An impurity characterization based approach for the rapid development of integrated downstream purification processes

Abstract

In this study, we describe a new approach for the characterization of process-related impurities along with an in silico tool to generate orthogonal, integrated downstream purification processes for biological products. A one-time characterization of process-related impurities from product expression in Pichia pastoris was first carried out using linear salt and pH gradients on a library of multimodal, salt-tolerant, and hydrophobic charge induction chromatographic resins. The Reversed-phase ultra-performance liquid chromatography (UPLC) analysis of the fractions from these gradients was then used to generate large data sets of impurity profiles. A retention database of the biological product was also generated using the same linear salt and pH gradients on these resins, without fraction collection. The resulting two data sets were then analyzed using an in silico tool, which incorporated integrated manufacturing constraints to generate and rank potential three-step purification sequences based on their predicted purification performance as well as whole-process "orthogonality" for impurity removal. Highly ranked sequences were further examined to identify templates for process development. The efficacy of this approach was successfully demonstrated for the rapid development of robust integrated processes for human growth hormone and granulocyte-colony stimulating factor.

A combined screening and in silico strategy for the rapid design of integrated downstream processes for process and product‐related impurity removal

“…Wildtype P. pastoris was also cultivated without further modification (null strain). Product‐producing and null strain cell culture fluid (CCF) production is described in detail elsewhere (Timmick et al, ).…”

“…Additional details describing CCF fractionation experiments and ultra‐performance reversed phase liquid chromatography (UP‐RPLC) sample analysis of process‐related impurities (HCPs, DNA, media components, etc.) can be found elsewhere (Timmick et al, ). Briefly, null strain CCF was titrated to pH 3.0, 4.0, 5.0, and 6.0 using 100 mM citric acid and to pH 7.0 using 100 mM Tris base.…”

“…Here, we extend upon our previous work to generate and rank integrated, straight‐through downstream processes for the efficient removal of both process‐related and product‐related impurities (Timmick et al, ). As a case study, partially purified Interferon alpha‐2b (IFN‐α2b) produced in P. pastoris containing a significant product variant challenge (N‐terminal extensions and C‐terminal truncations) was chromatographically screened on a set of 14 multimodal, ion exchange, and hydrophobic charge induction chromatography (HCIC) resins under various pH and salt conditions and used to generate a retention database for IFN‐α2b and its variants.…”

“…Liu et al () designed a two‐step integrated polishing train for the removal of fragments and aggregates from a recombinant Fc‐fusion protein. Recently, we used a custom in silico tool to generate and rank integrated, straight‐through processes for efficient HCP removal from Pichia pastoris‐ derived feed stocks (Timmick et al, ). These approaches are effective at identifying integrated, straight‐through processes to remove either process‐related (HCPs, DNA, media components, etc.)…”

“…As a case study, partially purified Interferon alpha‐2b (IFN‐α2b) produced in P. pastoris containing a significant product variant challenge (N‐terminal extensions and C‐terminal truncations) was chromatographically screened on a set of 14 multimodal, ion exchange, and hydrophobic charge induction chromatography (HCIC) resins under various pH and salt conditions and used to generate a retention database for IFN‐α2b and its variants. This database, along with a process‐related impurity database for P. pastoris previously constructed by our group (Timmick et al, ), were then used as inputs into a custom in silico tool that generated and ranked all possible integrated downstream processes for IFN‐α2b purification. Top‐ranking outputs were then used to guide the experimental refinement of two final three‐step purification processes, one consisting of all bind‐elute steps and the other having a flowthrough operation.…”