Process Analytical Technology PAT Control Tools for High Cell Density Cultivation of Glycoengineered Pichia pastoris for Human Interferon and#945;2b Production

Abstract

Interferons are a group of multifunctional secreted proteins categorized as cytokines involved in intracellular signalling. Human interferon and#945;2b (huIFN and#945;2b) is a type I interferon and one of 13 variants of interferon and#945; that have been reported. huIFN and#945;2b triggers many biological activities such as antiviral, antiproliferative, and immunomodulatory functions. As a result, it is used in the treatment of hepatitis B and C, hairy cell leukaemia, melanoma, and AIDS-related Kaposi s sarcoma. In the context of recombinant protein production, yeast expression systems, particularly Pichia pastoris, offer advantages over bacterial hosts. They facilitate the synthesis of glycosylated recombinant proteins through appropriate post-translational modifications and entail lower operational costs than mammalian cell lines. However, challenges arise in managing higher expression, leading to the accumulation of improperly folded proteins in the Endoplasmic Reticulum (ER). Inappropriately/unfolded protein fractions in culture supernatants require extensive purification steps and are strongly discouraged from the Quality by Design (QbD) perspective. In accordance with the Process Analytical Technology (PAT) initiative, the critical process parameters (CPPs) and critical quality attributes (CQAs) of the process need to be identified and monitored in real-time to achieve improved product quality. Therefore, optimization of the protein titer from P. pastoris could be achieved by developing relationships between the various CPPs/CQAs and product titers. Cultivation of P. pastoris by manipulating the specific growth rate newline, a CPP, has a stronger influence at the metabolic level. Therefore, controlling newlinein real-time leads to enhanced product output. However, deconvolution of the soft sensor/online sensor input into CPPs is at the nascent stage for most therapeutic protein production.