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Protein Expression and Purification 76 (2011) 7–14

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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-


dues may be attached to the ‘‘core’’ to generate further complex
Monoclonal antibodies (mAbs) are rapidly becoming key prod- biantennary glycan structures such as GalGlcNac2Man3GlcNac2
ucts for the biopharmaceutical industry. Most commercial thera- (G1), Gal2GlcNac2Man3GlcNac2 (G2), Neu5Ac2Gal2GlcNac2Man3Glc-
peutic antibodies are intact IgG molecules with IgG1 and IgG2 Nac2 [3,4].
being the common subclasses. These IgGs play a central role in Currently, therapeutic mAbs are produced in mammalian cells,
immunological processes by mediating the linkage between anti- mostly in Chinese hamster ovary (CHO) cell lines. The glycan pro-
gen and effector function. The binding of the IgG variable domain files of recombinant antibodies produced in mammalian cells are
to a specific antigen on a target cell directs the killing of the target predominantly heterogeneous. Heterogeneity can vary widely
cell by antibody-dependent cellular cytotoxicity (ADCC) and com- from clone to clone and is dependent on the mode of production
plement-dependent cytotoxicity (CDC). ADCC is triggered by the and culture conditions [3,4]. An antibody’s glycan profile can have
cross-linking of receptors for the Fc domain of the IgG antibody a significant effect on ADCC and CDC. Deglycosylated IgG shows no
(FccRs), particularly FccRI and FccRIII on immune effector cells. ability to activate ADCC and CDC [5]. Rituximab, a chimeric hu-
The effector cells that may mediate ADCC include natural killer man-murine humanized anti-CD20 mAb, produced in glycoengi-
cells, macrophages and neutrophils [1]. CDC is initiated by comple- neered Pichia pastoris has both higher binding affinity to FccRIIIa
ment component C1q binding to the Fc region of IgG, triggering a and greater B-cell depletion potency in comparison with the coun-
proteolytic cascade to activate complement [2]. terpart produced from CHO cells [6]. N-Glycans from therapeutic
IgG-type antibodies have two heavy chains and two light chains mAbs produced in mammalian cells are predominantly fucosylat-
held together by intra-molecular disulfide bonds to form a hetero- ed, in that a fucose residue is attached to the primary N-acetylglu-
tetramer. The heavy chains are glycosylated through covalent cosamine residue. It has been shown that the fucosylation of
attachment of an oligosaccharide at asparagine 297 (Asn-297). The glycans affects ADCC and CDC efficacy. For instance, afucosylated
main N-glycans of human IgG are complex biantennary type and glycans on human IgG1 can significantly increase IgG1 binding to
have a ‘‘core’’ heptasaccharide, GlcNac2Man3GlcNac2, which is FccRIII and enhance ADCC efficacy, but has no effect on binding
to FccRI or C1q [7]. These results indicate that antibody-mediated
⇑ Corresponding author. Fax: +1 (603) 643 8194. effector functions may be optimized by generating a specific glyco-
E-mail address: huijuan_li@merck.com (H. Li). 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. obtained from GE Healthcare (Piscataway, NJ, USA). POROS 50 HS
However, glycoproteins produced in wild-type yeast contain (50 lm particle size) was from Perseptive Biosystems (Framing-
potentially immunogenic high-mannose type N-glycans, limiting ham, MA, USA). Chemicals were from JT Baker (Phillipsburg, NJ,
the use of yeast expression systems for therapeutic protein and USA) or Sigma–Aldrich (St. Louis, MO, USA). All buffers were fil-
antibody production [8]. Another challenge for antibody expres- tered with Nalgene polyethersulfone membrane filters (0.2 lm
sion in yeast is the assembly of heavy and light chains to form a pore size) (Rochester, NY, USA). SARTOPORE 2 (0.8 + 0.45 lm)
heterotetramer. Generally, in yeast expression, the secreted anti- was from Sartorius (Göttingen, Germany). Vector pPICZA, Anti-hu-
body is predominately the heterodimer or monomers of heavy man IgG-AlexaFluor488, PicoGreenÒ reagent and 4-Methylumbel-
and light chains. This antibody assembly problem may be related liferyl phosphate (4-MUP) were from Invitrogen (Carlsbad, CA,
to a decreased efficiency of antibody folding, processing and secre- USA). Streptavidin–alkaline phosphatase (AP) conjugate was from
tion in yeast when there are high-mannose type N-glycans [9]. Promega (Madison, WI, USA). 4–20% Tris–HCl Ready Gels and Pre-
Human glycosylation pathways have been engineered into the stained SDS–PAGE standards (broad range) were from Bio-Rad Lab-
yeast P. pastoris to perform specific humanized N-glycosylation oratories (Hercules, CA, USA). FccRI was from R&D systems
reactions with high fidelity. The glycoengineered cell lines of P. (Minneapolis, MN, USA). Human C1q was from United States Bio-
pastoris have successfully produced recombinant proteins and logical (Swampscott, MA, USA). Anti-C1q polyclonal antibody-AP
mAbs with humanized N-glycan structures [6,10–13]. Rituximab conjugate was from Abcam (Cambridge, MA, USA).
