Appl Microbiol Biotechnol
DOI 10.1007/s00253-017-8301-x
BIOTECHNOLOGICAL PRODUCTS AND PROCESS ENGINEERING
Identification of multiple sources of the acidic charge variants
in an IgG1 monoclonal antibody
Shiwei Miao 1 & Panpan Xie 1 & Mao Zou 1 & Li Fan 1 & Xuping Liu 1 & Yan Zhou 1 &
Liang Zhao 1 & Ding Ding 2 & Haibin Wang 2 & Wen-Song Tan 1
Received: 14 February 2017 / Revised: 5 April 2017 / Accepted: 11 April 2017
# Springer-Verlag Berlin Heidelberg 2017
Abstract Charge variants, especially acidic charge variants,
of recombinant monoclonal antibodies are the major critical
quality attributes in the biotechnology industry due to their
potential influence on stability and biological activity. The
chemical properties of the acidic charge variants have been
challenging to fully characterize, and it is critical for process
development and optimization. To completely understand the
multiple sources of acidic charge variants, the major charge
forms of an IgG1 monoclonal antibody were firstly isolated
and then analyzed by a battery of characterization tools. It was
found that various degrees of disulfide bond reduction, the
deamination of HC-T8 Asn84 and HC-T35 Asn388 and ag-
gregation account for the majority of acidic charge heteroge-
neity and the terminal galactosylation content was in relation
to the acidic charge heterogeneity. The correlation between
acidic charge heterogeneity and galactosylation content was
further explored by weak cation exchange chromatography
with the use of β1-4 galactosidase digestion. The results
showed that galactosylation was not the source of the acidic
charge variants per se. Meanwhile, to gain insights into the
impact on binding affinity of monoclonal antibody to IgE and
FcRn, charge variants were also analyzed by competitive
ELISA and surface plasmon resonance, respectively. All
Electronic supplementary material The online version of this article
(doi:10.1007/s00253-017-8301-x) contains supplementary material,
which is available to authorized users.
* Wen-Song Tan
wstan@ecust.edu.cn
1
The State Key Laboratory of Bioreactor Engineering, East China
University of Science and Technology, No. 130 Meilong Road,
Shanghai 200237, China
2
Hisun Pharmaceutical (Hangzhou) Co., Ltd., Xialiancun, Xukou,
Fuyang, Hangzhou, Zhejiang 311404, China
isolated charge variants had similar affinity binding to IgE
and FcRn binding relative to the starting material.
Keywords mAb IgG1 . Acidic charge heterogeneity .
Deamidation . Galactosylation . Disulfide bond reduction
Introduction
Therapeutic recombinant monoclonal antibody (mAb) har-
vested from the Chinese hamster ovary (CHO) cell culture
typically exhibits extensive physicochemical heterogeneities
(Dorai and Ganguly 2014; Liu et al. 2008). As one of the most
commonly observed heterogeneities, charge variation is the
major critical quality attribute (CQA) of the mAb in the
biosimilar industry. The charge heterogeneity can optimize
the balance of gaining favorable electrostatic interactions, de-
termine mAb’s structure, and then affect the safety and effica-
cy of the products (Boswell et al. 2010; Gandhi et al. 2012;
Khawli et al. 2010). Therefore, it was closely monitored and
tightly controlled throughout the process development of
mAb therapeutics (Yang et al. 2014). These variants can be
classified as acidic or basic species as compared with the main
species. Charge variants with a relatively lower pI or eluted
early from cation exchange chromatography (CEX) are re-
ferred to as acidic variants, while charge variants with a rela-
tively higher pI or eluted late from cation exchange chroma-
tography are referred to as basic variants (Du et al. 2012).
Some known modifications have been reported to contribute
to the formation of acidic variants such as the sialylation
(Khawli et al. 2010; Lyubarskaya et al. 2006; Santora et al.
1999), deamidation (Dada et al. 2015; Harris et al. 2001;
Melissa Perkins 2000; Neill et al. 2015; Ponniah et al.
2015), and glycation (Khawli et al. 2010; Quan et al. 2008).
On the other hand, the C-terminal Lysine (Lys) clipping (Dada

et al. 2015; Khawli et al. 2010; Zhang et al. 2015b), pyro-
glutamate cyclization (Lyubarskaya et al. 2006), C-terminal
amidation (Kaschak et al. 2011), and the formation of
succinimide from isomerization of aspartate (Asp) residues
(Harris et al. 2001) are commonly observed modifications that
form basic variants.
Some published studies have reported how to control the
acidic charge variant levels of the mAb during the cell culture
process, such as the supplementation of members of the bio-
flavonoid chemical family into cell culture media (Hossler
et al. 2015), hyperosmotic stress (Schmelzer and Miller
2002), pH downshift in the stationary phase (Xie et al.
2016), cell culture temperatures (Kishishita et al. 2015;
Zhang et al. 2015a), and the ambient light exposure
(Mallaney et al. 2014). However, the contents of acidic charge
variants are still poorly controlled in the manufacturing pro-
duction. Thus, more effective methods are still needed to be
explored to control the acidic charge variant levels precisely.
