Protein Expression and Purification 76 (2011) 7–14

Contents lists available at ScienceDirect

Protein Expression and Purification

journal homepage: www.elsevier.com/locate/yprep

Purification process development of a recombinant monoclonal antibody

expressed in glycoengineered Pichia pastoris
Youwei Jiang a, Fang Li a, Dongxing Zha a, Thomas I. Potgieter a, Teresa Mitchell a, Renée Moore a,
Michael Cukan a, Nga Rewa Houston-Cummings a, Adam Nylen a, James E. Drummond b,
Troy W. McKelvey b, Marc d’Anjou a, Terrance A. Stadheim a, Natarajan Sethuraman a, Huijuan Li a,⇑
a
GlycoFi Inc., A Wholly-Owned Subsidiary of Merck & Co. Inc., 21 Lafayette Street, Suite 200, Lebanon, NH 03766, USA
b
Biologics Research, Merck Research Laboratories, West Point, PA 19486, USA

a r t i c l e i n f o a b s t r a c t

Article history: A robust and scalable purification process was developed to quickly generate antibody of high purity and
Received 18 March 2010 sufficient quantity from glycoengineered Pichia pastoris fermentation. Protein A affinity chromatography
and in revised form 4 November 2010 was used to capture the antibody from fermentation supernatant. A pH gradient elution was applied to
Available online 11 November 2010
the Protein A column to prevent antibody precipitation at low pH. Antibody from Protein A chromatog-
raphy contained some product related impurities, which were the misassembling of cleaved heavy chain,
Keywords: heavy chain and light chain. It also had some process related impurities, including Protein A residues,
Protein A chromatography
endotoxin, host cell DNA and proteins. Cation exchange chromatography with optimal NaCl gradient at
Ion exchange chromatography
Monoclonal antibody
pH 4.5–6.0 efficiently removed these product and process related impurities. The antibody from glycoen-
Purification gineered P. pastoris was comparable to its commercial counterpart in heterotetramer folding, physical
Pichia pastoris stability and binding affinity.
Glycosylation Ó 2010 Elsevier Inc. All rights reserved.

Introduction referred to as G0 in our work and literature. Additional sugar resi-

Monoclonal antibodies (mAbs) are rapidly becoming key prod-
ucts for the biopharmaceutical industry. Most commercial thera-
peutic antibodies are intact IgG molecules with IgG1 and IgG2
being the common subclasses. These IgGs play a central role in
immunological processes by mediating the linkage between anti-
gen and effector function. The binding of the IgG variable domain
to a specific antigen on a target cell directs the killing of the target
cell by antibody-dependent cellular cytotoxicity (ADCC) and com-
plement-dependent cytotoxicity (CDC). ADCC is triggered by the
cross-linking of receptors for the Fc domain of the IgG antibody
(FccRs), particularly FccRI and FccRIII on immune effector cells.
The effector cells that may mediate ADCC include natural killer
cells, macrophages and neutrophils [1]. CDC is initiated by comple-
ment component C1q binding to the Fc region of IgG, triggering a
proteolytic cascade to activate complement [2].
IgG-type antibodies have two heavy chains and two light chains
held together by intra-molecular disulfide bonds to form a hetero-
tetramer. The heavy chains are glycosylated through covalent
attachment of an oligosaccharide at asparagine 297 (Asn-297). The
main N-glycans of human IgG are complex biantennary type and
have a ‘‘core’’ heptasaccharide, GlcNac2Man3GlcNac2, which is
⇑ Corresponding author. Fax: +1 (603) 643 8194.
E-mail address: huijuan_li@merck.com (H. Li).
dues may be attached to the ‘‘core’’ to generate further complex
biantennary glycan structures such as GalGlcNac2Man3GlcNac2
(G1), Gal2GlcNac2Man3GlcNac2 (G2), Neu5Ac2Gal2GlcNac2Man3Glc-
Nac2 [3,4].
Currently, therapeutic mAbs are produced in mammalian cells,
mostly in Chinese hamster ovary (CHO) cell lines. The glycan pro-
files of recombinant antibodies produced in mammalian cells are
predominantly heterogeneous. Heterogeneity can vary widely
from clone to clone and is dependent on the mode of production
and culture conditions [3,4]. An antibody’s glycan profile can have
a significant effect on ADCC and CDC. Deglycosylated IgG shows no
ability to activate ADCC and CDC [5]. Rituximab, a chimeric hu-
man-murine humanized anti-CD20 mAb, produced in glycoengi-
neered Pichia pastoris has both higher binding affinity to FccRIIIa
and greater B-cell depletion potency in comparison with the coun-
terpart produced from CHO cells [6]. N-Glycans from therapeutic
mAbs produced in mammalian cells are predominantly fucosylat-
ed, in that a fucose residue is attached to the primary N-acetylglu-
cosamine residue. It has been shown that the fucosylation of
glycans affects ADCC and CDC efficacy. For instance, afucosylated
glycans on human IgG1 can significantly increase IgG1 binding to
FccRIII and enhance ADCC efficacy, but has no effect on binding
to FccRI or C1q [7]. These results indicate that antibody-mediated
effector functions may be optimized by generating a specific glyco-
form of antibody.