produced from different glycoengineered cell lines of P. pastoris
can properly assemble to form a heterotetramer and demonstrate Cell line and fermentation
same antigen-binding characteristics as commercial Rituximab
from CHO cells [6]. Anti-HER2 mAb P. pastoris expression cell line was constructed
In mammalian cell culture, Protein A affinity chromatography is according to methods described earlier [6,10–13]. Briefly, heavy
widely used as a first purification step to directly capture mAb and light chain genes were subcloned into the pPICZA vector and
product at a neutral pH. The product eluted from the Protein A col- transformed into the glycoengineered GFI5.0 strain. A cell line
umn at low pH usually contains process and product related impu- was selected after screening transformed strains for mAb expres-
rities. The process related impurities include endotoxin, leached sion titer and N-glycosylation profile.
Protein A, host cell protein and DNA. The product related impuri- Fermentations in 15 and 40 L bioreactors were conducted
ties mainly are aggregated and clipped mAb products. To remove according to methods described earlier [6]. Fermentation superna-
these impurities, second and third chromatographic steps are tant was recovered by centrifugation at 15,800g for 30 min and
generally employed, typically selected from cation exchange chro- clarified by filtration through SARTOPORE 2 (0.8 + 0.45 lm).
matography (CEX), anion exchange chromatography (AEX), hydro-
phobic interaction chromatography (HIC), hydrophobic charge
Column chromatography
induction chromatography (HCIC) and hydroxyapatite chromatog-
raphy [14]. In our studies, many mAb purification processes from
All chromatographic experiments were performed with an
mammalian cell culture did not work well to remove product
ÄKTA explorer 100 system from GE Healthcare (Piscataway, NJ,
related impurities from P. pastoris fermentation. We previously
USA) at room temperature. The flow rate was chosen to have
used Protein A affinity chromatography and Phenyl Sepharose of
5 min of column residence time, unless it was specifically indi-
HIC to purify small amounts of Rituximab from glycoengineered
cated. UV absorbance of proteins was monitored at 280 nm.
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- Protein A affinity chromatography
tation [15]. To determine Protein A chromatography binding capacity for
A humanized anti-HER2/neu receptor IgG1 mAb was used as an mAb from P. pastoris fermentation, STREAMLINE rProtein A and
example to study mAb production in glycoengineered P. pastoris. A MabSelect SuRe columns were equilibrated with 20 mM Tris, pH
robust and scalable purification process was developed by optimiz- 7.0 in five column bed volumes (CV) and loaded with fermentation
ing process conditions for Protein A and cation exchange chroma- supernatant, pH 7.0, to completely saturate the column binding
tographies. The process efficiently removed the product related capacity. Subsequently, the columns were washed with 20 mM
impurities and process related impurities of residual Protein A, Tris, pH 7.0, 1 M NaCl (3 CV) and eluted by a linear gradient
host cell DNA and endotoxin. Grams of highly pure anti-HER2 decreasing from pH 5.0 to 3.0 in 100 mM sodium citrate with or
mAb were easily generated from P. pastoris fermentation to sup- without 1 M arginine (10 CV). The pH at A280nm peak maxima
port biochemical characterization and animal studies. Anti-HER2 was measured in the effluent from the ÄKTA explorer 100 system.