Quality by design (QbD) has been demonstrated to be possible
solutions to the conundrum as an approach toward cell culture
process and mAb development (Brühlmann et al. 2015;
Rathore and Singh 2016). The aims of QbD were developing
products using a well-defined and controlled process based on
the deep understanding of the product itself (Rathore and
Singh 2016). Therefore, identification of the sources of the
acidic charge variants in an IgG1 monoclonal antibody was
necessary for controlling it. Although the molecular heteroge-
neities of the mAb had been already characterized, their cor-
relations among each other, such as glycosylation and charge
heterogeneities, were often less understood. Meanwhile, com-
plete understanding of acidic variants was lacking.
Furthermore, their effects on the biological activity and phar-
macokinetic behaviors often require further exploration.
To better understand the sources of the acidic charge vari-
ants in an IgG1 monoclonal antibody and their differences in
the IgE and FcRn binding affinity, the acidic charge variants,
basic charge variants, and main species were firstly prepared
from an intact antibody product by a semipreparative CEX
column in this study. And the three CEX fractions were then
further analyzed by a battery of characterization tools, such as
weak CEX to determine their purities, reduced mass analysis
by LC-MS to determine glycosylation distribution, LC-MS
analysis of molecular weights of intact antibody and capillary
electrophoresis-sodium dodecyl sulfate under non-reducing
conditions to respectively determine the discovery of disulfide
reduction and their degree, tryptic peptide mapping to deter-
mine the other post-translational modifications, size exclusion
chromatography to determine the aggregation and fragmenta-
tion percentage, boronate affinity chromatography to deter-
mine their glycation levels, and circular dichroism to provide
secondary structure information. As a result, we demonstrated
for the first time that the terminal galactosylation index (GI)
content of acidic charge variants was significantly higher than
Appl Microbiol Biotechnol
that in the main species. Besides, enrichments of disulfide
reduction, the deamination of HC-T8 Asn84 and HC-T35
Asn388, and aggregation species were also detected in the
acidic charge variants. The correlation between acidic charge
heterogeneity and GI was further explored by CEX with the
use of β1-4 galactosidase digestion. Meanwhile, the affinity
binding to IgE and FcRn binding activity of mAb charge
variants were also evaluated for the potential impact on bio-
logical activities and pharmacokinetic properties.
Materials and methods
Antibodies
The mAb is a recombinant DNA-derived humanized IgG1
monoclonal antibody that selectively binds to human immu-
noglobulin E (IgE) (Robblee et al. 2015). All purified samples
for isolation of charge variant fractions were supplied by
Hisun Pharmaceutical Co., Ltd. (Hangzhou, China). The hu-
manized IgG1 mAb was expressed in CHO cells. The weak
cation exchange chromatography profile of the purified mAb
showed the presence of three components: the acidic charge
variants (18%), the main species (69%), and the basic charge
variants (13%).
Charge fraction collection
CEX fractions were prepared using a ProPac WCX-10
Semipreparative Column, 22 mm × 250 mm (Dionex, CA,
USA) and Agilent 1260 HPLC system. The collected CEX
fractions were transferred into an Amicon Ultra-0.5 centrifuge
(Millipore, Billerica, MA) with a molecular weight cutoff of
30 kDa, concentrated and buffer exchanged with formulation
buffer (5 mM histidine acetate, 85 mM sucrose, 0.01% Tween
20) at pH 6.5.
Weak cation exchange chromatography
Weak cation exchange chromatography (WCX) was per-
formed to determine the purity of the isolated charge variants.
A ProPac® WCX-10 Analytical Column, 4 mm × 250 mm
(Dionex, CA, USA) was used in an Agilent 1260 HPLC sys-
tem. Mobile phase A was 20 mM MES, pH 6.5, and mobile
phase B was 150 mM sodium chloride, 20 mM MES, pH 6.5.
After sample injection, the column was equilibrated with 85%
mobile phase A and 15% mobile phase B in 5 min. And then,
a linear gradient was run from 15 to 45% B over 30 min
followed by a constant 100% B flow from 35.1 to 40 min.
The CEX fractions of the mAb were diluted with water to
approximately 1.2 mg/ml and the injection volume was
50 μl. The flow rate was set at 0.8 ml/min and the mAb charge
variants were detected at UV280. The column temperature was
Appl Microbiol Biotechnol
maintained at 35 °C. The purity of the isolated charge variants
was determined by calculating the percentage of total peak
area.
LC-MS analysis of molecular weights of reduced antibody
N-linked glycosylation was analyzed and quantified by LC-
MS. The purified CEX fractions (100 μg) were reduced by
adding 4 μl of 10 mM dithiothreitol (DTT) after incubation at
56 °C for 40 min prior to mass analysis. Samples were sepa-
rated on an ultraperformance liquid chromatography (UPLC)
system (Waters) with an ACQUITY UPLC C4 BEH300 col-
umn (1.7 μm, 2.1 × 50 mm). A Waters Xevo G2QTOF mass
spectrometer was used to measure the molecular weights of
the CEX fractions. Mobile phase A (water with 0.1% formic
acid) and mobile phase B (acetonitrile with 0.1% formic acid)
were used for all UPLC separations. The linear gradient was
from 5% B to 30% B in 11 min. The mass spectrometer runs in
positive mode with m/z range of 500–5000. Desolvation tem-
perature was set at 420 °C. Source temperature was set at
140 °C. Capillary voltage was set at 3 kV. Sampling cone
voltage was set at 40 V. Desolvation gas flow was set at
600 L/h. The mass spectra of heavy chain (HC) were
deconvoluted using BiopharmaLynx 1.3.
