Integration of Expanded Bed Adsorption and Hydrophobic Charge

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Integration of expanded bed adsorption and hydrophobic chargeinduction chromatography for monoclonal antibody separation Wei Shi, Dong Gao, Shan-Jing Yao, and Dong-Qiang Lin Ind. Eng. Chem. Res., Just Accepted Manuscript • DOI: 10.1021/acs.iecr.6b04108 • Publication Date (Web): 29 Dec 2016 Downloaded from http://pubs.acs.org on January 3, 2017

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Industrial & Engineering Chemistry Research

1

Integration of expanded bed adsorption and

2

hydrophobic charge-induction chromatography

3

for monoclonal antibody separation

4

Wei Shi a,b, Dong Gaoc, Shan-Jing Yaoa, Dong-Qiang Lin*a

5

a

6

Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China

7

b

8

318001, China

9

c

Key Laboratory of Biomass Chemical Engineering of Ministry of Education, College of

College of Pharmaceutical and Chemical Engineering, Taizhou University, Taizhou

Key Laboratory of Synthetic and Natural Functional Molecular Chemistry of Ministry

10

of Education, Institute of Modern Separation Science, Northwest University, Shaanxi

11

Key Laboratory of Modern Separation Science, Xi’an 710068, China

12

13

ABSTRACT: The integration of expanded bed adsorption (EBA) with hydrophobic

14

charge-induction chromatography (HCIC) affords a promising new technology to capture

15

antibody from the complex feedstock. New EBA resin T-ABI was prepared with

16

hydrophobic charge-induction ligand 5-aminobenzimidazole (ABI) and used to separate

17

monoclonal antibody (mAb) from CHO cell culture broth. The static and dynamic 1 ACS Paragon Plus Environment


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18

adsorption behaviors of hIgG and mAb were investigated, and the typical properties of

19

pH dependence and salt tolerance were found. High dynamic binding capacities at 10%

20

breakthrough (18.1~21.4 mg/ml resin) were obtained even for high operation velocities

21

(711~1203 cm/h) in expanded bed. By optimizing loading pH, elution pH and expansion

22

factor, hIgG could be separated from the protein mixture (2 mg/ml hIgG and 10 mg/ml

23

bovine serum albumin) with high efficiency. Finally, mAb was separated directly from

24

CHO cell culture broth with T-ABI EBA under optimized conditions (loading at pH 7.0,

25

elution at pH 4.0 or 4.5, expansion factor of 2.0), and the purity reached 93.7~97.7% with

26

the recovery of 72.6~79.4%. The results indicated that T-ABI resin was suitable to

27

capture hIgG from the complicated feedstock. New separation process with T-ABI EBA

28

showed a promising potential for antibody purification with high productivity and

29

process efficiency, which combined the advantages of HCIC and EBA.

30 31

1. INTRODUCTION

32

Expanded bed adsorption (EBA) is an integrated chromatographic technology,

33

which is suitable to capture proteins directly from crude feedstocks, such as cell culture

34

broth or homogenates 1. This technology combines solid-liquid separation, concentration

35

and primary purification into one unit, which can certainly reduce the processing times

36

and improve the process efficiency

37

relatively high operation velocity and low back-pressure. During the past two decades,

38

many kinds of EBA processes have been developed for primary protein separation, such

39

as ion-exchange

6, 7

2-5

. In addition, EBA has high productivity due to

, hydrophobic interaction

8-10

, affinity

11-14

etc. In recent years,

2 ACS Paragon Plus Environment

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mixed-mode chromatography (MMC) was introduced as a novel technology for protein

41

separation, which combines multiple binding modes, like ionic exchange, hydrophobic

42

interaction, hydrogen bonding, etc. Good selectivity, high capacity, salt-tolerance and

43

relatively low cost make MMC suitable for primary capture process

44

combining the advantages of EBA and MMC to develop new protein capture technology

45

- mixed-mode EBA, which has the potentials to improve the process efficiency and avoid

46

the frustrating pretreatments on the feedstock, such as clarification (solid-liquid

47

separation), dilution (reducing the conductivity for ion-exchange) and salt-adjustment

48

(salt addition to enhance the hydrophobic interactions).

