嵌段共聚温敏亲和色谱固定相的制备及其对抗体的分离纯化

doi:10.3724/SP.J.1123.2023.09028

摘要/Abstract

摘要：

抗体药物在癌症治疗和免疫诊断中起着重要作用,但抗体的分离纯化通常采用酸性洗脱,易导致抗体聚集失活等问题。本研究以硅胶为基质,以温敏嵌段聚合物聚[(N-异丙基丙烯酰胺)-b-4-乙烯基吡啶](P[NIPAM-b-4VP])为间隔臂,以4-巯基乙基吡啶(MEP)为配基,制备了一种嵌段共聚温敏亲和色谱固定相SiO₂-P[NIPAM-b-4VP]-MEP,并以牛血清白蛋白(BSA)和γ-球蛋白为模型蛋白,对制备的温敏亲和色谱固定相的色谱性能进行了表征。分别考察了流动相盐浓度和温度对二者分离性能的影响。结果表明,在40 ℃时该固定相只选择性保留γ-球蛋白,而不保留BSA;在5 ℃时采用含0.6 mol/L NaCl的Tris-HCl(pH 8.0)缓冲溶液进行洗脱,γ-球蛋白的质量回收率为92.7%。该固定相对γ-球蛋白的吸附量为(71.5±2.1) mg/g(n=3),是传统温敏亲和色谱固定相SiO₂-PNIPAM-MEP的2倍。将该固定相用于人血清中IgG的分离纯化,仅通过改变流动相温度和盐浓度即可一步实现对抗体的分离纯化,纯度大于97.4%±0.7%。上述结果表明该温敏亲和色谱固定相不仅对抗体具有特异的选择性,而且洗脱条件温和,绿色环保,从根本上解决了传统亲和色谱采用酸性洗脱易使抗体蛋白变性失活的难题。该技术在抗体药物的分离纯化和工业生产中具有重要的应用价值。

关键词: 温敏色谱, 亲和色谱, 抗体分离, 聚(N-异丙基丙烯酰胺), 4-乙烯基吡啶

Abstract:

Antibodies play an essential role in cancer diagnosis and treatment because of the specificity for target biomolecules and reduction of side effects. However, antibodies separation and purification still face some challenges. Antibody elution from columns using a low-pH aqueous solution leads to aggregation or loss of activity of the antibody drugs. In this paper, a block copolymer-based temperature-responsive affinity chromatography (TRAC) stationary phase, SiO₂-P[NIPAM-b-4VP]-MEP using the block temperature-responsive copolymer poly(N-isopropylacrylamide-b-4-vinylpyridine) (P[NIPAM-b-4VP]) as the space arms and 4-mercaptoethyl pyridine (MEP) as the ligand was prepared for antibody separation. The TRAC column was tested using bovine serum albumin (BSA) and γ-globulin as model proteins, and the effects of salt concentration in the mobile phase and temperature on their separation were studied in detail. At 40 ℃, the TRAC stationary phase only selectively retained γ-globulin due to the specific affinity interaction between antibodies and the ligand MEP. At 5 ℃, γ-globulin can be eluted from the column with a mass recovery of 92.7% using a Tris-HCl buffer (pH 8.0) solution containing 0.6 mol/L NaCl. The adsorption capacity of γ-globulin on this stationary phase was (71.5 ±2.1) mg/g (n=3), which was twice that of a traditional temperature-sensitive affinity chromatography stationary phase SiO₂-PNIPAM-MEP. The stationary phase was also used to separate and purify immunoglobulin (IgG) in human serum in one step by altering the temperature and ion strength of the mobile phase, resulting in a purity of 97.4%±0.7%. Thus, this new technology has specific selectivity for antibodies, as well as mild and green elution conditions, ultimately resolving the problem of traditional affinity chromatography using acid elution, which can lead to the antibodies aggregation/inactivation. This technology has great application potential for the industrial production of antibody drugs.

Key words: temperature-responsive chromatography, affinity chromatography, antibody separation, poly(N-isopropylacrylamide), 4-vinylpyridine

中图分类号:

O658

郭冬梅, 夏一然, Mujeeb ur RAHMAN, 王建忠, 刘家玮, 白泉. 嵌段共聚温敏亲和色谱固定相的制备及其对抗体的分离纯化[J]. 色谱, 2023, 41(12): 1045-1051.

GUO Dongmei, XIA Yiran, Mujeeb ur RAHMAN, WANG Jianzhong, LIU Jiawei, BAI Quan. Preparation of a block copolymer-based temperature-responsive affinity chromatography stationary phase for antibody separation and purification[J]. Chinese Journal of Chromatography, 2023, 41(12): 1045-1051.