The degree of terminal galactosylation index (GI)
was calculated according to Eq. (1) as described earlier
(Ivarsson et al. 2014):
2  ðG2 þ G2 FÞ þ G1 þ G1 F
GI ¼
100  2
In the same way, the degree of fucosylation (FI) was cal-
culated according to Eq. (2):
G0 F þ G1 F þ G0F−GlcNAc þ G2 F
FI ¼
100
β 1-4 galactosidase digestion
Acidic charge variant fractions and starting antibody materials
were de-galactosylated by incubation in 5 mM CaCl2 and
50 mM sodium acetate buffer (NEB, USA) at pH 5.5 with
2 units of β 1-4 galactosidase (NEB, USA) per microgram
substrate at 37 °C overnight.
LC-MS analysis of molecular weights of intact antibody
A UPLC system (Waters) with a Waters PREP™ micro
desalting column (20 μm, 2.1 × 5 mm, 1000 Å) and a
Waters Xevo G2QTOF mass spectrometer were used to mea-
sure the intact molecular weights of the CEX fractions.
Approximately 0.5 μg of each sample was injected into the
column at 95% mobile phase A (0.1% formic acid in water)
and 5% mobile phase B (0.1% formic acid in acetonitrile). The
flow rate was set at 0.5 ml/min and the column temperature
was set at 80 °C. The mass spectrometer was run in positive
mode with the following settings: a scan range of m/z 500–
5000, desolvation temperature was set at 420 °C, source tem-
perature was set at 140 °C, capillary voltage was set at 3 kV,
sampling cone voltage was set at 80 V, and desolvation gas
flow was set at 600 L/h. The raw data files were processed
with Biopharma Lynx 1.3.
Capillary electrophoresis-sodium dodecyl sulfate
under non-reducing conditions
Non-reduced capillary electrophoresis-sodium dodecyl sulfate
(CE-SDS) was performed for analysis on the fragmentation of
the mAb molecule induced by unmatched disulfide bridges. A
Beckman PA 800 high-performance capillary electrophoresis
system equipped with a 360 μm OD × 50 μm ID × 30.1 cm L
uncoated fused-silica capillary was used. 25-μl samples
(4 mg/ml) were mixed with 2 μl of a 10 kDa internal standard,
68 μl of SDS-MW sample buffer (Beckman coulter, A10663),
and 5 μl iodoacetamide and then boiled at 70 °C for 10 min.
After incubation, the samples were transferred into micro sam-
ple tubes. And then, samples were injected into the capillary
by applying voltage at −5 kV for 20 s. The separation was
achieved by applying a constant voltage of 15 kV over a 40-
min period (Beckman Coulter). The migration of proteins was
monitored at UV220. Data were acquired and processed by 32
Karat software to quantify the free light chain (L) and the
partial mAb missing one light chain (molecule containing
two heavy chains and one light chain, HHL).
Size exclusion chromatography
Size exclusion chromatography (SEC) was used for detection
aggregation level of the mAb sample. It was performed on an
Agilent 1260 HPLC system using a TSK G3000SWXL col-
umn, 7.8 mm × 300 mm (TOSOH, Yamaguchi, Japan) at
room temperature. Samples eluted during an isocratic flow
of 20 mM sodium phosphate, 200 mM sodium chloride, pH
7.4 at 0.5 ml/min for 30 min and monitored by a UV detector
at 280 nm. The level of aggregation was expressed as the
relative percentage of peak areas.
Boronate affinity chromatography
The glycation levels of charge variant fractions were deter-
mined by boronate affinity chromatography. The samples
were analyzed on an Agilent 1260 HPLC system using a
TSK-Gel boronate-5PW column, 7.5 mm × 75 mm
(TOSOH, Yamaguchi, Japan). The affinity-based separation
was performed at room temperature with the mobile phase
flow rate at 1.0 ml/min. The mobile phases used were as

follows: A, 100 mM HEPES, 25 mM Tris, 200 mM NaCl, at
pH 8.6; B, 500 mM sorbitol in mobile phase A. After sample
injection, the non-glycated antibodies flowed through the col-
umn equilibrated in mobile phase A. And then, the glycated
antibodies were eluted from the resin with the mobile phase B.
The UV detector was set at 280 nm with an 8-nm bandwidth.
The glycation level in each charge variant fraction was
expressed as the relative percentage of peak areas.
Circular dichroism
Purified CEX fractions of the IgG1 mAb were analyzed using
far and near-UV circular dichroism (CD) spectroscopy. The
CD spectroscopy was used here to explore the possibility of
conformational differences. Samples were buffer exchanged
in 50 mM phosphate buffer at pH 6.47. Experiments were
conducted on a Chirascan100 spectropolarimeter (Applied
Photophysics, England). Solution ellipticities for the near-
UV and the far-UV were measured from 320 to 250and from
180 to 260 nm in 50 mM phosphate buffer (pH 6.47), respec-
tively. Protein samples were diluted with 50 mM phosphate
buffer (pH 6.47) to 0.1 mg/ml for far-UV CD and 0.7 mg/ml
for near-UV CD. A 1-mm path length cell was used for far-
UV CD and a 10-mm path length cell for near-UV CD.
