Faculty of Molecular Chemistry and Engineering, Kyoto Institute of Technology, Kyoto 606-8585, Japan.
Research and Clinical Development, Nestlé Health Science, Lausanne 1000, Switzerland; Department of Chemical Engineering, University of Texas at Austin, Austin, TX 78712, USA.
J Biosci Bioeng. 2024 Apr;137(4):298-303. doi: 10.1016/j.jbiosc.2023.12.016. Epub 2024 Feb 1.
In this study we successfully developed an on-demand affinity chromatographic resin for manufacturing non-Fc-based biopharmaceuticals. Affinity chromatography columns with immobilized rabbit single-chain variable fragments (scFvs) were used for directly purifying the recombinant human kynureninase (KYNase) as a model target therapeutic protein from Escherichia coli cell lysates. Among the 38 different anti-KYNase scFv clones identified, four unique clones were selected as candidates for further characterization owing to their relatively low KYNase binding affinity at pH 4.0, thereby facilitating enzyme elution. Subsequently, all four clones were successfully produced and purified, followed by covalent coupling to NHS-activated HiTrap HP columns. While KYNase was specifically adsorbed to all four scFv-immobilized columns and was eluted at pH 4.0, the respective levels of static binding capacity (SBC) and recovery among the four scFv clones were different at this elution pH. That is, the scFv-immobilized columns captured KYNase with SBC ranging from 1.15 to 2.68 mg/cm-bed with clone R2-47 exhibiting the highest level of SBC, with a ligand utilization of 39.4 %. Moreover, using the scFv column of R2-47, 90.7 % of the captured human KYNase was recovered in the first elution step at pH 4.0, and approximately 67 % of enzymatic activity was retained. In summary, high-purity human KYNase was obtained from the E. coli cell lysate by one-step affinity purification, and 89.7 % of KYNase was recovered in the first elution step. The methodology demonstrated in the current study could be applied for the purification and development of various therapeutic proteins.
在这项研究中，我们成功开发了一种按需亲和色谱树脂，用于制造非 Fc 基生物制药。使用固定化兔单链可变片段（scFv）的亲和色谱柱，直接从大肠杆菌细胞裂解物中纯化重组人犬尿酸酶（KYNase）作为模型治疗蛋白。在鉴定的 38 个不同的抗 KYNase scFv 克隆中，由于其在 pH 4.0 时相对较低的 KYNase 结合亲和力，从而有利于酶洗脱，选择了四个独特的克隆作为进一步表征的候选物。随后，成功生产和纯化了所有四个克隆，并将其与 NHS 活化的 HiTrap HP 柱共价偶联。虽然 KYNase 特异性吸附到所有四个 scFv 固定化柱上，并在 pH 4.0 洗脱，但在该洗脱 pH 下，四个 scFv 克隆之间的静态结合容量（SBC）和回收率有所不同。也就是说，scFv 固定化柱以 SBC 为 1.15 至 2.68 mg/cm-床的范围捕获 KYNase，其中克隆 R2-47 的 SBC 最高，配体利用率为 39.4%。此外，使用 R2-47 的 scFv 柱，在 pH 4.0 的第一个洗脱步骤中回收了 90.7%的捕获的人 KYNase，并且保留了约 67%的酶活性。总之，通过一步亲和纯化从大肠杆菌细胞裂解物中获得了高纯度的人犬尿酸酶，并且在第一个洗脱步骤中回收了 89.7%的 KYNase。本研究中展示的方法可应用于各种治疗蛋白的纯化和开发。