mAb from glycoengineered P. pastoris is comparable to its commer- Fractions representing the mAb peak were pooled for protein con-
cial counterpart (Trastuzumab) produced by CHO cells in hetero- centration determination.
tetramer folding, physical stability and binding affinity for Protein A column binding capacities, (the amount of mAb bound
antigen, FccRI and C1q. Additionally, anti-HER2 mAb produced in per ml resin at the flow rate to have 5 min of column resident time)
P. pastoris is inherently afucosylated. were calculated from protein in the elute fractions according to the
following equation:

Binding capacity ¼ V E  C E =V R
Materials and methods
VE, volume of eluate (ml); CE, protein concentration in eluate
Materials fraction (mg/ml); VR, volume of Protein A resin in the column (ml).

STREAMLINE rProtein A (80–165 lm particle size), MabSelect Cation exchange chromatography


SuRe (85 lm particle size), SOURCE 30S (30 lm particle size), Cap- STREAMLINE rProtein A purified anti-HER2 mAb was used for
to S (90 lm particle size), SP Sepharose High Performance (HP) cation exchange chromatography development. The cation ex-
(34 lm particle size), SP Sepharose Fast Flow (FF) (45–165 lm change resins in Tricorn 10/200 (1 cm i.d.  19 cm) were equili-
particle size), XK26/40, XK16/40 and Tricorn 10/200 columns were 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 9

45 mg of IgG1 at pH 4.5 and washed with 25 mM sodium acetate, HPLC system (Hitachi, Ltd., Tokyo, Japan) with L7420 UV detector
pH 5.0 (2 CV) before the chromatographic separation. monitoring 280 nm. Sample volume was 90 ll and the mobile
Chromatographic separations using SOURCE 30S, SP Sepharose phase was 100 mM sodium phosphate, pH 6.8, 150 mM NaCl,
High Performance, Capto S and Poros 50 HS were performed in lin- 0.05% sodium azide at flow rate of 1.0 ml/min.
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 SDS polyacrylamide gel electrophoresis (SDS–PAGE) and two-
acetate buffer. dimensional electrophoresis
SOURCE 30S chromatographic separations were performed in
linear gradient from 0 to 300 mM NaCl (10 CV) in 25 mM sodium SDS–PAGE was carried out according to the Laemmli method
acetate at pH 4.5 and 5.0, respectively; and in 12.5 mM sodium with modified 4 non-reducing sample buffer (250 mM Tris, pH
acetate, 12.5 mM sodium phosphate at pH 6.0. After the linear gra- 6.8, 8% SDS, 40% v/v Glycerol, 0.4% Bromophenol Blue sodium salt,
dient elution, the column was continually eluted with 500 mM 100 mM N-ethylmaleimide) and 4 reducing sample buffer
NaCl (3 CV) in the buffer with the same pH. (250 mM Tris, pH 6.8, 8% SDS, 40% v/v Glycerol, 0.4% Bromophenol
SOURCE 30S stepwise gradient chromatography was performed Blue sodium salt, 20% v/v b-mercaptoethanol) [17,18]. Gels were
with NaCl at 100 mM (3 CV), 125 mM (3 CV), 150 mM (3 CV), stained with 0.025% Coomassie Brilliant Blue R 250 in 7% v/v acetic
175 mM (3 CV), 300 mM (2 CV) and 500 mM (2 CV) in 25 mM so- acid/40% v/v methanol and destained for 60 min in 10% v/v acetic
dium acetate, pH 5.0. acid.