Spectra were collected in continuous mode at a pitch of
0.5 nm, a response of 0.5 s, and a bandwidth of 1 nm. Three
scans were collected and averaged for each spectrum.
Measurements were performed at room temperature. The
buffer spectra were subtracted from the sample spectra, and
the resulting data were converted from mdeg to molar ellip-
ticity and plotted as a function of wavelength. The proportion
of the secondary structure was calculated by the CDPro
software.
Peptide mapping
Peptide mapping is widely used for analyzing post-
translational modifications in recombinant mAbs. The puri-
fied CEX fractions (1 mg) were denatured and reduced by
adding 300 μl of 6 M guanidine hydrochloride in 100 mM
Tris, pH 8.3, and 2 μl of 5 mM dithiothreitol (DTT) (final
volume, 500 μl), followed by incubation at 37 °C for 2.5 h.
Thirteen microliters of 13 mM iodoacetamide was then added
to each sample solution. The samples were then alkylated at
room temperature in the dark for 45 min. The reduced and
alkylated samples were then buffer exchanged into 0.1 M am-
monium bicarbonate using Amicon Ultra-0.5 centrifuge de-
vices with a molecular weight cutoff of 3 kDa (Millipore,
Billerica, MA). Each sample was digested using trypsin at a
final concentration of 1:50 (w:w) at 37 °C for 5 h. Then,
formic acid was added to stop the digestion by acidification.
The trypsin-digested products were underwent a UPLC sys-
tem (Waters) coupled with Xevo G2 Q-TOF mass
Appl Microbiol Biotechnol
spectrometer (waters). The column used for separation of the
peptides after trypsin digestion was an ACQUITY UPLC C18
BEH300 column (2.1×150 mm, 1.7 μm). Peptides were eluted
by a linear gradient of 1–40% of mobile phase B (mobile
phase A, 0.1% formic acid in water; mobile phase B, 0.1%
formic acid in acetonitrile) at a flow rate of 0.2 ml/min for
105 min at 60 °C. Desolvation temperature was set at 420 °C.
Source temperature was set at 100 °C. Capillary voltage was
set at 3 kV. Sampling cone voltage was set at 40 V.
Desolvation gas flow was set at 600 L/h. Raw data were col-
lected by MassLynx4.1. The raw data files were processed
with BiopharmaLynx 1.3.
Affinity measurements binding to IgE by competitive
ELISA assay
A 96-well plate was coated 2 h at 37 °C with recombinant
human Fc epsilon RI alpha (1 μg/ml) in 35 mM sodium car-
bonate buffer. The coat solutions were then removed from the
wells and the wells were blocked with 200 μl blocking buffer
(3 g BSA in 100 ml water). Blocking treatment was performed
at 2–8 °C for 16–18 h. After blocking and three wash steps,
50 μl human IgE full length protein (0.2 μg/ml) and 50 μl
samples (serially diluted controls or CEX fractions) were
added to the wells. And then, the mixtures were incubated
for 2 h at 37 °C. After three washings, biotin-labeled affinity
purified antibody to human IgE was diluted 1:2000 in assay
buffer (1 g BSA in 100 ml water) and added to wells at 100 μl.
The plates were incubated for 2 h at 37 °C. The wells were
then washed three times with 270 μl of PBS buffer. HRP-
labeled streptavidin was diluted 1:40,000 in assay buffer
(1 g BSA in 100 ml water) and added to wells at 100 μl.
The plates were incubated for 45 h at room temperature.
After washing, 100 μl 3, 30, 5, 50-tetramethylbenzidine
(TMB) was added as a substrate and color was developed
for 10 min. The reaction was stopped by adding 100 μl of
2 M H2SO4. The absorbance was read at 450 nm using a
Spectra Max M5 (Molecular Devices, Californie, USA). The
median effective dose (ED50) values were calculated by four
parameter equation. The control was starting antibody which
used for charge fractions collection. The relative affinity ac-
tivity of tested CEX fraction was calculated according to the
following formula: relative affinity activity (%) = (ED50 of
tested CEX fraction)/ (ED50 of control) × 100.
FcRn binding by surface plasmon resonance
The pharmacokinetic properties of the CEX fractions were de-
termined by surface plasmon resonance (SPR). A Biacroe™ X-
100 instrument (GE Healthcare, Piscataway, USA) was used to
analysis the FcRn binding affinity. His-tagged recombinant hu-
man FcRn (0.5 μg/ml) was immobilized on a CM5 sensor chip
using His Capture Kit (GE Healthcare, Piscataway, USA). All
Appl Microbiol Biotechnol
test samples were diluted by HBS-EP Buffer (GE Healthcare,
Piscataway, USA) and injected at a flow rate of 30 μl/min. A
series of mAb concentration including 3333, 1667, 833, 417,
and 208 nm were injected consecutively, each with a contact
time of 120 s and a dissociation time of 300 s. Data was ana-
lyzed by the Biacore X100 Evaluation Software (GE
Healthcare, Piscataway, USA).