1046-5928/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.pep.2010.11.004
8 Y. Jiang et al. / Protein Expression and Purification 76 (2011) 7–14
Yeast is a widely used recombinant protein expression system.
However, glycoproteins produced in wild-type yeast contain
potentially immunogenic high-mannose type N-glycans, limiting
the use of yeast expression systems for therapeutic protein and
antibody production [8]. Another challenge for antibody expres-
sion in yeast is the assembly of heavy and light chains to form a
heterotetramer. Generally, in yeast expression, the secreted anti-
body is predominately the heterodimer or monomers of heavy
and light chains. This antibody assembly problem may be related
to a decreased efficiency of antibody folding, processing and secre-
tion in yeast when there are high-mannose type N-glycans [9].
Human glycosylation pathways have been engineered into the
yeast P. pastoris to perform specific humanized N-glycosylation
reactions with high fidelity. The glycoengineered cell lines of P.
pastoris have successfully produced recombinant proteins and
mAbs with humanized N-glycan structures [6,10–13]. Rituximab
produced from different glycoengineered cell lines of P. pastoris
can properly assemble to form a heterotetramer and demonstrate
same antigen-binding characteristics as commercial Rituximab
from CHO cells [6].
In mammalian cell culture, Protein A affinity chromatography is
widely used as a first purification step to directly capture mAb
product at a neutral pH. The product eluted from the Protein A col-
umn at low pH usually contains process and product related impu-
rities. The process related impurities include endotoxin, leached
Protein A, host cell protein and DNA. The product related impuri-
ties mainly are aggregated and clipped mAb products. To remove
these impurities, second and third chromatographic steps are
generally employed, typically selected from cation exchange chro-
matography (CEX), anion exchange chromatography (AEX), hydro-
phobic interaction chromatography (HIC), hydrophobic charge
induction chromatography (HCIC) and hydroxyapatite chromatog-
raphy [14]. In our studies, many mAb purification processes from
mammalian cell culture did not work well to remove product
related impurities from P. pastoris fermentation. We previously
used Protein A affinity chromatography and Phenyl Sepharose of
HIC to purify small amounts of Rituximab from glycoengineered
P. pastoris [6]. More recently, a small scale, non-optimized version
of the method presented herein was used to purify small amounts
of a monoclonal antibody from glycoengineered P. pastoris fermen-
tation [15].
A humanized anti-HER2/neu receptor IgG1 mAb was used as an
example to study mAb production in glycoengineered P. pastoris. A
robust and scalable purification process was developed by optimiz-
ing process conditions for Protein A and cation exchange chroma-
tographies. The process efficiently removed the product related
impurities and process related impurities of residual Protein A,
host cell DNA and endotoxin. Grams of highly pure anti-HER2
mAb were easily generated from P. pastoris fermentation to sup-
port biochemical characterization and animal studies. Anti-HER2
mAb from glycoengineered P. pastoris is comparable to its commer-
cial counterpart (Trastuzumab) produced by CHO cells in hetero-
tetramer folding, physical stability and binding affinity for
antigen, FccRI and C1q. Additionally, anti-HER2 mAb produced in
P. pastoris is inherently afucosylated.
Materials and methods
Materials
STREAMLINE rProtein A (80–165 lm particle size), MabSelect
SuRe (85 lm particle size), SOURCE 30S (30 lm particle size), Cap-
to S (90 lm particle size), SP Sepharose High Performance (HP)
(34 lm particle size), SP Sepharose Fast Flow (FF) (45–165 lm
particle size), XK26/40, XK16/40 and Tricorn 10/200 columns were
obtained from GE Healthcare (Piscataway, NJ, USA). POROS 50 HS
(50 lm particle size) was from Perseptive Biosystems (Framing-
ham, MA, USA). Chemicals were from JT Baker (Phillipsburg, NJ,
USA) or Sigma–Aldrich (St. Louis, MO, USA). All buffers were fil-
tered with Nalgene polyethersulfone membrane filters (0.2 lm
pore size) (Rochester, NY, USA). SARTOPORE 2 (0.8 + 0.45 lm)
was from Sartorius (Göttingen, Germany). Vector pPICZA, Anti-hu-
man IgG-AlexaFluor488, PicoGreenÒ reagent and 4-Methylumbel-
liferyl phosphate (4-MUP) were from Invitrogen (Carlsbad, CA,
USA). Streptavidin–alkaline phosphatase (AP) conjugate was from
Promega (Madison, WI, USA). 4–20% Tris–HCl Ready Gels and Pre-