Two-dimensional electrophoresis was performed by Kendrick
Preparative chromatographic run Labs, Inc. (Madison, WI, USA) in the standard format. Briefly, iso-
STREAMLINE rProtein A resin was packed in a XK26/40 column electric focusing (IEF) was carried out in a glass tube using 2% pH
(2.6 cm i.d.  28 cm) and equilibrated with 20 mM Tris pH 7.0 (5 3.5–10 mixed ampholines 4 L. The pH gradient plot was deter-
CV). The column was loaded with 7 L of fermentation supernatant, mined with surface pH electrode and six IEF tube gels (run with
pH 7.0, then washed according to the following washing protocol: buffer). Tropomyosin (isoelectric point, pI, 5.2) was included in
(1) 20 mM Tris pH 7.0, 1 M NaCl (4 CV); (2) 20 mM Tris, pH 7.0 (2 the sample as an internal standard. The tube gel from IEF was then
CV); (3) 20 mM Tris pH 7.0, 10 mM EDTA, 10 mM CHAPS (6 CV); (4) sealed onto the top of 10% acrylamide slab gel to run a SDS slab gel
20 mM Tris pH 7.0 (4 CV). The column was then eluted with a lin- electrophoresis. The acrylamide slab gel was stained with Coomas-
ear gradient decreasing from pH 4.0 to 3.0 in 100 mM sodium cit- sie blue and dried between sheets of cellophane. Protein’s pI was
rate (3 CV) followed with pH 3.0 (5 CV). The flow rate was 21 ml/ determined using the pH gradient plot provided by the vendor.
min. The eluted fractions were adjusted to pH 5.0 with 1 M sodium
phosphate, dibasic, pH 8.9. Fractions representing the IgG1 peak Endotoxin analysis
were pooled for next step purification.
SOURCE 30S resin was packed in an XK26/40 column (2.6 cm Endotoxin was determined using the end-point chromogenic
i.d.  28 cm) and equilibrated with 25 mM sodium acetate, pH Limulus Amebocyte Lysate (LAL) method provided by Charles River
4.5 (5 CV). Anti-HER2 mAb from STREAMLINE rProtein A pool Endosafe (Wilmington, MA, USA).
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- Residual Protein A analysis
umn. SOURCE 30S chromatography with a stepwise gradient was
conducted according to the above protocol. The flow rate was Residual Protein A was determined as described [19] with some
10 ml/min. modifications. Protein A standard and samples were captured in
SP Sepharose Fast Flow resin was packed in an XK16/40 column 96-well microplates coated with anti-Protein A polyclonal antibod-
(1.6 cm i.d.  35 cm) and equilibrated with buffer A (5 CV). Anti- ies. Biotinylated anti-Protein A polyclonal antibodies were added
HER2 mAb from SOURCE 30S was diluted five times with buffer to the plate to form an immune complex with captured Protein
A and loaded on an SP Sepharose column. After buffer A washing A. The complex was detected with streptavidin-AP conjugate, fol-
(5 CV), mAb was eluted from the column with 5 CV of formulation lowed by the substrate 4-MUP. The product was quantified by
buffer (10 mM histidine, pH 6.0, 150 mM NaCl, 0.01% polysorbate reading fluorescence with excitation at 360 nm, emission at
20). Fractions of the peak were pooled as final product, filtered 485 nm.
through 0.2 lm polyethersulfone membrane filter and stored asep-
tically at 4 °C. Host cell genomic DNA analysis

Protein concentration Host cell genomic DNA was determined by using PicoGreenÒ re-
agent according to instruction from Invitrogen (Carlsbad, CA, USA).
Anti-HER2 mAb concentration was determined by measuring
the absorption in a 1 cm quartz cuvette at 280 nm with a UV spec- N-Glycan analysis
trophotometer. The theoretical extinction coefficient of
1.42 L g1 cm1 calculated from amino acid sequence was used N-Glycan profile was determined by matrix-assisted laser
[16]. desorption/ionization/time-of-flight mass spectrometry (MALDI-
TOF MS) using a Voyager-DE™ BioSpectrometry™ Workstation
Physical stability analysis (Applied Biosystems, Foster City, CA, USA) as previously described
[12].