Results
Purity analysis by CEX
The starting mAb was firstly analyzed using a CEX method. As
shown in Fig. 1 (starting materials), the main peak (MP) was
observed at the retention time of 18.5 min. Species eluting before
the main peak are defined as acidic peaks (AP). Species around
21.5 min are defined as basic peaks (BP). After all fractions
collected, samples were reanalyzed by CEX. The results were
shown in Table 1. The purities of the CEX fractions were all
higher than 95%. The charge variants were cumulative effects
of chemical reactions changes the structure and conformation of
antibody. In order to understand the multiple sources of the
charge variants in the IgG1 monoclonal antibody, three fractions
(AP, MP, BP) were further analyzed by LC-MS, CE-SDS, SEC,
CD, and boronate affinity chromatography.
N-linked glycosylation
The differences of N-linked glycosylation between charge
variants (AP and BP) and main species (MP) were identified
by analyzing reduced mass of mAb. Take MP for example,
multiple molecular weights of the heavy chain with different
Fig. 1 The CEX chromatograms of the mAb (Khawli et al. 2010). Chromatograms from reanalysis of the collected charge variant fractions were shown
for AP, MP, and BP as labeled
types of oligosaccharides were observed (Fig. 2a). The mass at
50695 Da was assigned to the G0F type in a heavy chain.
According to the mass difference from heavy chain with
G0F, these glycan types were identified as G1F (+162 Da
for galactose from G0F), G1 (+16 Da for fucose and galactose
from G0F), G2 (+178 Da for fucose and galactose from G0F),
G0F-GlcNAc (−203 Da for N-acetyl-glucosamine from G0F),
G2F (+324 Da for galactose from G0F), G0 (−146 Da for
fucose from G0F), and Man5 (−228 Da for fucose, high man-
nose and N-acetyl-glucosamine from G0F), G0-GlcNAc
(−349 Da for N-acetyl-glucosamine and fucose from G0F),
respectively. The structure schematics of each glycoform were
also inserted into Fig. 2a. Besides, there was no oligosaccha-
ride containing sialic acid detected in the reduced antibody
analysis. The proportion of sialylation was under the limita-
tion of detection. By normalizing to the sum, the relative pro-
portion of each glycoform in individual fraction could be de-
termined. The N-glycosylation distribution varied among
CEX fractions (Fig. 2b). Compared with MP, a decrease
(4.7%) of the G0F proportion was observed in AP, whereas
an increase (2.8%) of G0F proportion was detected in BP
(Fig. 2b). Furthermore, the proportion of G1F in AP was
higher (5.5%) than that in MP and BP, but the proportion of
G1 in MP was higher than that in AP and BP (Fig. 2b). In
order to further verify the above conclusion, the data of charge
variants and glycoslation from more than 20 different produc-
tions was used to do the statistical correlation analyses. Plots
of charged variant levels versus G0F or G1F proportion were
shown in Fig. 3. When most of the data points are located
around a straight line, there is correlation between them.
And the slope of the straight line is the correlation coefficient
R2. The stronger correlation is with R2 value closing to 1. The
levels of acidic variants were linearly decreased with
 Appl Microbiol Biotechnol

Table 1 Analytical results to assess the purities of charge variant Besides, the levels of acidic variants were approximately lin-
fractions
early increased with increasing G1F proportion (R2 = 0.796)
Fraction Acidic (%) Main (%) Basic (%) (Fig. 3c), suggesting an involvement of N-glycosylation (es-
pecially terminal galactose) in mediating the charge profile of
AP 97.646 0.63 ND the mAb.
MP ND 97.222 2.78 The degree of GI and FI of CEX fractions were further
BP ND 4.752 95.248 calculated. The terminal GI content of AP was significantly
higher than that in MP (p = 0.003 < 0.01) (Fig. 4a). But there
ND not detected
were no significant differences of GI content between BP and
MP (Fig. 4a). Besides, basically, similar FI contents were also
increasing G0F proportion (R2 = 0.871) (Fig. 3a), whereas the observed in the three CEX fractions (Fig. 4b). The results
levels of basic variants were approximately linearly increased shown above all suggested that the GI may be an important
with increasing G0F proportion (R2 = 0.628) (Fig. 3b). factor and have a direct relationship with charge distribution.

Fig. 2 Analysis of N-linked glycosylation by LC-MS. a The deconvoluted mass spectra of heavy chain (HC) of MP. b N-linked glycosylation
distribution of CEX fractions. White bars: acidic peak (AP); backs lash bars: main peak (MP); black bars: basic peak (BP)
Appl Microbiol Biotechnol
In order to confirm the relationship between GI modification
and acidic variant content, acidic fraction was digested with β
1-4 galactosidase and then reanalyzed by CEX. The CEX
analysis on the acidic fractions treated or not treated by β 1-
4 galactosidase (Fig. 4c) revealed that the acidic fraction
digested with β 1-4 galactosidase and the control (the acidic
fractions not digested by β 1-4 galactosidase) were nearly the
same. The retention time of the acidic fraction were not
changed after digestion. Meanwhile, the acidic fractions were
not turned into main species. Therefore, the GI modification
was not a direct reason of the acidic charge distribution per se.