stained SDS–PAGE standards (broad range) were from Bio-Rad Lab-
oratories (Hercules, CA, USA). FccRI was from R&D systems
(Minneapolis, MN, USA). Human C1q was from United States Bio-
logical (Swampscott, MA, USA). Anti-C1q polyclonal antibody-AP
conjugate was from Abcam (Cambridge, MA, USA).
Cell line and fermentation
Anti-HER2 mAb P. pastoris expression cell line was constructed
according to methods described earlier [6,10–13]. Briefly, heavy
and light chain genes were subcloned into the pPICZA vector and
transformed into the glycoengineered GFI5.0 strain. A cell line
was selected after screening transformed strains for mAb expres-
sion titer and N-glycosylation profile.
Fermentations in 15 and 40 L bioreactors were conducted
according to methods described earlier [6]. Fermentation superna-
tant was recovered by centrifugation at 15,800g for 30 min and
clarified by filtration through SARTOPORE 2 (0.8 + 0.45 lm).
Column chromatography
All chromatographic experiments were performed with an
ÄKTA explorer 100 system from GE Healthcare (Piscataway, NJ,
USA) at room temperature. The flow rate was chosen to have
5 min of column residence time, unless it was specifically indi-
cated. UV absorbance of proteins was monitored at 280 nm.
Protein A affinity chromatography
To determine Protein A chromatography binding capacity for
mAb from P. pastoris fermentation, STREAMLINE rProtein A and
MabSelect SuRe columns were equilibrated with 20 mM Tris, pH
7.0 in five column bed volumes (CV) and loaded with fermentation
supernatant, pH 7.0, to completely saturate the column binding
capacity. Subsequently, the columns were washed with 20 mM
Tris, pH 7.0, 1 M NaCl (3 CV) and eluted by a linear gradient
decreasing from pH 5.0 to 3.0 in 100 mM sodium citrate with or
without 1 M arginine (10 CV). The pH at A280nm peak maxima
was measured in the effluent from the ÄKTA explorer 100 system.
Fractions representing the mAb peak were pooled for protein con-
centration determination.
Protein A column binding capacities, (the amount of mAb bound
per ml resin at the flow rate to have 5 min of column resident time)
were calculated from protein in the elute fractions according to the
following equation:
Binding capacity ¼ V E  C E =V R
VE, volume of eluate (ml); CE, protein concentration in eluate
fraction (mg/ml); VR, volume of Protein A resin in the column (ml).
Cation exchange chromatography
STREAMLINE rProtein A purified anti-HER2 mAb was used for
cation exchange chromatography development. The cation ex-
change resins in Tricorn 10/200 (1 cm i.d.  19 cm) were equili-
brated with 25 mM sodium acetate, pH 4.5 (5 CV), loaded with
 Y. Jiang et al. / Protein Expression and Purification 76 (2011) 7–14
45 mg of IgG1 at pH 4.5 and washed with 25 mM sodium acetate,
pH 5.0 (2 CV) before the chromatographic separation.
Chromatographic separations using SOURCE 30S, SP Sepharose
High Performance, Capto S and Poros 50 HS were performed in lin-
ear gradient from 0 to 300 mM NaCl (10 CV) in 25 mM sodium ace-
tate, pH 5.0, followed by 500 mM NaCl (3 CV) in the same sodium
acetate buffer.
SOURCE 30S chromatographic separations were performed in
linear gradient from 0 to 300 mM NaCl (10 CV) in 25 mM sodium
acetate at pH 4.5 and 5.0, respectively; and in 12.5 mM sodium
acetate, 12.5 mM sodium phosphate at pH 6.0. After the linear gra-
dient elution, the column was continually eluted with 500 mM
NaCl (3 CV) in the buffer with the same pH.
SOURCE 30S stepwise gradient chromatography was performed
with NaCl at 100 mM (3 CV), 125 mM (3 CV), 150 mM (3 CV),
175 mM (3 CV), 300 mM (2 CV) and 500 mM (2 CV) in 25 mM so-
dium acetate, pH 5.0.
Preparative chromatographic run
STREAMLINE rProtein A resin was packed in a XK26/40 column
(2.6 cm i.d.  28 cm) and equilibrated with 20 mM Tris pH 7.0 (5
CV). The column was loaded with 7 L of fermentation supernatant,
pH 7.0, then washed according to the following washing protocol:
(1) 20 mM Tris pH 7.0, 1 M NaCl (4 CV); (2) 20 mM Tris, pH 7.0 (2
CV); (3) 20 mM Tris pH 7.0, 10 mM EDTA, 10 mM CHAPS (6 CV); (4)
20 mM Tris pH 7.0 (4 CV). The column was then eluted with a lin-
ear gradient decreasing from pH 4.0 to 3.0 in 100 mM sodium cit-
rate (3 CV) followed with pH 3.0 (5 CV). The flow rate was 21 ml/
min. The eluted fractions were adjusted to pH 5.0 with 1 M sodium
phosphate, dibasic, pH 8.9. Fractions representing the IgG1 peak
were pooled for next step purification.
SOURCE 30S resin was packed in an XK26/40 column (2.6 cm
i.d.  28 cm) and equilibrated with 25 mM sodium acetate, pH
