基于琼脂糖微球金属螯合层析介质的制备及性能评价

doi:10.16085/j.issn.1000-6613.2023-1528

摘要/Abstract

摘要：

以琼脂糖凝胶微球为研究对象，通过对微球进行化学交联与配体连接，制备系列金属螯合亲和层析介质，并测试其在模型蛋白中的吸附性能。首先，探究不同工艺条件对微球尺寸的影响。结果表明，在最优条件下，琼脂糖微球粒径集中在45~160μm之间，收率为95%，平均粒径为91.2μm。随后，以琼脂糖微球4B和6B为原料制备交联琼脂糖微球4FF（4 Fast Flow）和6FF（6 Fast Flow），调控交联剂滴加方式，探究不同因素对4FF微球机械强度的影响，所得4FF和6FF的最大流速分别达1190cm/h和2228cm/h。此外，本工作对制备的4FF微球进行复原性能测试及形貌表征，借助凝胶色谱法测定不同标准蛋白的保留体积，并计算有效分配系数。最后，以交联琼脂糖微球6FF为基质，制备葡聚糖接枝型金属螯合亲和层析介质，以单克隆抗体Ig G为模型蛋白，探究不同条件对介质动态蛋白载量的影响。结果表明，介质的最高动态蛋白载量可达44.8mg/mL gel。本工作为琼脂糖微球的可控制备、改性，以及在生物蛋白分离纯化中的应用提供了新思路，也为后续扩大化生产奠定了基础。

关键词: 琼脂糖微球, 金属螯合, 层析填料, 优化, 蛋白质

Abstract:

Agarose gel microspheres were used to prepare a series of metal-chelating affinity chromatography media by chemically cross-linking the microspheres and ligand attachment. Subsequently, their adsorption properties in model proteins were examined. Firstly, the effects of different reaction conditions on microsphere size were explored. The results showed that under the optimal conditions, the particle size of agarose microspheres was concentrated in the range of 45—160μm, with a yield of 95% and an average particle size of 91.2μm. Then, the crosslinked agarose microspheres 4FF(4 Fast Flow) and 6FF(6 Fast Flow) were prepared from agarose microspheres 4B and 6B, and the cross-linking agent dropping method was regulated to explore the effects of different factors on the mechanical strength of the cross-linked agarose microsphere 4FF. The maximum flow rates of 4FF and 6FF could reach 1190cm/h and 2228cm/h, respectively. In addition, the recovery performance and morphological characterization of the prepared 4FF microspheres were tested, and the retention volumes of different standard proteins were determined with the help of size-exclusion chromatography and the effective distribution coefficients were calculated. Finally, dextran-grafted metal chelating affinity chromatography media were prepared by using cross-linked agarose microspheres 6FF as a matrix, and monoclonal antibody Ig G was used as a model protein to explore the effects of different conditions on the dynamic protein loading of the media. The results showed that the highest dynamic protein loading of the medium could reach 44.8mg/mL gel. The present work provides a new idea for the controllable preparation and modification of agarose microspheres, as well as their application in the separation and purification of biological proteins, and lays a foundation for the subsequent scaled-up production.

Key words: agarose microspheres, metal chelation, chromatographic packing, optimization, protein

中图分类号:

TQ033

曾泽阳, 徐偲, 金子杰, 王晓钟, 陈英奇, 戴立言. 基于琼脂糖微球金属螯合层析介质的制备及性能评价[J]. 化工进展, 2024, 43(10): 5693-5703.

ZENG Zeyang, XU Cai, JIN Zijie, WANG Xiaozhong, CHEN Yingqi, DAI Liyan. Preparation and performance evaluation of metal chelating chromatography media based on agarose microspheres[J]. Chemical Industry and Engineering Progress, 2024, 43(10): 5693-5703.

图/表 21

表1 悬浮搅拌法现有工艺参数

油相	水油比（质量比）	乳化剂种类	乳化剂用量（质量分数）/%	搅拌转速/r·min^-1	参考文献
液体石蜡和石油醚	1∶3.6	PO-500	4	1000	[3]
甲苯	1∶6	吐温60	4	1500	[4]
液体石蜡	1∶3	复合乳化剂	6~12	500~600	[9]
液体石蜡和石油醚	1∶4	复合乳化剂	4	800	[10]
液体石蜡和石油醚	1∶8	PO-500	4	1000	[11]

图1 琼脂糖微球制备和交联过程示意图

图2 琼脂糖微球机械强度测试示意图

表2 不同品牌来源的琼脂糖性能指标

琼脂糖编号	外观	干燥失重/%	灰分/%	硫酸根/%	凝胶强度（1.0%）/g·cm^-2
GS001	白色粉末	≤10	≤0.5	≤0.3	≥1200
SL901	淡黄-白	<10	<1.0	<0.2	800~1000
SL91	淡黄-白	<10	<1.0	<0.2	800~1000
LP-900	淡黄-白	<10	<1.0	<0.2	800~1000
ML900	淡黄-白	<18	<1.0	<0.3	800~1000
M900	淡黄-白	<18	<1.5	<0.4	800~1000

图3 不同来源琼脂糖原料黏度对比图

图4 不同来源琼脂糖制备微球4B的显微镜（物镜4X）照片

图5 不同搅拌方式制备琼脂糖微球4B显微镜（物镜4X）照片

图6 不同HLB值乳化剂制备琼脂糖微球4B显微镜（物镜4X）照片

图7 250mL反应瓶中不同乳化剂浓度制备琼脂糖微球4B显微镜（物镜4X）照片

图8 3L反应瓶中不同乳化剂浓度制备琼脂糖微球4B显微镜（物镜4X）照片

图9 不同乳化温度制备琼脂糖微球4B显微镜（物镜4X）照片

图10 不同搅拌速度制备琼脂糖微球4B显微镜（物镜4X）照片

图11 不同工艺参数对交联琼脂糖微球4FF流通性能的影响

图12 激光粒度仪测定的微球粒径分布

图13 真空冷冻干燥琼脂糖微球复原性能测试显微镜（物镜4X）照片

图14 交联琼脂糖扫描电子显微镜照片

图15 不同分子在琼脂糖微球4B、4FF、6FF中的凝胶过滤图谱

表3 不同分子在琼脂糖微球4B、4FF、6FF中的保留体积和分配系数

分子名称	4B/mL	K_av,4B	4FF/mL	K_av,4FF	6FF/mL	K_av,6FF
蓝葡聚糖2000	18.67	0	23.19	0	24.22	0
甲状腺球蛋白	42.87	0.558	42.34	0.540	38.15	0.370
γ球蛋白	52.00	0.769	49.44	0.740	48.40	0.642
牛血清白蛋白	52.16	0.773	50.01	0.756	49.73	0.678
核糖核酸酶 A	57.36	0.892	54.80	0.891	56.87	0.867
氯化钠	62.01	1	58.67	1	61.86	1

图16 激光共聚焦扫描显微镜下FITC-葡聚糖接枝微球照片

图17 不同活化剂对葡聚糖接枝量的影响

图18 不同工艺参数对介质动态载量的影响

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