The purified anti-HER2 mAb was concentrated and buffer ex-
changed in the formulation buffer. Antibody in formulation buffer Binding assays
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- Antigen-binding assay was performed using SKBR3 cells
tography (SEC) to determine aggregation and degradation for six (American Type Culture Collection) as described [6] with minor
weeks. The analytical SEC was conducted using a Zorbax GF-250 modifications. The mAb–antigen complex was stained with anti-
(4 lm particle size) column, 9.4  250 mm, on a Hitachi D-7000 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- Results and discussion
ward, CA, USA) using excitation at 488 nm and emission at 525 nm.
C1q-binding assay was carried out as described [20] with minor Protein A affinity chromatograph for anti-HER2 mAb purification
modifications. The mAb-C1q complex was detected with anti-C1q
polyclonal antibody-AP conjugate, followed by the substrate 4- Protein A affinity chromatography is a common method for
MUP. The product was quantified by reading relative fluorescence mAb purification. STREAMLINE rProtein A is designed for antibody
units (RFU) with excitation at 360 nm, emission at 485 nm. purification directly from crude feedstock by expanded bed
FccRI-binding assays were conducted as previously described adsorption chromatography. MabSelect SuRe is genetically engi-
[6]. 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 −
decreasing from pH 5.0 to 3.0 in 100 mM sodium citrate. The pH
115 − maxima was shifted to 3.7 in STREAMLINE rProtein A chromatogra-
98 −
phy when 1 M arginine was included. Over 90% of mAb was recov-
54 − ered from the Protein A column in each of these elution conditions
37 − (Table 1). No mAb was detected in the post-elution resin (data not
29 −
shown). Antibody precipitation was prevented by eluting with a
20 − decreasing pH linear gradient in the presence or absence of 1 M argi-
7−
nine. Considering its low pressure drop in column chromatography,
we chose to use STREAMLINE rProtein A in scale-up purification pro-
Fig. 2. (A) SDS–PAGE analysis of anti-HER2 mAb from scale-up purification process.
cess and perform pH gradient elution without arginine in the elution
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 buffer.
(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
Cation exchange chromatography to remove product related
(lane 2, NR; lane 4, R), II (lane 6, NR; lane 8, R), III (lane 10, NR; lane 12, R) and impurities
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). In Protein A purified anti-HER2 mAb from P. pastoris, there are
some product related impurities which are different from impuri-
for column regeneration and sanitization. MabSelect SuRe exhibits ties observed in other expression systems. After evaluating differ-
a comparable binding capacity to wild-type Protein A resins, ent chromatographies including CEX, AEX, HIC, HCIC and
including MabSelect Xtra and ProSep-vA Ultra [21]. We compared hydroxyapatite chromatography, we found that cation exchange
binding capacity of STREAMLINE rProtein A and MabSelect SuRe chromatography can perform well to remove these impurities.
columns for anti-HER2 mAb purification from P. pastoris fermenta- Fig. 1A compares cation exchange chromatograms performed with
tion supernatant. Both columns had similar binding capacity of the same NaCl linear gradient elution at pH 5.0. The chromato-
30 mg/ml (Table 1), which were comparable to their binding grams of SOURCE 30S, Poros 50 HS and SP Sepharose HP all dem-
capacity determined with purified human IgG [21]. Therefore, we onstrated good peak resolution. However, the peak resolution
used both columns to capture anti-HER2 mAb from P. pastoris fer- disappeared in the Capto S chromatogram which might be due to
mentation supernatant and evaluate their elution conditions. its large particle size, suggesting that SOURCE 30S, Poros 50 HS
Antibody is conventionally eluted from Protein A affinity chro- and SP Sepharose HP could perform well to remove the impurities.