4.5 (5 CV). Anti-HER2 mAb from STREAMLINE rProtein A pool
was diluted five times with water (conductivity 10 mS/cm), ad-
justed to pH 4.5 with 1 M acetic acid, and then loaded on the col-
umn. SOURCE 30S chromatography with a stepwise gradient was
conducted according to the above protocol. The flow rate was
10 ml/min.
SP Sepharose Fast Flow resin was packed in an XK16/40 column
(1.6 cm i.d.  35 cm) and equilibrated with buffer A (5 CV). Anti-
HER2 mAb from SOURCE 30S was diluted five times with buffer
A and loaded on an SP Sepharose column. After buffer A washing
(5 CV), mAb was eluted from the column with 5 CV of formulation
buffer (10 mM histidine, pH 6.0, 150 mM NaCl, 0.01% polysorbate
20). Fractions of the peak were pooled as final product, filtered
through 0.2 lm polyethersulfone membrane filter and stored asep-
tically at 4 °C.
Protein concentration
Anti-HER2 mAb concentration was determined by measuring
the absorption in a 1 cm quartz cuvette at 280 nm with a UV spec-
trophotometer. The theoretical extinction coefficient of
1.42 L g1 cm1 calculated from amino acid sequence was used
[16].
Physical stability analysis
The purified anti-HER2 mAb was concentrated and buffer ex-
changed in the formulation buffer. Antibody in formulation buffer
was adjusted to 10 mg/ml and stored at different temperatures of
4, 25 and 37 °C. It was analyzed weekly by size-exclusion chroma-
tography (SEC) to determine aggregation and degradation for six
weeks. The analytical SEC was conducted using a Zorbax GF-250
(4 lm particle size) column, 9.4  250 mm, on a Hitachi D-7000
9
HPLC system (Hitachi, Ltd., Tokyo, Japan) with L7420 UV detector
monitoring 280 nm. Sample volume was 90 ll and the mobile
phase was 100 mM sodium phosphate, pH 6.8, 150 mM NaCl,
0.05% sodium azide at flow rate of 1.0 ml/min.
SDS polyacrylamide gel electrophoresis (SDS–PAGE) and two-
dimensional electrophoresis
SDS–PAGE was carried out according to the Laemmli method
with modified 4 non-reducing sample buffer (250 mM Tris, pH
6.8, 8% SDS, 40% v/v Glycerol, 0.4% Bromophenol Blue sodium salt,
100 mM N-ethylmaleimide) and 4 reducing sample buffer
(250 mM Tris, pH 6.8, 8% SDS, 40% v/v Glycerol, 0.4% Bromophenol
Blue sodium salt, 20% v/v b-mercaptoethanol) [17,18]. Gels were
stained with 0.025% Coomassie Brilliant Blue R 250 in 7% v/v acetic
acid/40% v/v methanol and destained for 60 min in 10% v/v acetic
acid.
Two-dimensional electrophoresis was performed by Kendrick
Labs, Inc. (Madison, WI, USA) in the standard format. Briefly, iso-
electric focusing (IEF) was carried out in a glass tube using 2% pH
3.5–10 mixed ampholines 4 L. The pH gradient plot was deter-
mined with surface pH electrode and six IEF tube gels (run with
buffer). Tropomyosin (isoelectric point, pI, 5.2) was included in
the sample as an internal standard. The tube gel from IEF was then
sealed onto the top of 10% acrylamide slab gel to run a SDS slab gel
electrophoresis. The acrylamide slab gel was stained with Coomas-
sie blue and dried between sheets of cellophane. Protein’s pI was
determined using the pH gradient plot provided by the vendor.
Endotoxin analysis
Endotoxin was determined using the end-point chromogenic
Limulus Amebocyte Lysate (LAL) method provided by Charles River
Endosafe (Wilmington, MA, USA).
Residual Protein A analysis
Residual Protein A was determined as described [19] with some
modifications. Protein A standard and samples were captured in
96-well microplates coated with anti-Protein A polyclonal antibod-
ies. Biotinylated anti-Protein A polyclonal antibodies were added
to the plate to form an immune complex with captured Protein
A. The complex was detected with streptavidin-AP conjugate, fol-
lowed by the substrate 4-MUP. The product was quantified by
reading fluorescence with excitation at 360 nm, emission at
485 nm.
Host cell genomic DNA analysis
Host cell genomic DNA was determined by using PicoGreenÒ re-
agent according to instruction from Invitrogen (Carlsbad, CA, USA).
N-Glycan analysis
N-Glycan profile was determined by matrix-assisted laser
desorption/ionization/time-of-flight mass spectrometry (MALDI-
TOF MS) using a Voyager-DE™ BioSpectrometry™ Workstation
(Applied Biosystems, Foster City, CA, USA) as previously described
[12].
Binding assays
Antigen-binding assay was performed using SKBR3 cells
(American Type Culture Collection) as described [6] with minor
modifications. The mAb–antigen complex was stained with anti-
human IgG-AlexaFluor488. Mean fluorescence intensity (MFI)
10 Y. Jiang et al. / Protein Expression and Purification 76 (2011) 7–14