Disulfide bond reduction
The intact mass of isolated AP and MP were determined by
LC-MS. Each deconvoluted mass spectra were shown in
Fig. 5. The molecular weight of the dominant species in the
MP was 149,183 Da, which was in agreement with the theo-
retical molecular weight (149,183 Da) of the intact mAb with
G0F on both of the heavy chains (Fig. 5a). The molecular
weight of the major peak in AP was 149,360 Da, which
corresponded to the intact mAb with G0F on one heavy chain
and G1F on the other heavy chain (Fig. 5b). In AP, there was
another peak with molecular weight of 125,597 Da, indicating
one light chain missing in the mAb molecule induced by un-
matched disulfide bonds between light chain and heavy chain
(approximately −25,000 Da compared to the intact molecule)
Fig. 3 The linear correlation of a G0F proportion and acidic charge variant content, b G0F proportion and basic charge variant content, or c G1F
proportion and acidic charge variant content of mAb from more than 20 different productions
(Fig. 5b). The purity and fragmentation under denaturing con-
dition of the CEX fractions were then analyzed by CE-SDS.
As shown in Table 2, the contents of L and HHL were signif-
icantly higher in AP (p = 0.00018 < 0.01) and BP
(p = 0.00018 < 0.01) than that in MP, which was further
reflected in the decrease of the intact antibody proportion from
93.58% (MP) to 85.87% (AP) or 85.60% (BP). Therefore, the
partial mAb molecule induced by the unpaired disulfide
bridge between light chain and heavy chain was one of the
causes of the acidic charge variants. However, the partial mol-
ecule of mAb was not observed in basic charge variants using
LC-MS, indicating other cycstein-linkage variants were
existed in basic variants.
Aggregation and fragmentation
The CEX fractions were analyzed by SEC and typical elution
profiles were shown in Fig. S1. The significantly higher ag-
gregate levels were in the AP and BP as compared to the MP
(Table 3). Meanwhile, the analysis of CEX fractions by SEC
indicated that the fragments eluted mainly in the AP (4.18%)
versus 1.26% in the MP. Also, 3.08% fragments eluted in the
BP. HHL was one type of the low molecular weight frag-
ments. So the SEC results shown above were consistent with
the non-reduced CE-SDS analysis. The low molecular weight
fragments and aggregates also contributed to the formation of
acidic charge variants.

Fig. 4 The degree of a GI or b FI among IgG1 CEX fractions. c The
CEX profiles of starting materials and the acidic charge fraction treated
with β 1-4 galactosidase or without β 1-4 galactosidase (control) as
Glycation
The glycation levels of CEX fractions were then determined
by boronate chromatography analysis. No statistically signif-
icant differences of glycation contents were observed between
the AP and MP as shown in Fig. S2. The glycation contents of
AP, MP, and BP were 3.03, 2.72, and 3.24%, respectively.
Secondary structure
CD was used to investigate potential conformational differ-
ences among CEX fractions. The far-UV CD spectra sug-
gested comparable secondary structure for the CEX fractions
(Fig. S3a). The calculated protein secondary structure contents
for the three fractions were very similar and given in Fig. S3b.
In general, the results suggested that the mAb had a predom-
inant content of β sheet (40–42.4%) and random coil (31.6–
32.3%). No major differences in the tertiary structure were
detected in the near-UV CD spectra (data not shown here).
Therefore, the glycation and high-order structure differences
were not the source of the acidic charge variants.
Appl Microbiol Biotechnol
labeled in each line. The error bars indicate the standard deviations from
three independent experiments. **p < 0.01 relative to MP (two-tailed
Student’s t test)
Deamidation
The CEX fractions were also analyzed by tryptic peptide map-
ping (LC-MSE) to identify their differences at the peptide
level. Tryptic peptides were analyzed by searching for known
post-modifications with minimal molecular weight differ-
ences such as Asn deamidation (+1 Da), C-terminal Lys clip-
ping variants (−128 Da), Gln deamidation (+1 Da), cyclization
of the N-terminal glutamine (Gln) to pyro glutamic acid
(pyroGlu) (−17 Da), Met oxidation (+16 Da), deamidation
succinimide D (−17 Da), and deamidation succinimide N
(−17 Da). Apart from small proportion in succinimide and
Met oxidation, Asn deamidation was the major modification
identified among CEX fractions (data not shown here). The
sites and percentages of Asn deamination of each CEX frac-
tions were summarized in Table 4. The levels of Asn
deamidation in the HC-T8 Asn84 and HC-T35 Asn388 were
significantly higher in AP than that in MP. Meanwhile, the
levels of Asn deamidation in the HC-T8 Asn84 and HC-T24
Asn319 were significantly lower in BP than that in MP.
Therefore, the Asn deamidation in HC-T8 Asn84 and HC-
T35 Asn388 was the source of the acidic charge variants.
Appl Microbiol Biotechnol
Fig. 5 Deconvoluted mass spectra from analysis of the intact antibody (a MP and b AP), as labeled in each spectrum
Affinity binding to IgE
To determine whether CEX fractions had similar binding af-
finity to IgE, recombinant human Fc epsilon RI alpha coated
in plate was used to compete with CEX fractions for binding
to IgE. The competitive ELISA revealed that the relative af-
finity activity of AP, MP, and BP were 80.45, 91.01, and
76.36%, respectively (Table S1). These results suggested that
the formation of the charge variants would reduce the binding
affinity to IgE.
FcRn binding
In order to assess whether IgG1 charge variations altered the
FcRn binding activities, the binding properties of each isolat-
ed CEX fractions to immobilized recombinant human FcRn
was analyzed by SPR. The dissociation constants (KD) calcu-
lated from the kinetic analyses was summarized in Table 5.
