Preparation of a block copolymer-based temperature-responsive affinity chromatography stationary phase for antibody separation and purification

doi:10.3724/SP.J.1123.2023.09028

Abstract

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

CLC Number:

O658

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.

Figures/Tables 8

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.

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

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

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

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.

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.

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.

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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