Purification process development of a recombinant monoclonal antibody expressed in glycoengineered Pichia pastoris

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 b

GlycoFi Inc., A Wholly-Owned Subsidiary of Merck & Co. Inc., 21 Lafayette Street, Suite 200, Lebanon, NH 03766, USA Biologics Research, Merck Research Laboratories, West Point, PA 19486, USA

a r t i c l e

i n f o

Article history: Received 18 March 2010 and in revised form 4 November 2010 Available online 11 November 2010 Keywords: Protein A chromatography Ion exchange chromatography Monoclonal antibody Purification Pichia pastoris Glycosylation

a b s t r a c t A robust and scalable purification process was developed to quickly generate antibody of high purity and sufficient quantity from glycoengineered Pichia pastoris fermentation. Protein A affinity chromatography was used to capture the antibody from fermentation supernatant. A pH gradient elution was applied to the Protein A column to prevent antibody precipitation at low pH. Antibody from Protein A chromatography contained some product related impurities, which were the misassembling of cleaved heavy chain, heavy chain and light chain. It also had some process related impurities, including Protein A residues, endotoxin, host cell DNA and proteins. Cation exchange chromatography with optimal NaCl gradient at pH 4.5–6.0 efficiently removed these product and process related impurities. The antibody from glycoengineered P. pastoris was comparable to its commercial counterpart in heterotetramer folding, physical stability and binding affinity. Ó 2010 Elsevier Inc. All rights reserved.

Introduction Monoclonal antibodies (mAbs) are rapidly becoming key products for the biopharmaceutical industry. Most commercial therapeutic 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 antigen 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 complement-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 complement 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 heterotetramer. 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: [email protected] (H. Li). 1046-5928/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.pep.2010.11.004

referred to as G0 in our work and literature. Additional sugar residues may be attached to the ‘‘core’’ to generate further complex biantennary glycan structures such as GalGlcNac2Man3GlcNac2 (G1), Gal2GlcNac2Man3GlcNac2 (G2), Neu5Ac2Gal2GlcNac2Man3GlcNac2 [3,4]. Currently, therapeutic mAbs are produced in mammalian cells, mostly in Chinese hamster ovary (CHO) cell lines. The glycan profiles 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 human-murine humanized anti-CD20 mAb, produced in glycoengineered Pichia pastoris has both higher binding affinity to FccRIIIa and greater B-cell depletion potency in comparison with the counterpart produced from CHO cells [6]. N-Glycans from therapeutic mAbs produced in mammalian cells are predominantly fucosylated, in that a fucose residue is attached to the primary N-acetylglucosamine 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 glycoform of antibody.

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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 expression in yeast is the assembly of heavy and light chains to form a heterotetramer. Generally, in yeast expression, the secreted antibody 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 secretion 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 column at low pH usually contains process and product related impurities. The process related impurities include endotoxin, leached Protein A, host cell protein and DNA. The product related impurities mainly are aggregated and clipped mAb products. To remove these impurities, second and third chromatographic steps are generally employed, typically selected from cation exchange chromatography (CEX), anion exchange chromatography (AEX), hydrophobic interaction chromatography (HIC), hydrophobic charge induction chromatography (HCIC) and hydroxyapatite chromatography [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 fermentation [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 optimizing process conditions for Protein A and cation exchange chromatographies. 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 support biochemical characterization and animal studies. Anti-HER2 mAb from glycoengineered P. pastoris is comparable to its commercial counterpart (Trastuzumab) produced by CHO cells in heterotetramer 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

obtained from GE Healthcare (Piscataway, NJ, USA). POROS 50 HS (50 lm particle size) was from Perseptive Biosystems (Framingham, MA, USA). Chemicals were from JT Baker (Phillipsburg, NJ, USA) or Sigma–Aldrich (St. Louis, MO, USA). All buffers were filtered 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-human IgG-AlexaFluor488, PicoGreenÒ reagent and 4-Methylumbelliferyl 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 Prestained SDS–PAGE standards (broad range) were from Bio-Rad Laboratories (Hercules, CA, USA). FccRI was from R&D systems (Minneapolis, MN, USA). Human C1q was from United States Biological (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 expression titer and N-glycosylation profile. Fermentations in 15 and 40 L bioreactors were conducted according to methods described earlier [6]. Fermentation supernatant 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 indicated. 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 concentration 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

Materials

VE, volume of eluate (ml); CE, protein concentration in eluate fraction (mg/ml); VR, volume of Protein A resin in the column (ml).