15-17

. Therefore,

49

For mixed-mode EBA, the critical point is the specially-designed EBA matrices with

50

MMC ligands. Based on this concept, some commercial resins have been developed, such

51

as Streamline Direct HST, Fastline PRO, Fastline HSA and Fastline MabDirect MM.

52

Charoenrat et al.18 reported that Streamline Direct HST could separate β-glucosidase

53

from Pichia pastoris high-cell-density culture broth with higher recovery (74%)

54

compared with ion-exchange EBA resin Streamline SP (about 48%). In addition,

55

Streamline Direct HST could be operated at higher feedstock concentration and flow

56

velocity, and tolerate high conductivity and hash elution condition. Yong et al.

57

used Streamline Direct HST to isolate lipase from Burkholderia pseudomallei, and high

58

efficiency was found even in 4.5% biomass concentration. Lu et al. 20 used Fastline PRO

59

to capture nattokinase from Bacillus subtilis broth and found that the existence of cells in

60

the feedstock did not affect the stability of bed. In addition, the salt-tolerant property was

61

confirmed, and high adsorption capacity could be maintained in 400 mM salt

62

concentration. Kelly et al.

21

19

also

used Fastline HSA and Fastline MabDirect MM to capture 3 ACS Paragon Plus Environment


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recombinant protein from high-cell-density yeast broth, and high binding capacities (54.7

64

mg/ml and 72 mg/ml settled adsorbent) were obtained with the recovery of 96%. As

65

shown above，mixed-mode EBA shows outstanding performance for protein capture, and

66

more resins with special designed-ligands should be developed for the individual target

67

proteins.

68

Hydrophobic charge-induction chromatography (HCIC), as one special MMC, was 22-24

69

introduced for antibody purification

70

interactions at neutral pH and elute effectively by electrostatic repulsion at acidic pH 16.

71

The increasing clinical demand of monoclonal antibodies (mAbs) has promoted the

72

revolution of antibody production techniques, and HCIC was developed as a

73

cost-effective alternative to Protein A-based affinity chromatography for the capture of

74

antibodies.

75

4-mercaptoethyl-pyridine (MEP) ligand to separation mAb from CHO cell culture broth.

76

Brenac et al.

77

the purity was as high as 99%. Chen et al.

78

kinds of mAb from CHO cell culture supernatants. These results indicated that HCIC is a

79

potential technology for antibody separation with high efficiency and productivity.

80

However, due to weak hydrophobic interactions between target proteins and ligands, the

81

dynamic binding capacities are relatively low, especially under high operation velocity.

82

Therefore, HCIC processes are mainly operated as packed bed rather than expanded bed.

83

New HCIC ligands need to be developed by enhancing the affinity for antibodies and

84

then combined with EBA, which would certainly improve the process efficiency.

For

26

instance,

Ghose

, which can bind antibody through hydrophobic

et

al.

25

used

MEP

HyperCel

with

used MBI HyperCel to capture mAb from recombinant cell culture, and 27

also used MEP-based resin to purify two


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Based our previous work

85

28

, new HCIC ligand, 5-aminobenzimidazole (ABI), was

86

coupled onto tungsten carbide-densified agarose matrix for EBA. Compared with MEP

87

ligand with a pyridine ring, ABI ligand has two cyclic structures including one benzene

88

ring and one imidazole ring, which improves the hydrophobic binding with target protein

89

29

90

and was suitable for high-velocity EBA. The process efficiencies had also been verified

91

by the separation of bovine IgG from the protein mixture and crude bovine whey 28. In

92

this work, ABI-based EBA resin was further challenged for hIgG separation and the

93

purification of one IgG1-type mAb would also be tested. The static and dynamic

94

adsorption behaviors of hIgG and mAb would be investigated. The effects of pH and salt

95

on binding capacity and adsorption kinetics would be focused. Then the separation

96

conditions, including loading pH, elution pH and expansion factor, would be optimized

97

and used for mAb purification from CHO cell culture broth. The potential of HCIC-based

98

EBA process for mAb purification would be discussed.