Table 1
Protein A affinity chromatography of anti-HER2 mAb from glycoengineered P. pastoris fermentation supernatant.

Resins Binding capacity (mg/ml) Elution buffers pH at maximal A280nm Normalized recovery (%)
STREAMLINE rProtein A 32 100 mM sodium citrate pH gradient 5–3 3.3 100
STREAMLINE rProtein A 32 1 M arginine 100 mM sodium citrate pH gradient 5–3 3.7 97
MabSelect SuRe 28 100 mM sodium citrate pH gradient 5–3 3.7 90

was detected in a Guava Express Plus (Guava Technologies, Hay-
ward, CA, USA) using excitation at 488 nm and emission at 525 nm.
C1q-binding assay was carried out as described [20] with minor
modifications. The mAb-C1q complex was detected with anti-C1q
polyclonal antibody-AP conjugate, followed by the substrate 4-
MUP. The product was quantified by reading relative fluorescence
units (RFU) with excitation at 360 nm, emission at 485 nm.
FccRI-binding assays were conducted as previously described
[6].
Results and discussion
Protein A affinity chromatograph for anti-HER2 mAb purification
Protein A affinity chromatography is a common method for
mAb purification. STREAMLINE rProtein A is designed for antibody
purification directly from crude feedstock by expanded bed
adsorption chromatography. MabSelect SuRe is genetically engi-
neered Protein A affinity resin which has enhanced alkali stability

A
mAU
2500
B
mAU
Capto S

2000
2000

1500 SP Sepharose HP
1500

1000 pH 6.0
1000

POROS 50 HS

500 pH 5.0
500

SOURCE 30S pH 4.5

0 0

0 50 100 150 200 250 300 ml 0 50 100 150 200 250 300 ml

C D 1 2 3 4 5 6 7 8 9
KDa

2 − mAb
mAU 205 −

600 115 −
98 −
400 1 − Hc
54 −
200
3 37 − − Lc
0 29 −
0 50 100 150 200 250 300 350 ml
20 −

7−

Fig. 1. Anti-HER2 mAb cation exchange chromatography. The columns (1 cm i.d.  19 cm) were equilibrated with 25 mM sodium acetate, pH 4.5 (5 CV), loaded with 45 mg of
Protein A purified anti-HER2 mAb at pH 4.5 and washed with 25 mM sodium acetate, pH 5.0 (2 CV) before the following chromatographic separations. (A) Chromatograms of
SOURCE 30S, Poros 50 HS, SP Sepharose HP, and Capto S. These chromatograms were conducted in linear gradient from 0 to 300 mM NaCl (10 CV) in 25 mM sodium acetate,
pH 5.0, followed with 500 mM NaCl in the buffer. (B) Chromatograms at variant pH. SOURCE 30S chromatographies were compared in a linear gradient from 0 to 300 mM
NaCl (10 CV) in 25 mM sodium acetate at pH 4.5 and 5.0 respectively, and in 12.5 mM sodium acetate, 12.5 mM sodium phosphate at pH 6.0. Five hundred millimolar NaCl (3
CV) in the same pH buffers followed after the linear gradient. (C) NaCl stepwise chromatogram of SOURCE 30S. It was performed with NaCl at 100 mM (3 CV), 125 mM (3 CV),
150 mM (3 CV), 175 mM (3 CV), 300 mM (2 CV) and 500 mM (2 CV) in 25 mM sodium acetate, pH 5.0. (D) SDS–PAGE analysis of samples from SOURCE 30S stepwise
chromatography in non-reducing (NR) and reducing (R) conditions. Prestained SDS–PAGE standard (lane 1), empty (lanes 2 and 6), peak 1 pool (lane 3, NR; lane 7, R), peak 2
pool (lane 4, NR; lane 8, R), peak 3 pool (lane 5, NR; lane 9, R). Hc, heavy chain; Lc, light chain. Product related impurities are indicated with arrows.
 Y. Jiang et al. / Protein Expression and Purification 76 (2011) 7–14 11