STREAMLINE rProtein A (80–165 lm particle size), MabSelect SuRe (85 lm particle size), SOURCE 30S (30 lm particle size), Capto 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

Cation exchange chromatography STREAMLINE rProtein A purified anti-HER2 mAb was used for cation exchange chromatography development. The cation exchange resins in Tricorn 10/200 (1 cm i.d.  19 cm) were equilibrated 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 linear gradient from 0 to 300 mM NaCl (10 CV) in 25 mM sodium acetate, 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 gradient 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 sodium 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 linear gradient decreasing from pH 4.0 to 3.0 in 100 mM sodium citrate (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), adjusted to pH 4.5 with 1 M acetic acid, and then loaded on the column. 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). AntiHER2 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 aseptically 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 spectrophotometer. 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 exchanged 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 chromatography (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 twodimensional 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, isoelectric focusing (IEF) was carried out in a glass tube using 2% pH 3.5–10 mixed ampholines 4 L. The pH gradient plot was determined 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 Coomassie 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 antibodies. 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, followed 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 reagent 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 (MALDITOF 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 antihuman IgG-AlexaFluor488. Mean fluorescence intensity (MFI)

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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 STREAMLINE rProtein A MabSelect SuRe

32 32 28

100 mM sodium citrate pH gradient 5–3 1 M arginine 100 mM sodium citrate pH gradient 5–3 100 mM sodium citrate pH gradient 5–3

3.3 3.7 3.7

100 97 90

was detected in a Guava Express Plus (Guava Technologies, Hayward, 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 4MUP. 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 engineered 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 pH 5.0

500

500

SOURCE 30S

0

pH 4.5

0

0

50

100

150

200

250

300

0

ml

50

D

C

1

100 2

3

150 4

5

200 6

7

250 8

300

ml

9

KDa 2

mAU

205 −

− mAb

115 − 98 −

600 400

1

54 −

200

37 − 29 −

3

0 0

50

100

150

200

250

300

350 ml

− Hc

− Lc

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.

11

A

1

2

3

4

5

6

7

8 9

10

Aggregation (%) Degradation (%)

Y. Jiang et al. / Protein Expression and Purification 76 (2011) 7–14

11

KDa 205 − 115 − 98 − 54 − 37 − 29 −

1.0

A

0.5 0.0 1.0

B

0.5 0.0 0

1

2

20 − 7−

B

3 4 Time (Weeks)

5

6

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.

1

2

3

4

5

6

7

8

9 10 11 12 13

KDa 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 fermentation 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 fermentation supernatant and evaluate their elution conditions. Antibody is conventionally eluted from Protein A affinity chromatography 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 chromatography 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 chromatography and at pH 3.7 in MabSelect SuRe chromatography when

anti-HER2 mAb was eluted from both columns by a linear gradient 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 chromatography when 1 M arginine was included. Over 90% of mAb was recovered 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 arginine. Considering its low pressure drop in column chromatography, we chose to use STREAMLINE rProtein A in scale-up purification process 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 impurities observed in other expression systems. After evaluating different 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 chromatograms of SOURCE 30S, Poros 50 HS and SP Sepharose HP all demonstrated 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 compared SOURCE 30S chromatograms with NaCl linear gradient elution 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 purification 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 SOURCE 30S SP Sepharose Fast Flow

100 60 60

81 99 99

0.6 1.7 0.3

290 3 0.6

<7 <6 <0.3

12

Y. Jiang et al. / Protein Expression and Purification 76 (2011) 7–14

A 100

1648.22

1486.18

90

- fucosylated G0

80

Intensity (%)

