Computational prediction of blend time in a large-scale viral inactivation process for monoclonal antibodies biomanufacturing.

Viral inactivation (VI) is a process widely used across the pharmaceutical industry to eliminate the cytotoxicity resulting from trace levels of viruses introduced by adventitious agents. This process requires adding Triton X-100, a non-ionic detergent solution, to the protein solution and allowing sufficient time for this agent to inactivate the viruses. Differences in process parameters associated with vessel designs, aeration rate, and many other physical attributes can introduce variability in the process, thus making predicting the required blending time to achieve the desired homogeneity of Triton X-100 more critical and complex. In this study we utilized a CFD model based on the lattice Boltzmann method (LBM) to predict the blend time to homogenize a Triton X-100 solution added during a typical full-scale commercial VI process in a vessel equipped with an HE-3-impeller for different modalities of the Triton X-100 addition (batch vs. continuous). Although direct experimental progress of the blending process was not possible because of GMP restrictions, the degree of homogeneity measured at the end of the process confirmed that Triton X-100 was appropriately dispersed, as required, and as computationally predicted here. The results obtained in this study were used to support actual production at the biomanufacturing site.

Generation of diploid Pichia pastoris strains by mating and their application for recombinant protein production.

BACKGROUND: Yeast mating provides an efficient means for strain and library construction. However, biotechnological applications of mating in the methylotrophic yeast Pichia pastoris have been hampered because of concerns about strain stability of P. pastoris diploids. The aim of the study reported here is to investigate heterologous protein expression in diploid P. pastoris strains and to evaluate diploid strain stability using high cell density fermentation processes. RESULTS: By using a monoclonal antibody as a target protein, we demonstrate that recombinant protein production in both wild-type and glycoengineered P. pastoris diploids is stable and efficient during a nutrient rich shake flask cultivation. When diploid strains were cultivated under bioreactor conditions, sporulation was observed. Nevertheless, both wild-type and glycoengineered P. pastoris diploids showed robust productivity and secreted recombinant antibody of high quality. Specifically, the yeast culture maintained a diploid state for 240 h post-induction phase while protein titer and N-linked glycosylation profiles were comparable to that of a haploid strain expressing the same antibody. As an application of mating, we also constructed an antibody display library and used mating to generate novel full-length antibody sequences. CONCLUSIONS: To the best of our knowledge, this study reports for the first time a comprehensive characterization of recombinant protein expression and fermentation using diploid P. pastoris strains. Data presented here support the use of mating for various applications including strain consolidation, variable-region glycosylation antibody display library, and process optimization.

In vivo anti-tumor efficacy of afucosylated anti-CS1 monoclonal antibody produced in glycoengineered Pichia pastoris.

Monoclonal antibody (mAb) therapy has been successfully used for the treatment of B-cell lymphomas and is currently extended for the treatment of multiple myeloma (MM). New developments in MM therapeutics have achieved significant survival gains in patients but the disease still remains incurable. Elotuzumab (HuLuc63), an anti-CS1 monoclonal IgG1 antibody, is believed to induce anti-tumor activity and MM cytotoxicity through antibody dependent cellular cytotoxicity (ADCC) and inhibition of MM cell adhesion to bone marrow stromal cells (BMSCs). Modulations of the Fc glycan composition at the N297 site by selective mutations or afucosylation have been explored as strategies to develop bio-better therapeutics with enhanced ADCC activity. Afucosylated therapeutic antibodies with enhanced ADCC activity have been reported to possess greater efficacy in tumor growth inhibition at lower doses when compared to fucosylated therapeutic antibodies. The N-linked glycosylation pathway in Pichia pastoris has been engineered to produce human-like N-linked glycosylation with uniform afucosylated complex type glycans. The purpose of this study was to compare afucosylated anti-CS1 mAb expressed in glycoengineered Pichia pastoris with fucosylated anti-CS1 mAb expressed in mammalian HEK293 cells through in vitro ADCC and in vivo tumor inhibition models. Our results indicate that Fc glycosylation is critical for in vivo efficacy and afucosylated anti-CS1 mAb expressed in glycoengineered Pichia pastoris shows a better in vivo efficacy in tumor regression when compared to fucosylated anti-CS1 mAb expressed in HEK293 cells. Glycoengineered Pichia pastoris could provide an alternative platform for generating homogeneous afucosylated recombinant antibodies where Fc mediated immune effector function is important for efficacy.

Maximizing recombinant human serum albumin production in a Mut(s) Pichia pastoris strain.

Human serum albumin (HSA) is a cysteine rich molecule that is most abundant in human blood plasma. To remain viable in the market due to lower marketing costs for HSA, it is important to produce a large quantity in an economical manner by recombinant technology. The objective of this study was to maximize recombinant HSA (rHSA) production using a Mut(s) Pichia pastoris strain by fermentation process optimization. We evaluated the impact of process parameters on the production of rHSA, including induction cell density (wet cell weight, g/L) and the control of specific growth rate at induction. In this study, we demonstrated that induction cell density is a critical factor for high level production of rHSA under controlled specific growth rate. We observed higher specific productivities at higher induction cell densities (285 g/L) and at lower specific growth rates (0.0022-0.0024/h) during methanol induction phase, and achieved the broth titer of rHSA up to 10 g/L. The temperature shift from 24 to 28(o) C was effective to control the specific growth rate at low level (≤0.0024/h) during methanol induction phase while maintaining high specific productivity [0.0908 mgrHSA /(gwcw h)].