The results demonstrated that FcRn binds to the AP, MP, and
BP with an KD value of 1.6720 × 10−6, 1.1860 × 10−6, and
0.8486 × 10−6 M, respectively. These values were comparable
to the starting materials, which had an average KD value of
1.2900 × 10−6 M. No major discrepancy was observed be-
tween the experimental data and the fitted curves, as the low
chi-squared values (ranging from 0.974–13.9 RU2), suggest-
ing that the sample 1:1 binding model adequately describes
the interaction of the human FcRn and the IgG1 charge vari-
ants. Representative sensorgrams for all CEX fractions were
shown in Fig. S4. The three CEX fractions sensorgrams were
of high similarity. Comparable FcRn binding capacities for
AP and MP indicate that the pharmacokinetic properties and
half-life of the IgG1 mAb are likely not impacted by the for-
mation of acidic charge variants.
Discussion
With the primary goal to identify the sources of acidic charge
variants of the mAb used in this study, a number of analytical
tools were employed, including N-glycosylation pattern, di-
sulfide bridge variants, aggregation and fragmentation,
glycation properties, Asn Deamidation, binding affinities to
IgE and FcRn receptor. Different levels of sialylation of Fc
glycan of IgG1 have been commonly reported to be an
 Appl Microbiol Biotechnol

Table 2 Distribution of L, HHL, and intact antibody in CEX fractions Table 4 Asn deamination of each peptide in the CEX fractions as
as determined by non-reduced CE-SDS. The error margin indicates the detected using LC-MSE
standard deviations from three independent experiments
Label Sequence Deamidation (%)
Fraction L (%) HHL (%) Intact antibody (%)
AP MP BP
AP 2.00 ± 0.02 9.41 ± 0.18 85.87 ± 0.25
MP 0.81 ± 0.01 4.32 ± 0.14 93.58 ± 0.14 HC-T8 NTFYLQMN84SLR 3.5 2.3 0
BP 1.98 ± 0.02 9.39 ± 0.10 85.60 ± 0.05 HC-T21 FN280WYVDGVEVHN290AK 0.6 0.6 0
HC-T24 VVSVLTVLHQDWLN319GK 10.8 10.2 9.1
HC-T34 N365QVSLTCLVK 0.7 0.6 0
important factor that influences charge distribution (due to the HC-T35 GFYPSDIAVEWESN388GQPENNYK 28.2 24.6 25
presence of the carboxyl group within sialic acid) (Khawli LC-T10 SGTASVVCLLN141NFYPR 5 5 5
et al. 2010; Lyubarskaya et al. 2006; Scallon et al. 2007; SGTASVVCLLNN142FYPR 0.9 0.8 0.8
Vlasak and Ionescu 2008). N-linked glycosylation of the three
CEX fractions was firstly analyzed by LC-MS. However, si-
alic acid species were not observed in our mAb. Vlasak and of enzyme-catalyzing reactions in endoplasmic reticulum
Ionescu (2008) had also reported that the level of sialylation of (ER) and Golgi apparatus (Brühlmann et al. 2015; MD
the mAb expressed in the CHO cell lines was usually very 2000). Meanwhile, our previous studies have shown the in-
low. Instead, several types of neutral glycans, including G0F, formation that the formation of acidic charge variants is oc-
G1F, and G1, were found to vary in association with charge curred inside and outside the cell at the same time (data not
characteristics (Fig. 2b). More importantly, the terminal GI shown here). Thus, the influence on the accessibility of
content was significantly higher in AP than that in MP and galactosyltransferase to the effecting site by other modifica-
BP (Fig. 4a). To our best knowledge, this is the first report on tions (the sources of the acidic charge variants) might be one
the correlation between GI content and charge variants. Yang of the possible explanations for the observed degree of termi-
et al. (2014) found that N-linked glycosylation patterns were nal GI among charge variants. Another explanation is that
in relation to the terminal modifications (N-terminal pyro- other modifications may be affected by different GI in CEX
glutamate formation and C-terminal lysine cleavage) in their fractions. So the levels of GI may be indirect causes to the
study. Gandhi et al. (2012) also noted the differences in gly- formation of acidic charge variant. Nevertheless, this conjec-
cosylation between the purified pre-peak and main peak frac- ture needs to be verified with further experimental study, and
tions. But the specific relationship and reason were not well the root cause needs to be clearly understood. It was also
explored. In order to confirm the relationship between GI worth noting that the proportions of FI were not changed
modification and acidic variant content, acidic fraction was among charge variants (Fig. 4b). Yang et al. (2014) also re-
digested with β 1-4 galactosidase and then reanalyzed by ported that the contents of core fucose were the same in charge
CEX. The CEX-based separation was not only dependent on variants.
the differences of the overall charge, but also on local spatial The disulfide bond reduction contents of the three CEX
structure. Galactose was a neutral glycan. So the overall fractions were analyzed by non-reduced CE-SDS. It showed
charge of the mAb could not be changed by the different the higher enrichment of L and HHL species in the AP and BP
degree of GI. If the GI could mediate the charge profile, the compared to the MP (Table 2). As we know, the information
most likely reason was that the GI modification had changed of the mAb will be significantly changed by missing a light
the local spatial structure (which can lead the difference of the chain. Besides, the surface charge of the mAb can also be
CEX distribution) of the mAb. The results of β 1-4 galactosi- redistribution. Khawli et al. (2010) also found that 29% re-
dase digestion experiment further indicated that GI was not duced disulfide enriched in the acidic charge variants.
the source of the acidic charge variant per se. It is well known Therefore, the disulfide bond reduction between heavy chain
that the biosynthesis of N-glycans of IgG1 involves a number and light chain was the source of the acidic charge variants.

