目的研究陶瓷羟基磷灰石(ceramic hydroxyapatite,CHT)I型和II型对2种单克隆抗体(monoclonal Ab,m Ab)1和2聚集体的去除工艺。方法层析仪为AKTA AVANT 150,层析柱为Tricon 10/50,先进行CHT I、CHT II 2种填料的动态载样量研究,然后选取合适的载量进行分离纯化研究。上样条件为5 mmol/L磷酸二氢钠(Na H2PO4),p H 6.5,上样,然后用10 mmol/L Na H2PO4,p H6.5和10 mmol/L Na H2PO4,2 mol/L Na Cl,p H 6.5梯度洗脱来分离单体和聚集体。用分子尺寸排阻高效液相色谱(SEC-HPLC)测定上样蛋白溶液和洗脱峰单体及聚集体的含量。使用20 cm高的XK 16/40的层析柱进行工艺放大研究。结果 m Ab 1在CHT I型的载量为40 mg/m L,去除后单体含量为98.6%,工艺收率为92.5%;m Ab 2在CHT I型的载量为45 mg/m L,去除后单体含量为98.8%,工艺收率为91.5%;m Ab 1在CHT II型载量为16 mg/m L,去除后单体含量为99.8%,工艺收率为91.8%;m Ab 2在CHT II型载量为20 mg/m L,去除后单体含量为99.9%,工艺回收率为92.2%。结论 2种类型的陶瓷羟基磷灰石填料在聚集体含量高于10%的情况下,都有很好的去除能力,去除结果符合法规要求。该方法操作简单,能够很顺利的进行工艺放大,满足中试和生产需求。
Currently,display-based methods are well established and widely used in antibody engineering for affinity maturation and structural stability improvement.We obtained a novel anti-human programmed death 1(PD-1)antibody using computer-aided design and a mammalian cell display technology platform.We used computer-aided modeling and distance geometry methods to predict and assign the key residues that contributed to the binding of human PD-L1 to PD-1.Then,we analyzed the sequence of nivolumab(an anti-human PD-1 antibody,referred to as MIL75 in the article)to determine the template for antibody design and library construction.We identified a series of potential substitutions on the obtained template and constructed a virtual epitope-targeted antibody library based on the physicochemical properties and each possible location of the assigned key residues.The virtual antibody libraries were displayed on the surface of mammalian cells as the antigen-binding fragments of full-length immunoglobulin G.Then,we used flow cytometry and sequencing approaches to sort and screen the candidates.Finally,we obtained a novel anti-human PD-1 antibody named FV78.FV78 competitively recognized the PD-1 epitopes that interacted with MIL75 and possessed an affinity comparable to MIL75.Our results implied that FV78 possessed equivalent bioactivity in vitro and in vivo compared with MIL75,which highlighted the probability and prospect of FV78 becoming a new potential antibody therapy.