Aggregation (%) Degradation (%)

A 1 2 3 4 5 6 7 8 9 10 11 1.0 A
KDa
0.5

205 − 0.0

115 − 1.0
B
98 −
0.5
54 −
37 − 0.0
29 −
0 1 2 3 4 5 6
20 − Time (Weeks)
7−
Fig. 3. The physical stability of anti-HER2 mAb from glycoengineered P. pastoris.
Percent of degradation (A) and aggregation (B) formed after storing anti-HER2 mAb
product (10 mg/ml) at 4 °C (d), 25 °C (.) and 37 °C (j) up to six weeks.
B 1 2 3 4 5 6 7 8 9 10 11 12 13
KDa

anti-HER2 mAb was eluted from both columns by a linear gradient

205 −
115 −
98 −
54 −
37 −
29 −
20 −
7−
Fig. 2. (A) SDS–PAGE analysis of anti-HER2 mAb from scale-up purification process.
Prestained SDS–PAGE standards (Lane 1), empty (Lanes 2 and 7), Protein A pool
(lane 3, NR; lane 8, R), SOURCE 30S pool (lane 4, NR; lane 9, R), SP Sepharose pool
(lane 5, NR; lane 10, R), Trastuzumab from CHO cells (lane 6, NR; lane 11, R). (B)
SDS–PAGE analysis of IgG1 (I), IgG2(II) and IgG4 (III) from glycoengineered P.
pastoris. Prestained SDS–PAGE standards (lane 1), Protein A purified mAb product I
(lane 2, NR; lane 4, R), II (lane 6, NR; lane 8, R), III (lane 10, NR; lane 12, R) and
SOURCE 30S purified mAb product I (lane 3, NR; lane 5, R), II (lane 7, NR; 9, R), III
(lane 11, NR; lane 13, R).
for column regeneration and sanitization. MabSelect SuRe exhibits
a comparable binding capacity to wild-type Protein A resins,
including MabSelect Xtra and ProSep-vA Ultra [21]. We compared
binding capacity of STREAMLINE rProtein A and MabSelect SuRe
columns for anti-HER2 mAb purification from P. pastoris fermenta-
tion supernatant. Both columns had similar binding capacity of
30 mg/ml (Table 1), which were comparable to their binding
capacity determined with purified human IgG [21]. Therefore, we
used both columns to capture anti-HER2 mAb from P. pastoris fer-
mentation supernatant and evaluate their elution conditions.
Antibody is conventionally eluted from Protein A affinity chro-
matography at low pH (3.0). However, antibody is not stable at
low pH (2.5–3.5) and tends to aggregate [22]. An elution buffer with
higher pH was applied to reduce the aggregation, which led to
incomplete elution of anti-HER2 mAb. Arginine has been used as
an alternative strategy to elevate elution pH to prevent antibody
aggregation in Protein A affinity chromatography [22]. To optimize
elution conditions for anti-HER2 mAb, we compared Protein A chro-
matography with and without arginine in a decreasing gradient of
elution pH (5.0–3.0). As summarized in Table 1, the pH at A280nm peak
maxima appeared at pH 3.3 in STREAMLINE rProtein A chromatogra-
phy and at pH 3.7 in MabSelect SuRe chromatography when
decreasing from pH 5.0 to 3.0 in 100 mM sodium citrate. The pH
maxima was shifted to 3.7 in STREAMLINE rProtein A chromatogra-
phy when 1 M arginine was included. Over 90% of mAb was recov-
ered from the Protein A column in each of these elution conditions
(Table 1). No mAb was detected in the post-elution resin (data not
shown). Antibody precipitation was prevented by eluting with a
decreasing pH linear gradient in the presence or absence of 1 M argi-
nine. Considering its low pressure drop in column chromatography,
we chose to use STREAMLINE rProtein A in scale-up purification pro-
cess and perform pH gradient elution without arginine in the elution
buffer.
Cation exchange chromatography to remove product related
impurities
In Protein A purified anti-HER2 mAb from P. pastoris, there are
some product related impurities which are different from impuri-
ties observed in other expression systems. After evaluating differ-
ent chromatographies including CEX, AEX, HIC, HCIC and
hydroxyapatite chromatography, we found that cation exchange
chromatography can perform well to remove these impurities.
Fig. 1A compares cation exchange chromatograms performed with
the same NaCl linear gradient elution at pH 5.0. The chromato-
grams of SOURCE 30S, Poros 50 HS and SP Sepharose HP all dem-
onstrated good peak resolution. However, the peak resolution
disappeared in the Capto S chromatogram which might be due to
its large particle size, suggesting that SOURCE 30S, Poros 50 HS
and SP Sepharose HP could perform well to remove the impurities.
To determine the optimal pH for NaCl gradient elution, we com-
pared SOURCE 30S chromatograms with NaCl linear gradient elu-
tion at different pH of 4.5, 5.0 and 6.0. As shown in Fig. 1B, there
were good resolutions at pH 4.5 and 5.0 but some overlap in the
first two elution peaks at pH 6.0. To develop an easy scale-up puri-
fication process, we optimized SOURCE 30S chromatography with
NaCl stepwise gradient elution. As shown in Fig. 1C, the elution
peaks were well separated at different NaCl concentrations in
25 mM sodium acetate buffer, pH 5.0. Fig. 1D depicts the SDS–
PAGE analysis of samples in these peaks. The first elution peak at
100 mM NaCl contained misassembled and cleaved anti-HER2

