Figure and Tables
Creators: name: Jalalirad, Reza
name: Doroud, Delaram
name: Reza Aghasadeghi, Mohammad
name: Hozouri, Hamidreza
Publisher: name: figshare
Year: 2024
Subjects: classificationCode: 310602
subject: Bioprocessing, bioproduction and bioproducts

description: <b>Project Objectives:</b>The main objective of this study was rHBsAg purification with high purity from the <i>P. pastoris</i> industrial feedstock by multimodal Capto adhere resin in different purification modes. For this purpose, the important factors and responses were first defined using OFAT non-binding experiments. Then the target protein binding and non-binding conditions were defined using the DOE method. The optimal conditions were finally utilized to purify rHBsAg from <i>P. pastoris</i> industrial feedstock in two purification modes (i.e., bind-elute and flow-through) and the purified rHBsAg was assessed for purity and SEC-HPLC profile. Finally, the protein purity, purification yield, and capacity of Capto adhere were discussed to evaluate its efficacy and selectivity for rHBsAg purification in a single-step.<b>Conclusions</b>The multimodal bioprocess resins for production-scale chromatography provide an opportunity for improvement of the established downstream processes. Capto adhere, a strong ion-exchanger with multimodal functionality, was used in this study for the final aim of rHBsAg purification from the industrial <i>P. pastoris</i> feedstock. Due to the number and complexity of protein-ligand interactions in multimodal resins, optimization works were conducted for binding and elution steps based on the DOE method.In the binding optimization process using CCF design, conditions for high rHBsAg binding capacity were identified. The obtained higher Capto adhere binding capacity compared to single-mode resin reported in other studies[38], being attributed to the multimodal interactions of the resin. Although strong multimodal interactions were involved in the protein binding to Capto adhere, a reasonable recovery (ca. 60%) was achieved using 1 M arginine, pH 6.5, after elution optimization.The obtained optimal conditions were finally used for purification of rHBsAg from <i>P. pastoris</i> industrial feedstock in both bind-elute and flow-through purification modes. The same rHBsAg binding capacity and elution recovery figures were achieved during rHBsAg purification from the industrial feedstock. In the flow-through purification mode, the target protein recovery was substantially increased up to 97%, without significant decrease in rHBsAg purity. These findings were in agreement with other studies which encourage the usage of this resin in the flow-through mode.Comparison of this study with our previous study[41]highlights the complexity of various multimodal resins, their unpredictable behavior in interactions with rHBsAg and necessity of using the DOE approach to deconvolute complex protein- resin interactions.The SEC-HPLC of the purified rHBsAg (with 100% purity) showed that the retention times of the elution step were similar to those reported elsewhere. The binding capacity, recovery and purity of rHBsAg obtained from these optimization studies show that Capto adhere can be considered as prospective resin in downstream processing of rHBsAg originated from recombinant <i>P. pastoris</i>.