matography at low pH (3.0). However, antibody is not stable at To determine the optimal pH for NaCl gradient elution, we com-
low pH (2.5–3.5) and tends to aggregate [22]. An elution buffer with pared SOURCE 30S chromatograms with NaCl linear gradient elu-
higher pH was applied to reduce the aggregation, which led to tion at different pH of 4.5, 5.0 and 6.0. As shown in Fig. 1B, there
incomplete elution of anti-HER2 mAb. Arginine has been used as were good resolutions at pH 4.5 and 5.0 but some overlap in the
an alternative strategy to elevate elution pH to prevent antibody first two elution peaks at pH 6.0. To develop an easy scale-up puri-
aggregation in Protein A affinity chromatography [22]. To optimize fication process, we optimized SOURCE 30S chromatography with
elution conditions for anti-HER2 mAb, we compared Protein A chro- NaCl stepwise gradient elution. As shown in Fig. 1C, the elution
matography with and without arginine in a decreasing gradient of peaks were well separated at different NaCl concentrations in
elution pH (5.0–3.0). As summarized in Table 1, the pH at A280nm peak 25 mM sodium acetate buffer, pH 5.0. Fig. 1D depicts the SDS–
maxima appeared at pH 3.3 in STREAMLINE rProtein A chromatogra- PAGE analysis of samples in these peaks. The first elution peak at
phy and at pH 3.7 in MabSelect SuRe chromatography when 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 which may introduce protein degradation and aggregation. Fig. 2A
chains, as confirmed by N-terminal amino acid sequencing (data shows the SDS–PAGE analysis of anti-HER2 mAb products in each
not shown). The second elution peak at 125 mM NaCl contained in- step of the purification process and its commercial counterpart
tact anti-HER2 mAb with few product related impurities. The small product, Trastuzumab produced in CHO cells. The data indicate that
elution peaks at or above 150 mM NaCl were misassembled mAb, our two-step purification process can produce anti-HER2 mAb from
which were composed of a mixture of heavy chains, cleaved heavy P. pastoris fermentation and achieve the same purity as Trast-
chains and light chains. These results indicate that in Protein A uzumab. This purification process was generally utilized to purify
purified anti-HER2 mAb from glycoengineered P. pastoris, product other IgG1, IgG2 and IgG4 mAbs which target different antigens.
related impurities come from the misassembling of cleaved heavy Fig. 2B is the SDS–PAGE analysis of three other representative mAbs
chain, heavy chain and light chain. These impurities can be effi- from glycoengineered P. pastoris and shows the efficient removal of
ciently removed by cation exchange chromatography in optimal product related impurities to achieve a high purity.
NaCl gradient at pH 4.5–6.0. The results of the anti-HER2 mAb purification process are sum-
marized in Table 2. Protein A purified anti-HER2 product contained
Scale-up purification process about 20% of product related impurities, which were completely
removed from the intact antibody product by using SOURCE 30S
Based on the above optimized conditions in small column purifi- chromatography. The purification process had been shown to pro-
cation, we have developed a robust and scalable purification process duce final anti-HER2 mAb product in 99% purity and achieve 60% of
to produce grams of high quality anti-HER2 mAb from 10 to 40 L of P. total recovery from Protein A captured proteins. Anti-HER2 mAb
pastoris fermentations. After passing the fermentation supernatant from Protein A affinity chromatography contained 290 ppm of
through the STREAMLINE rProtein A column, non-specific binding residual Protein A, which co-eluted with antibody from the col-
impurities were removed from the column by washing, and the anti- umn. After SOURCE 30S and SP Sepharose chromatography, Resid-
body was eluted with a gradient decreasing from pH 4.0 to 3.0. Anti- ual Protein A was reduced almost 500-fold to 0.6 ppm in the final
body pool from Protein A chromatography was diluted with water by product (Table 2). Protein A purified anti-HER2 mAb had low levels
5-fold volume to reduce conductivity to 10 mS/cm and directly ap- of host cell DNA (<7 ppm) and endotoxin (0.6 EU/mg). In the final
plied to a SOURCE 30S column to remove product related impurities. product, host DNA was further reduced over 20-fold to <0.3 ppm,
Antibody was eluted using a stepwise NaCl gradient at pH 5.0. Final- and endotoxin was reduced 2-fold to 0.3 EU/mg (Table 2). Host cell
ly, antibody from the SOURCE 30S column was applied to a SP protein impurities in the final product were barely detected with
Sepharose Fast Flow column and eluted with formulation buffer as an in-house Western blot assay using a polyclonal antibody. The
the final product. This purification process eliminates the time-con- polyclonal antibody was raised in rabbits using Pichia host cell pro-
suming buffer exchange process between each chromatography step 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.