. The results demonstrated that ABI-based resin had higher dynamic binding capacity

99

100

2. EXPERIMENTAL SECTION

101

2.1. Materials. 3% crosslinked agarose beads containing tungsten carbide were

102

prepared with similar methods as published in our previous work 24. The average tungsten

103

carbide in volume ratio was about 20%. Agarose and ABI were purchased from J&K

104

technology Co., Ltd (China). Human immunoglobulin for intravenous injection (hIgG,

105

purity >98%, 150 kDa, pI 6.0~8.0) was purchased from Sichuan Yuanda Shuyang

106

Pharmaceutical Co., Ltd. (China). Bovine serum albumin (BSA, 67 kDa, pI 4.7) was



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obtained from Sigma (Milwaukee, WI, USA). The CHO cell culture broth was

108

generously provided by a local biopharmaceutical company (China), containing one kind

109

of IgG1-type mAb (150 kDa, pI 6.8~7.0), named MAB in the present work. MAB

110

concentration in the broth was 0.65 mg/ml. Protein Marker for SDS-PAGE was

111

purchased from Takara Biomedical Technology Co., Ltd (Beijing). Other reagents were

112

of analytical grade and were provided by local suppliers.

113

T-ABI resin was prepared as published previously

28

. The main process was as

114

follows. Crosslinked agarose beads containing tungsten carbide were activated with allyl

115

bromide, then the allyl-activated matrices were brominated by N-bromosuccinimide.

116

Finally, HCIC ligand, ABI, was coupled onto the brominated matrices.

117

2.2. Adsorption equilibrium and adsorption kinetics experiments. The static

118

adsorption behaviors of hIgG or MAB on T-ABI resin were evaluated by the batch

119

experiments. 20 mM phosphate buffer (pH 6.0, 7.0 and 8.0) and 20 mM glycine buffer

120

(pH 9.0 and 10.0) were used as liquid phases for different pH conditions. Sodium

121

chloride was added into the buffer with different concentrations (0.2, 0.4 and 0.8 M) to

122

investigate the influence of salt concentration. The adsorption data were fitted with the

123

Langmuir equation, and the saturated adsorption capacity (Qm) and the dissociation

124

constant (Kd) were obtained. The adsorbed protein density (Qc) was calculated with the

125

correlated Langmuir equation at the equilibrium protein concentration of 5 mg/ml as

126

suggested by Sun and coworkers 30-33.

127

The adsorption kinetics curves of hIgG or MAB on T-ABI at pH 7.0 were measured

128

as follows. T-ABI resins were pre-equilibrated and 2 g drained resins (corresponding to


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129

0.7 ml) were mixed with 50 ml of 2 mg/ml hIgG or MAB in the 20 mM phosphate buffer

130

(pH 7.0). The mixture was stirred at 200 rpm and 25 oC. At various time intervals, 40 µl

131

of solution was removed to determine the protein concentration. The apparent effective

132

diffusivity De was obtained through fitting experiments data with the pore diffusion

133

model (PDM) as described in the previous work 34.

134

2.3. Residence time distribution experiments. The residence time distribution

135

(RTD) experiments were performed in 1 cm-diameter home-made column with 13.5 ml

136

T-ABI resins (about 19 cm). 20 mM sodium phosphate buffer (pH 7.0) was used as the

137

mobile phase. Seven flow velocities (372, 550, 711, 889, 1050, 1203 and 1355 cm/h,

138

corresponding to expansion factors 1.4 to 2.6) were tested. The settled bed was expanded

139

firstly and a stable bed height was maintained for 20–30 min. Then 0.5 ml 10% acetone

140

solution

141

Chuangxintongheng Science & Technology Co., Ltd., Beijing, China) monitored the

142

response signal at 254 nm and the RTD curve was recorded. The RTD test for each flow

143

velocity was repeated 3 to 4 times. The number of theoretical plates (N), the height

144

equivalent of theoretical plate (HETP) and the axial dispersion coefficients (Dax) were

145

calculated based on the RTD curves as published previously 28.

was

injected

as

the

tracer.

The

UV

monitor

LC3000

(Beijing

146

2.4. Frontal adsorption experiments. The dynamic binding capacity of hIgG was

147

measured through frontal adsorption experiments in packed and expanded beds. 20 mM

148

sodium phosphate buffer (pH 7.0) was used as the equilibrium buffer. For packed bed

149

mode, 5-mm I.D column (Tricorn 5/100, GE Healthcare, Uppsala, Sweden) was packed

150

with 2.0 ml T-ABI resins (about 10 cm settled bed height). hIgG solution (2 mg/ml) was

151

loaded into the column at the superficial linear velocity of 200 cm/h. For expanded bed 7 ACS Paragon Plus Environment


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mode, 1 cm-diameter home-made column was used, and 13.5 ml T-ABI resins were

153

packed with a settled-bed height of 19 cm. hIgG solution (2 mg/ml) was loaded at 711,

154

889, 1050 and 1203 cm/h (corresponding to expansion factors 1.8, 2.0, 2.2 and 2.4). The

155

protein concentration in the effluent was monitored by absorbance at 280 nm. The

156

dynamic binding capacity at 10% breakthrough (Q10%) was calculated.