70

- fucosylated G1

60 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

Intensity (%)

70 60

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 intact 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 efficiently 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 purification, 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 antibody was eluted with a gradient decreasing from pH 4.0 to 3.0. Antibody pool from Protein A chromatography was diluted with water by 5-fold volume to reduce conductivity to 10 mS/cm and directly applied to a SOURCE 30S column to remove product related impurities. Antibody was eluted using a stepwise NaCl gradient at pH 5.0. Finally, 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-consuming 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 Trastuzumab. 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 summarized 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 produce 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 column. After SOURCE 30S and SP Sepharose chromatography, Residual 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 proteins as antigens (data not shown).

Y. Jiang et al. / Protein Expression and Purification 76 (2011) 7–14

A

400

MFI

300

200

100

0 -2

-1 0 Log[mAb] (µg/ml)

1

B 40000 35000

RFU

30000 25000 20000

13

N-Glycan profiles of Trastuzumab from CHO cells and anti-HER2 mAb from glycoengineered P. pastoris were compared by MALDITOF MS (Fig. 4A and B). Trastuzumab N-glycans consisted of predominantly fucosylated G0 and G1 structures. N-Glycans of antiHER2 mAb from glycoengineered P. pastoris are inherently afucosylated and consisted mainly of G0 and some G1 and G2. O-Linked glycans for anti-HER2 mAb from glycoengineered P. pastoris was detected at low level (data not shown). Trastuzumab from CHO cells and anti-HER2 mAb from glycoengineered P. pastoris were also analyzed by two-dimensional electrophoresis. Both products migrated at the same position and had experimental pI values of 7.7. N-terminal amino acid sequencing by Edman degradation confirmed that the N-termini of both heavy and light chains were intact. Peptide mapping confirmed the same amino acid sequence in both products (data not shown). Anti-HER2 mAb product from glycoengineered P. pastoris was characterized for its binding to antigen, FccRI and C1q in comparison with Trastuzumab. Both products demonstrated the same affinity to cell surface expressed target antigen (Fig. 5A). They also had similar binding affinity for FccRI and C1q (Fig. 5B and C), which is consistent with a previous report that afucosylation has no effect on mAb binding to FccRI or C1q [7].

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A robust and scalable purification process has been developed to efficiently produce large quantities of anti-HER2 mAb from P. pastoris fermentation. In addition, process related impurities were removed from the product. Anti-HER2 mAb from glycoengineered P. pastoris was comparable to its commercial counterpart (Trastuzumab) from CHO cells with correct heterotetramer folding and good physical stability, similar binding affinity to its antigen, FccRI and C1q. Glycoengineered cell lines of P. pastoris are expected to play a significant role in the manufacture of therapeutic antibodies, substantially reducing production time while improving glycan uniformity and decreasing production costs.

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Disclosure statement

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The authors declare no conflict of interests.

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Fig. 5. Binding activity of Trastuzumab from CHO cells (d), anti-HER2 mAb from glycoengineered P. pastoris (.) and off target control IgG1 (j). (A) Cell surface target antigen binding, (B) FccRI binding, (C) C1q binding. Fitted curves were calculated using sigmoidal dose–response (variable slope) from SigmaPlot (Systat Software, Inc., San Jose, CA, USA). (Goodness of fit, R2 P0.99).

Acknowledgments We thank BBR Automated Process Monitoring Facility in MERCK & Co. Inc. for analysis of residual Protein A and host cell genomic DNA. We are also grateful to Karen M. Page for editing of the manuscript. References

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 degradation. 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 another commonly used formulation buffer (10 mM sodium phosphate, pH 6.0, 150 mM NaCl, 0.01% polysorbate 80), 0.7% of degradation and 0.3% of aggregation were detected, after storage at 37 °C for six weeks. Anti-HER2 mAb concentration was also constant over the same time period (data not shown). These data indicate that anti-HER2 mAb from glycoengineered P. pastoris has good physical stability at different temperatures.

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