References
Characterization of anti-HER2 mAb
[1] J.E. Gessner, H. Heiken, A. Tamm, R.E. Schmidt, The IgG Fc receptor family, Ann.
Hematol. 76 (1998) 231–248.
The physical stability of anti-HER2 mAb from glycoengineered [2] N.C. Hughes-Jones, B. Gardner, Reaction between the isolated globular sub-
P. pastoris was assessed by using SEC to detect aggregation or deg- units of the complement component C1q and IgG-complexes, Mol. Immunol.
16 (1979) 697–701.
radation. When anti-HER2 mAb in formulation buffer was stored at [3] R. Jefferis, Glycosylation of recombinant antibody therapeutics, Biotechnol.
4, 25 and 37 °C for a period of six weeks, no significant amount of Prog. 21 (2005) 11–16.
aggregation or degradation was detected by SEC analysis. Only [4] R. Jefferis, Glycosylation of natural and recombinant antibody molecules, Adv.
Exp. Med. Biol. 564 (2005) 143–148.
0.3% of degradation was detected after storage at 37 °C for 6 weeks [5] Y. Mimura, S. Church, R. Ghirlando, P.R. Ashton, S. Dong, M. Goodall, J. Lund, R.
(Fig. 3A and B). In parallel stability studies of anti-HER mAb in an- Jefferis, The influence of glycosylation on the thermal stability and effector
other commonly used formulation buffer (10 mM sodium phos- function expression of human IgG1-Fc: properties of a series of truncated
glycoforms, Mol. Immunol. 37 (2000) 697–706.
phate, pH 6.0, 150 mM NaCl, 0.01% polysorbate 80), 0.7% of
[6] H. Li, N. Sethuraman, T.A. Stadheim, D. Zha, B. Prinz, N. Ballew, P. Bobrowicz,
degradation and 0.3% of aggregation were detected, after storage B.K. Choi, W.J. Cook, M. Cukan, N.R. Houston-Cummings, R. Davidson, B. Gong,
at 37 °C for six weeks. Anti-HER2 mAb concentration was also con- S.R. Hamilton, J.P. Hoopes, Y. Jiang, N. Kim, R. Mansfield, J.H. Nett, S. Rios, R.
stant over the same time period (data not shown). These data indi- Strawbridge, S. Wildt, T.U. Gerngross, Optimization of humanized IgGs in
glycoengineered Pichia pastoris, Nat. Biotechnol. 24 (2006) 210–215.
cate that anti-HER2 mAb from glycoengineered P. pastoris has good [7] R.L. Shields, J. Lai, R. Keck, L.Y. O’Connell, K. Hong, Y.G. Meng, S.H. Weikert, L.G.
physical stability at different temperatures. Presta, Lack of fucose on human IgG1 N-linked oligosaccharide improves
14 Y. Jiang et al. / Protein Expression and Purification 76 (2011) 7–14

binding to human Fcgamma RIII and antibody-dependent cellular toxicity, J. [14] A.A. Shukla, B. Hubbard, T. Tressel, S. Guhan, D. Low, Downstream processing
Biol. Chem. 277 (2002) 26733–26740. of monoclonal antibodies – application of platform approaches, J. Chromatogr.
[8] R. Daly, M.T. Hearn, Expression of heterologous proteins in Pichia pastoris: a B Analyt Technol. Biomed. Life Sci. 848 (2007) 28–39.
useful experimental tool in protein engineering and production, J. Mol. [15] T.I. Potgieter, M. Cukan, J.E. Drummond, N.R. Houston-Cummings, Y. Jiang, F. Li,
Recognit. 18 (2005) 119–138. H. Lynaugh, M. Mallem, T.W. McKelvey, T. Mitchell, A. Nylen, A. Rittenhour,
[9] B. Gasser, D. Mattanovich, Antibody production with yeasts and T.A. Stadheim, D. Zha, M. D’Anjou, Production of monoclonal antibodies by
filamentous fungi: on the road to large scale?, Biotechnol Lett. 29 (2007) glycoengineered Pichia pastoris, J. Biotechnol. 139 (2009) 318–325.