Table 3 Distribution of fractions, aggregates, and monomer in purified Table 5 The kinetics constants of recombinant human FcRn binding of
CEX fractions. The error margin indicates the standard deviations from the CEX fractions by SPR assay (Khawli et al. 2010)
three independent experiments
Fractions KD (×10−6 M) Chi2 (RU2)
Fractions Aggregates (%) Monomer (%) Fragments (%)
Starting materials 1.2900 1.70
AP 0.24 ± 0.052 95.58 ± 0.075 4.18 ± 0.068 AP 1.6720 7.55
MP 0.069 ± 0.012 98.67 ± 0.032 1.26 ± 0.024 MP 1.1860 0.974
BP 0.34 ± 0.032 96.58 ± 0.054 3.08 ± 0.047 BP 0.8486 13.9
Appl Microbiol Biotechnol
Higher aggregation and fragmentation levels in AP and BP
compared to MP were then discovered by SEC. Khawli et al.
(2010) and Gandhi et al. (2012) also reported that antibody
aggregation and fragmentation were enriched in BP. Besides,
Du et al. (2012) speculated that aggregates may tend to have
higher affinity for cation exchangers and then elute as the
basic species. However, aggregates and fragments were also
enriched in AP in our study. So the types of the aggregates or
fragments could play an important role in affecting the elution
order of charge variants by CEX. Meanwhile, because aggre-
gates constituted only a small percentage (0.069–0.34%) of
the mAb, its contribution in the formation of acidic charge
variants could be ignored.
Contrary to the statistically significant differences of frag-
mentation, similar glycation contents and high-order structure
were observed among the three CEX fractions. Quan et al.
(2008) firstly demonstrated that glycation could contribute to
the formation of acidic charge variants (due to neutralization
of the positive charges). However, glycation was not the
source of the acidic charge variants of the mAb used in this
study. And the formation of acidic charge variants was also
not due to secondary or tertiary structural changes as deter-
mined by CD.
Asn deamidation is considered as the most common source
of acidic charge heterogeneity due to the introduction of one
negative charge (Dada et al. 2015; Du et al. 2012).
Significantly higher deamination proportions of HC-T8
Asn84 and HC-T35 Asn388 in acidic charge variants were
detected by tryptic peptide mapping in our study. Therefore,
the deamination of HC-T8 Asn84 and HC-T35 Asn388 was
the source of acidic charge variants. Several literatures had
also reported that the Asn deamination of the peptide
GFYPSDIAVEWESNGQPENNYK in the Fc domain mainly
enriched in acidic charge variants (Gandhi et al. 2012; Neill
et al. 2015; Ponniah et al. 2015). The IgG1 mAb used in this
study is a humanized monoclonal anti-IgE antibody directed
at the FcεRI-binding domain of human IgE (Qian et al. 2010).
The competitive ELISA results showed that the formation of
acidic charge variants would have a reduced effect on IgE
binding properties. It is well known that FcRn is responsible
for regulating the level of circulating IgG and increasing se-
rum half-life (Israel et al. 1996; Petkova et al. 2006; Yeung
et al. 2009). Analysis of FcRn binding affinity in vitro is a
good indicator of pharmacokinetic properties of the IgG1
mAb in vivo. Therefore, the contribution of charge differences
to IgG interaction with human FcRn was then tested with the
isolated CEX fractions by SPR. Although slight changes in
binding of acidic charge variants and basic charge variants to
human FcRn were detected, these changes did not translate to
incomparable affinity and kinetic properties relative to the
main species. It was suggested that the formation of acidic
charge variants did not have a significant effect on FcRn bind-
ing properties.
As we all know, acidic charge variants are generated by
various post-translational modifications and chemical degra-
dations. Generally, more than one modification will lead to the
acidic charge variants in one mAb. The sources of the acidic
charge variants were the disulfide bond reduction,
deamidation in HC-T8 Asn84 and HC-T35 Asn388, aggrega-
tion and fragmentation in our mAb. Several literatures had
been reported that culture medium or process conditions could
significantly affect recombinant protein quality attributes. The
reduction of the antibody’s interchain disulfide bonds could be
prevented by modifying the cell culture medium with cupric
sulfate, EDTA, and L-cystine, as well as lowering the pH and
air sparging of the harvested cell culture fluid (Trexler-
Schmidt et al. 2010). Xie et al. (2016) found that the
deamidation was significantly decreased by pH downshift.
Therefore, the in-depth study of the sources of the acidic
charge variants that we have presented here can provide guid-
ance on the reduction of product charge heterogeneity under
the quality by design (QbD) scope.
Acknowledgements This work was supported by the National Natural
Science Foundation of China (Nos. 21206040 and 21406066).
Compliance with ethical standards This article does not contain any
studies with human participants or animals performed by any of the
authors.
Conflict of interest The authors declare that they have no conflict of
interest.
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