图/表 8

图1 嵌段共聚温敏亲和色谱固定相SiO2-P[NIPAM-b-4VP]-MEP的制备流程图

Fig. 1 Scheme of preparation of block copolymer-based temperature-responsive affinity chromatography stationary phase SiO2-P[NIPAM-b-4VP]-MEP NIPAM: N-isopropylacrylamide; DDTTC: S-1-dodecyl-S'-(1-α'-dimethyl-α″-acetic acid) trithiocarbonate; AIBN: azobisisobutyronitrile; 4-VP: 4-vinyl pyridine; PNIPAM-CTA: poly(N-isopropylacrylamide)-chain transfer agent; P[NIPAM-b-4VP]-CTA: poly[N-isopropylacrylamide-b-4VP]-chain transfer agent; TCEP: tris(2-carboxyethyl) phosphine hydrochloride; MEP: 4-mercapto-ethylpyridine; EDC: N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide hydrochloride; NHS: N-hydroxysuccinimide.

图2 P[NIPAM-b-4VP]-MEP和SiO2-P[NIPAM-b-4VP]-MEP的FT-IR图

Fig. 2 FT-IR spectra of P[NIPAM-b-4VP]-MEP and SiO2-P[NIPAM-b-4VP]-MEP

图3 SiO2、SiO2-NH2和SiO2-P[NIPAM-b-4VP]-MEP的XPS图

Fig. 3 XPS spectra of SiO2, SiO2-NH2 and SiO2- P[NIPAM-b-4VP]-MEP

表1 SiO2-NH2、SiO2-PNIPAM-MEP和SiO2-P[NIPAM- b-4VP]-MEP的元素分析

Table 1 Elemental analysis of SiO2-NH2, SiO2-PNIPAM- MEP and SiO2-P[NIPAM-b-4VP]-MEP

Sample	Atomic percents/%
Sample	C 1s	O 1s	Si 2p	S 2p	N 1s
SiO₂-NH₂	14.49	25.44	58.28	0	1.79
SiO₂-PNIPAM-MEP	38.17	37.7	14.94	2.10	7.09
SiO₂-P[NIPAM-b-4VP]-MEP	40.13	36.25	13.04	2.06	8.52

图4 温敏亲和色谱对BSA和γ-球蛋白的分离色谱图

Fig. 4 Chromatograms of bovine serum albumin (BSA) and γ-globulin separated with temperature-responsive chromatography The column was equilibrated at 40 ℃ with 100% mobile phase A (50 mmol/L Tris-HCl, pH 8.0) for 30 min, 10 μL of sample was injected and then the pump was stopped after 10 min. Move the column to 5 ℃ water bath for 10 min, and elute it with pre-cooled mobile phase B (50 mmol/L Tris HCl+0.6 mol/L NaCl, pH 8.0) at 1.0 mL/min. The detection wavelength was 280 nm.

图5 洗脱温度对γ-球蛋白和BSA色谱分离的影响

Fig. 5 Effects of elution temperature on the separation of γ-globulin and BSA The column was equilibrated at 40 ℃ with 100% mobile phase A for 30 min, 10 μL of sample was injected and then the pump was stopped after 10 min. Move the column to water bath at constant temperature for 10 min under different temperature, and elute it with mobile phase B at 1.0 mL/min. The detection wavelength was 280 nm.

图6 流动相中NaCl浓度对γ-球蛋白洗脱的影响

Fig. 6 Effect of the concentration of NaCl in the mobile phase on γ-globulin elution The column was equilibrated at 40 ℃ with 100% mobile phase A for 30 min, 10 μL of sample was injected and then the pump was stopped after 10 min. Move the column to 5 ℃ water bath for 10 min, and elute it with pre-cooled mobile phase B at 1.0 mL/min. The detection wavelength was 280 nm.

图7 温敏亲和色谱固定相SiO2-P[NIPAM-b-4VP]-MEP 对人血清中IgG的(a)色谱分离图及(b)SDS-PAGE电泳分析结果

Fig. 7 (a) Chromatogram and (b) SDS-PAGE assay of IgG from human serum with temperature-responsive affinity chromatography stationary phase SiO2-P[NIPAM-b-4VP]-MEP The column was equilibrated at 40 ℃ with 100% mobile phase A for 30 min, 10 μL of sample was injected and then the pump was stopped after 10 min. Move the column to 5 ℃ water bath for 10 min, and elute it with pre-cooled mobile phase B at 1.0 mL/min. The detection wavelength was 280 nm. Lane 1: human serum; Lane 2-4: purified IgG in triplicate.

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