Table 2
Summary of anti-HER2 mAb scale-up purification process from P. pastoris fermentation supernatant.

Process step Accumulated yield (%) Purity (%) Endotoxin (EU/mg) Protein A (ppm) Host DNA (ppm)
STREAMLINE rProtein A 100 81 0.6 290 <7
SOURCE 30S 60 99 1.7 3 <6
SP Sepharose Fast Flow 60 99 0.3 0.6 <0.3
12 Y. Jiang et al. / Protein Expression and Purification 76 (2011) 7–14

A 1648.22
1486.18
100
90
- fucosylated G0
80
70
- fucosylated G1
60
Intensity (%) 50
40
30
20
10

849.0 1319.4 1789.8 2260.2 2730.6 3201.0

Mass (m/z)

B 1342.73
100
90
80 - afucosylated G0
70
60
Intensity (%)

50
40
30 1504.92
20 - afucosylated G1
10

849.0 1319.4 1789.8 2260.2 2730.6 3201.0

Mass (m/z)

Fig. 4. MALDI-TOF analysis of N-glycan profiles in Trastuzumab from CHO cells (A), and anti-HER2 mAb from glycoengineered P. pastoris (B). Respective glycan structures are
indicated.

mAb, which was composed of light chains and cleaved heavy
chains, as confirmed by N-terminal amino acid sequencing (data
not shown). The second elution peak at 125 mM NaCl contained in-
tact anti-HER2 mAb with few product related impurities. The small
elution peaks at or above 150 mM NaCl were misassembled mAb,
which were composed of a mixture of heavy chains, cleaved heavy
chains and light chains. These results indicate that in Protein A
purified anti-HER2 mAb from glycoengineered P. pastoris, product
related impurities come from the misassembling of cleaved heavy
chain, heavy chain and light chain. These impurities can be effi-
ciently removed by cation exchange chromatography in optimal
NaCl gradient at pH 4.5–6.0.
Scale-up purification process
Based on the above optimized conditions in small column purifi-
cation, we have developed a robust and scalable purification process
to produce grams of high quality anti-HER2 mAb from 10 to 40 L of P.
pastoris fermentations. After passing the fermentation supernatant
through the STREAMLINE rProtein A column, non-specific binding
impurities were removed from the column by washing, and the anti-
body was eluted with a gradient decreasing from pH 4.0 to 3.0. Anti-
body pool from Protein A chromatography was diluted with water by
5-fold volume to reduce conductivity to 10 mS/cm and directly ap-
plied to a SOURCE 30S column to remove product related impurities.
Antibody was eluted using a stepwise NaCl gradient at pH 5.0. Final-
ly, antibody from the SOURCE 30S column was applied to a SP
Sepharose Fast Flow column and eluted with formulation buffer as
the final product. This purification process eliminates the time-con-
suming buffer exchange process between each chromatography step
which may introduce protein degradation and aggregation. Fig. 2A
shows the SDS–PAGE analysis of anti-HER2 mAb products in each
step of the purification process and its commercial counterpart
product, Trastuzumab produced in CHO cells. The data indicate that
our two-step purification process can produce anti-HER2 mAb from
P. pastoris fermentation and achieve the same purity as Trast-
uzumab. This purification process was generally utilized to purify
other IgG1, IgG2 and IgG4 mAbs which target different antigens.
Fig. 2B is the SDS–PAGE analysis of three other representative mAbs
from glycoengineered P. pastoris and shows the efficient removal of
product related impurities to achieve a high purity.
The results of the anti-HER2 mAb purification process are sum-
marized in Table 2. Protein A purified anti-HER2 product contained
about 20% of product related impurities, which were completely
removed from the intact antibody product by using SOURCE 30S
chromatography. The purification process had been shown to pro-
duce final anti-HER2 mAb product in 99% purity and achieve 60% of
total recovery from Protein A captured proteins. Anti-HER2 mAb
from Protein A affinity chromatography contained 290 ppm of
residual Protein A, which co-eluted with antibody from the col-
umn. After SOURCE 30S and SP Sepharose chromatography, Resid-
ual Protein A was reduced almost 500-fold to 0.6 ppm in the final
product (Table 2). Protein A purified anti-HER2 mAb had low levels
of host cell DNA (<7 ppm) and endotoxin (0.6 EU/mg). In the final
product, host DNA was further reduced over 20-fold to <0.3 ppm,
and endotoxin was reduced 2-fold to 0.3 EU/mg (Table 2). Host cell
protein impurities in the final product were barely detected with
an in-house Western blot assay using a polyclonal antibody. The
polyclonal antibody was raised in rabbits using Pichia host cell pro-
teins as antigens (data not shown).
 Y. Jiang et al. / Protein Expression and Purification 76 (2011) 7–14 13