157

2.5. Separation of hIgG from the protein mixture with packed bed and

158

expanded bed. The separation of hIgG from BSA containing feedstock was evaluated

159

firstly with the Tricorn 5/100 column and the ÄKTA explorer 100 system (GE Healthcare,

160

Uppsala, Sweden). Loading and elution pH were screened. 2.0 ml T-ABI resins were

161

packed with a bed height of about 10 cm. 20 mM phosphate buffer (pH 7.0 and 8.0) and

162

20 mM glycine buffer (pH 9.0) were used as the loading buffer, and 20 mM acetate

163

buffer (pH 3.5, 4.0, 4.5, 5.0 and 5.5) were used as the elution buffer. The protein mixture

164

containing 2 mg/ml hIgG and 10 mg/ml BSA was used as the loading feedstock, and the

165

flow rate was 0.5 ml/min (about 153 cm/h, 4 min retention time). The chromatographic

166

run was monitored on-line at 280 nm. The fractions were collected and analyzed with the

167

SEC-HPLC and SDS-PAGE. Then 13.5 ml T-ABI resins were packed into 1 cm-diameter

168

expanded bed as mentioned above (about 19 cm settled bed height) to investigate the

169

influences of the expansion factor (1.8, 2.0, 2.2 and 2.4) for the separation of hIgG and

170

BSA.

171

2.6. Separation of MAB from cell culture broth with expanded bed. Same

172

expanded bed and 13.5 ml T-ABI resins as mentioned above were used for MAB

173

separation from CHO cell culture broth. The pH and conductivity of broth were 7.5 and

174

7.12 mS/cm, respectively. The concentration and purity of MAB in the broth were 8 ACS Paragon Plus Environment

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175

analyzed with SEC-HPLC. The dynamic binding capacity of MAB was also evaluated

176

through frontal adsorption experiments as mentioned above at the expansion factor of 2.0.

177

For MAB separation from cell culture broth, the bed was expanded to 2.0 expansion

178

factor with 20 mM sodium phosphate buffer (pH 7.0). 130 ml broth (about 10 column

179

volume) was loaded. After sample loading, the column was washed with the equilibrium

180

buffer. Then MAB was eluted with 20 mM acetate buffer (pH 4.0 or 4.5). The column

181

was regenerated with 0.1 M NaOH and re-equilibrated with the equilibrium buffer. The

182

fractions were collected during the process and analyzed with the SEC-HPLC and

183

SDS-PAGE to determine the recovery and purity of MAB.

184

2.7. SEC-HPLC analysis. The analytical SEC-HPLC was performed on LC3000

185

system (Beijing Chuangxintongheng Science & Technology Co., Ltd., Beijing, China)

186

with TSK G3000SWXL column (7.8 mm × 30.0 cm, TOSOH, Japan). The mobile phase

187

was 0.1 M sodium phosphate buffer containing 0.1 M Na2SO4 (pH 6.7). The buffer was

188

filtrated with 0.22 µm membrane and degassed before use. The flow rate was 0.5 ml/min.

189

The purities of hIgG or MAB were defined as the percentage of the peak area of

190

monomers to the total integrated peak areas. The recoveries of hIgG or MAB were

191

calculated as the percentage of monomer in the elution fraction to that in the loading

192

sample during the separation process.

193

194

The recovery (R) and purity (P) of hIgG were calculated as,

R（%）=

AEVE × 100% ALVL

（1）



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P（%）=

195

AE × 100% Aall

Page 10 of 34

（2）

196

where AE is the HPLC peak area of hIgG or MAB in the elution faction; VE is the volume

197

of elution faction; AL is the HPLC peak area of hIgG or MAB in loading sample; VL is the

198

volume of loading sample; Aall is the HPLC peak area of all components in the elution

199

faction.