201–212. [16] S.C. Gill, P.H. von Hippel, Calculation of protein extinction coefficients from
[10] P. Bobrowicz, R.C. Davidson, H. Li, T.I. Potgieter, J.H. Nett, S.R. Hamilton, T.A. amino acid sequence data, Anal. Biochem. 182 (1989) 319–326.
Stadheim, R.G. Miele, B. Bobrowicz, T. Mitchell, S. Rausch, E. Renfer, S. Wildt, [17] U.K. Laemmli, Cleavage of structural proteins during the assembly of the head
Engineering of an artificial glycosylation pathway blocked in core of bacteriophage T4, Nature 227 (1970) 680–685.
oligosaccharide assembly in the yeast Pichia pastoris: production of complex [18] H. Liu, G. Gaza-Bulseco, C. Chumsae, A. Newby-Kew, Characterization of lower
humanized glycoproteins with terminal galactose, Glycobiology 14 (2004) molecular weight artifact bands of recombinant monoclonal IgG1 antibodies
757–766. on non-reducing SDS–PAGE, Biotechnol. Lett. 29 (2007) 1611–1622.
[11] S.R. Hamilton, R.C. Davidson, N. Sethuraman, J.H. Nett, Y. Jiang, S. Rios, P. [19] C. Lucas, C. Nelson, M.L. Peterson, S. Frie, D. Vetterlein, T. Gregory, A.B. Chen,
Bobrowicz, T.A. Stadheim, H. Li, B.K. Choi, D. Hopkins, H. Wischnewski, J. Roser, Enzyme-linked immunosorbent assays (ELISAs) for the determination of
T. Mitchell, R.R. Strawbridge, J. Hoopes, S. Wildt, T.U. Gerngross, Humanization contaminants resulting from the immunoaffinity purification of recombinant
of yeast to produce complex terminally sialylated glycoproteins, Science 313 proteins, J. Immunol. Methods 113 (1988) 113–122.
(2006) 1441–1443. [20] E.E. Idusogie, L.G. Presta, H. Gazzano-Santoro, K. Totpal, P.Y. Wong, M. Ultsch,
[12] S.R. Hamilton, P. Bobrowicz, B. Bobrowicz, R.C. Davidson, H. Li, T. Mitchell, J.H. Y.G. Meng, M.G. Mulkerrin, Mapping of the C1q binding site on rituxan, a
Nett, S. Rausch, T.A. Stadheim, H. Wischnewski, S. Wildt, T.U. Gerngross, chimeric antibody with a human IgG1 Fc, J. Immunol. 164 (2000) 4178–4184.
Production of complex human glycoproteins in yeast, Science 301 (2003) [21] R. Hahn, P. Bauerhansl, K. Shimahara, C. Wizniewski, A. Tscheliessnig, A.
1244–1246. Jungbauer, Comparison of protein A affinity sorbents II. Mass transfer
[13] B.K. Choi, P. Bobrowicz, R.C. Davidson, S.R. Hamilton, D.H. Kung, H. Li, R.G. properties, J. Chromatogr. A 1093 (2005) 98–110.
Miele, J.H. Nett, S. Wildt, T.U. Gerngross, Use of combinatorial genetic libraries [22] K. Tsumoto, M. Umetsu, I. Kumagai, D. Ejima, J.S. Philo, T. Arakawa, Role of
to humanize N-linked glycosylation in the yeast Pichia pastoris, Proc. Natl. arginine in protein refolding, solubilization, and purification, Biotechnol. Prog.
Acad. Sci. USA 100 (2003) 5022–5027. 20 (2004) 1301–1308.