A N-Glycan profiles of Trastuzumab from CHO cells and anti-HER2

400 mAb from glycoengineered P. pastoris were compared by MALDI-
TOF MS (Fig. 4A and B). Trastuzumab N-glycans consisted of pre-
dominantly fucosylated G0 and G1 structures. N-Glycans of anti-
300
HER2 mAb from glycoengineered P. pastoris are inherently afucosy-
lated and consisted mainly of G0 and some G1 and G2. O-Linked
glycans for anti-HER2 mAb from glycoengineered P. pastoris was
MFI

200
detected at low level (data not shown).
Trastuzumab from CHO cells and anti-HER2 mAb from glycoen-
100 gineered P. pastoris were also analyzed by two-dimensional elec-
trophoresis. Both products migrated at the same position and
had experimental pI values of 7.7. N-terminal amino acid sequenc-
0 ing by Edman degradation confirmed that the N-termini of both
-2 -1 0 1
Log[mAb] (µg/ml) heavy and light chains were intact. Peptide mapping confirmed
the same amino acid sequence in both products (data not shown).
B 40000 Anti-HER2 mAb product from glycoengineered P. pastoris was
characterized for its binding to antigen, FccRI and C1q in compar-
35000
ison with Trastuzumab. Both products demonstrated the same
30000 affinity to cell surface expressed target antigen (Fig. 5A). They also
had similar binding affinity for FccRI and C1q (Fig. 5B and C), which
25000
RFU

is consistent with a previous report that afucosylation has no effect

20000 on mAb binding to FccRI or C1q [7].
15000

10000 Conclusions

5000
A robust and scalable purification process has been developed
0 to efficiently produce large quantities of anti-HER2 mAb from P.
-4 -3 -2 -1 0
pastoris fermentation. In addition, process related impurities were
Log[mAb] (µg/ml)
removed from the product. Anti-HER2 mAb from glycoengineered
P. pastoris was comparable to its commercial counterpart (Trast-
C 40000
uzumab) from CHO cells with correct heterotetramer folding and
35000 good physical stability, similar binding affinity to its antigen, FccRI
30000 and C1q. Glycoengineered cell lines of P. pastoris are expected to
play a significant role in the manufacture of therapeutic antibodies,
25000
RFU

substantially reducing production time while improving glycan

20000 uniformity and decreasing production costs.
15000
Disclosure statement
10000

5000 The authors declare no conflict of interests.

0
-1 0 1 2 Acknowledgments
Log[mAb] (µg/ml)

Fig. 5. Binding activity of Trastuzumab from CHO cells (d), anti-HER2 mAb from We thank BBR Automated Process Monitoring Facility in MERCK
glycoengineered P. pastoris (.) and off target control IgG1 (j). (A) Cell surface target & Co. Inc. for analysis of residual Protein A and host cell genomic
antigen binding, (B) FccRI binding, (C) C1q binding. Fitted curves were calculated
DNA. We are also grateful to Karen M. Page for editing of the
using sigmoidal dose–response (variable slope) from SigmaPlot (Systat Software,
Inc., San Jose, CA, USA). (Goodness of fit, R2 P0.99).
manuscript.

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Characterization of anti-HER2 mAb
The physical stability of anti-HER2 mAb from glycoengineered
P. pastoris was assessed by using SEC to detect aggregation or deg-
radation. When anti-HER2 mAb in formulation buffer was stored at
4, 25 and 37 °C for a period of six weeks, no significant amount of
aggregation or degradation was detected by SEC analysis. Only
0.3% of degradation was detected after storage at 37 °C for 6 weeks
(Fig. 3A and B). In parallel stability studies of anti-HER mAb in an-
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degradation and 0.3% of aggregation were detected, after storage
at 37 °C for six weeks. Anti-HER2 mAb concentration was also con-
stant over the same time period (data not shown). These data indi-
cate that anti-HER2 mAb from glycoengineered P. pastoris has good
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