200

2.8. SDS-PAGE analysis. The samples and fractions collected during the separation

201

process were analyzed by non-reducing SDS-PAGE. The concentration of resolving gel

202

and condensing gel were 12% and 8%, respectively. Protein migration was performed

203

under 180V for 50 min. The gel was stained with Coomassie Blue R-250 and destained.

204

The Gel Doc 2000 imaging system (Bio-Rad, Hercules, CA, USA) was used to image the

205

protein gel.

206

207

3. RESULTS AND DISCUSSION

208

3.1. Adsorption equilibrium of hIgG and MAB on T-ABI resin. The properties of

209

T-ABI resin are listed in Table 1. The adsorption isotherms of hIgG and MAB with

210

T-ABI resin at different pH values and NaCl concentrations were determined. The

211

correlated Qms of hIgG and MAB with the Langmuir equation were compared in Figure

212

1.

213


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214


Table 1. Main properties of T-ABI resin prepared. Crosslinked agarose (%)

Tungsten carbide (%)

Partical size (µm)

Mean partical size （µm）

Mean density (g/ml)

Porosity (%)

Ligand density (µmol/ml)

3

~20

50~230

104

2.8

74

70

215

216



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Page 12 of 34

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218

Figure 1. The Qms (a) of hIgG and MAB with T-ABI at different pH values and the Qcs

219

(b) at the equilibrium liquid-phase concentration of 5 mg/ml (pH 7.0) as different NaCl

220

concentrations.

221

As shown in Figure 1 (a), both hIgG and MAB showed similar pH-dependent

222

adsorption behaviors on T-ABI resins. High adsorption capacities were found at the range

223

of pH 7.0-9.0, and the acidic and strong basic condition significantly reduced the

224

adsorption of hIgG and MAB. For hIgG, the highest Qm (76.83 mg/ml resin) was at pH

225

9.0, and for MAB it was 64.09 mg/ml resin at pH 7.0. This result indicated that the

226

neutral and weak basic condition is suitable for T-ABI resin to adsorb IgG. As we know,

227

the pH values of serum and mammalian cell culture are both about 7.0-8.0, so the

228

feedstock do not need to be adjusted pH and can be used directly for the chromatographic

229

separation.

230

The reason to explain the phenomenon above is as follows. The hydrophobic and 12 ACS Paragon Plus Environment

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231

electrostatic interactions are main forces between HCIC resin and protein, which are

232

observably affected by pH. Therefore, pH plays an essential role during the adsorption

233

and desorption process of protein on HCIC resin

234

protein (pH 8.0 for hIgG and pH 8.6 for MAB) and above the pKa of ABI ligand (6.5),

235

both protein and ligand have no or few charges and the protein would bind to the

236

ABI-based resins through the hydrophobic interactions. When pH is below pI of protein

237

and pKa of ABI ligand, both protein and ligand would be predominantly positively

238

charged and result in the desorption of protein from the resin through the electrostatic

239

repulsion.

22, 35, 36

. When pH is around the pI of

240

The effects of salt addition on the adsorption of hIgG and MAB on T-ABI resin

241

were also determined. It was found that some adsorption isotherms did not reach the

242

plateaus, especially for high salt concentration, so the fitted Qm values would be not

243

suitable to describe adsorption ability. As suggested by Sun and coworkers

244

adsorbed protein density (Qc) at an equilibrium liquid-phase concentration calculated by

245

Langmuir equation was introduced. The Qc values at the equilibrium liquid-phase

246

concentration of 5 mg/ml were calculated and are compared in Figure 1 (b). It could be

247

found that Qcs of hIgG and MAB had similar “U” shape trends with the increase of salt

248

concentration. Qc declined slightly when NaCl concentration increased from 0 to 0.2 M.

249

Then the adsorption capacity increased gradually with the increase of NaCl concentration

250

from 0.2 to 0.8 M. So the lowest Qc s was found at around 0.2 M NaCl. For hIgG, Qc

251

values at high salt concentrations (>0.4 M NaCl) were even higher than that without salt

252

addition.

253

30-33

the

The influences of salt addition could be explained as follows. When NaCl 13 ACS Paragon Plus Environment


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254

concentration was low (

Integration of Expanded Bed Adsorption and Hydrophobic Charge

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