肽配基仿生亲和介质的制备和抗体纯化性能研究

Synthesis of Affinity Chromatography with Biomimetic Peptide Ligand and the Performance in Antibody Purification

寻找选择性高、稳定性强、价格低廉的蛋白A替代配基是抗体分离纯化领域的研究热点.针对L型氨基酸组成的线性多肽配基易被酶降解的问题,以D型非天然氨基酸为原料合成了配基D-FYWHCLDE,通过将游离的多肽配基与α-糜蛋白酶共同孵育研究了多肽配基的酶稳定性.通过将多肽配基偶联到Sepharose 6FF基质上制备了用以分离纯化抗体的多肽亲和介质,以细胞培养上清液和人血清为实际分离体系,考察了仿生多肽亲和介质的分离效果,通过测定多次从人血清中纯化hIgG及用0.1 mol/L NaOH长时间原位清洗后多肽亲和介质的动态吸附容量(DBC)变化来考察多肽亲和介质的可重复利用性和稳定性.结果表明,与酶共同孵育8 h后,D型多肽配基的降解率(8%)远低于L型多肽配基(55%),说明D型多肽配基具有更高的酶稳定性.使用D型多肽亲和介质从细胞培养上清液中纯化的单克隆抗体(mAb)的纯度高达98%,从人血清中纯化hIgG纯度为91.37%,说明多肽亲和介质具有从复杂的实际体系中分离抗体的能力,但不具备分离抗体不同亚型的能力.经0.1 mol/L NaOH原位清洗12 h后,两种多肽亲和介质对抗体的吸附量下降均小于8%,以人血清为原料,经过40次上样后,L型多肽亲和介质DBC下降14.51%,而D型多肽亲和介质仅下降5.92%,表明D型多肽亲和介质具有更高的操作稳定性.

Antibody isolation and purification focus on searching for protein A substitute ligand with high selectivity,high stability,and low cost.Focusing on the problem of the easy degradation of linear peptide ligands composed of natural amino acids by enzymes,a biomimetic peptide affinity ligand D-FYWHCLDE composed of Dtype amino acids was synthesized.The two peptide ligands were investigated for stability againstα-chymotrypsin before being coupled to the matrix Sepharose 6FF to prepare a peptide affinity chromatography for the separation and purification of antibodies.The separation effect of the biomimetic peptide affinity chromatography was investigated using cell culture supernatant and human serum as the actual separation system.The peptide affinity chromatography’s reusability and stability were measured using the dynamic binding capacity(DBC)change measurement of the peptide affinity chromatography after repeated purification of the human immunoglobulins(hIgG)from human serum and long-term in situ cleaning-in-place with 0.1 mol/L NaOH.The results revealed a much lower degradation rate of the Dtype peptide affinity ligand(8%)than that of the L-type peptide affinity ligand(55%)after 8 hours of coincubation with the enzyme,indicating that the D-type peptide affinity ligand had higher enzyme stability.The purity of monoclonal antibody(mAb)purified from cell culture supernatant using the D-type peptide affinity chromatography was as high as 98%,and hIgG purified from human serum had 91.37%purity,indicating that the peptide affinity chromatography could separate antibodies from actual complex systems but not different subtypes of antibodies.After washing in situ with 0.1 mol/L of NaOH for 12hours,the two peptide affinity chromatography’s DBC on the antibody decreased by less than 8%.Using human serum as the raw material,the DBC of the L-type peptide affinity chromatography de-creased by14.51%after 40 cycles,whereas the D-type peptide affinity chromatography only reduced by 5.92%,indicating a more stable D-type peptide affinity chromatography.