissociation (CID)
were placed in an exclusion list. Gen-
erated MS/MS spectra were subjected
to database searching (yeast proteins
and therapeutic enzyme sequence)
and relative protein quantif ication
was performed from total protein
intensities computed by the Spectrum
Mill search engine. Total intensity
is the sum of intensities for all spec-
tra of peptides belonging to a given
protein. Figure 4 shows the evolu-
tion of the therapeutic enzyme and
some selected HCPs throughout the
f inal stages of purif ication. Of par-
ticular interest, during downstream
manufacturing, a nonyeast-derived
glycosidase was added to shape the
glycosylation profile of the therapeu-
tic enzyme (in between stage 1 and 2).
This glycosidase temporarily reduced
the purity of the therapeutic enzyme
but was rapidly cleared. The HCPs
detected were mainly proteases, which
inf luenced stability of the therapeu-
tic enzyme. While some were clearly
reduced throughout the process (ser-
ine carboxypeptidase 1 and aspartyl
peptidase), others were enriched (ser-
ine carboxypeptidase 2 and metallo-
peptidase). While these proteases were
present at low levels (<0.1%), stability
studies have shown that they act on
the protein. With the identity of these
proteases revealed, they could be the
subject of a gene knockout to increase
product stability.
It is important to note that none of
the HCPs reported could be identi-
f ied using 1D-LC–MS/MS operated
under exactly the same conditions
as reported in the legend of Figure
3. Column load was evidently much
lower compared to the 2D-LC–MS/
MS analysis (4 μg versus 115 μg).
In conclusion, off-line 2D-LC–MS/
MS represents a valuable new tool for
the characterization of HCPs and their
monitoring throughout downstream
processing. The use of multidimen-
sional chromatography substantially
increases peak capacity and improves
the dynamic range providing access to
otherwise unmined HCPs. Based on the
output of the 2D-LC–MS/MS experi-
ment, processes can be adjusted and
identified HCPs can be incorporated
in single product ELISAs or in targeted
multiple reaction monitoring (MRM)
MS assays for routine monitoring.
References
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Shameem, BioPharm Int. 28 , 32–38
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CMC R e gu l a t o r y Comp l i anc e for
Biopharmaceuticals and Other Biologics
(Springer Science & Business Media,
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Perkins, A. Buko, S. Bai, V. Nguyen, and
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(2012).
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L. Tie, Y. Lu, N. Aboulaich, R. Strouse,
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Koen Sandra is Director at the
Research Institute for Chromatography
(RIC) in Kortrijk, Belgium.
Alexia Ortiz is a Proteomics Re-
searcher at the Research Institute for
Chromatography (RIC).
Pat Sandra is Chairman at the
Research Institute for Chromatography
(RIC) and Emeritus Professor at Ghent
University in Ghent, Belgium. ◾
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