Glycoengineered Pichia Pastoris Research Articles

Protective RBD-dimer vaccines against SARS-CoV-2 and its variants produced in glycoengineered Pichia pastoris.

Protective vaccines are crucial for preventing and controlling coronavirus disease 2019 (COVID-19). Updated vaccines are needed to confront the continuously evolving and circulating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants. These vaccines should be safe, effective, amenable to easily scalable production, and affordable. Previously, we developed receptor binding domain (RBD) dimer-based protein subunit vaccines (ZF2001 and updated vaccines) in mammalian cells. In this study, we explored a strategy for producing RBD-dimer immunogens in Pichia pastoris. We found that wild-type P. pastoris produced hyperglycosylated RBD-dimer protein containing four N-glycosylation sites in P. pastoris. Therefore, we engineered the wild type P. pastoris (GS strain) into GSΔOCH1pAO by deleting the OCH1 gene (encoding α-1,6-mannosyltransferase enzyme) to decrease glycosylation, as well as by overexpressing the HIS4 gene (encoding histidine dehydrogenase) to increase histidine synthesis for better growth. In addition, RBD-dimer protein was truncated to remove the R328/F329 cleavage sites in P. pastoris. Several homogeneous RBD-dimer proteins were produced in the GSΔOCH1pAO strain, demonstrating the feasibility of using the P. pastoris expression system. We further resolved the cryo-EM structure of prototype-Beta RBD-dimer complexed with the neutralizing antibody CB6 to reveal the completely exposed immune epitopes of the RBDs. In a murine model, we demonstrated that the yeast-produced RBD-dimer induces robust and protective antibody responses, which is suitable for boosting immunization. This study developed the yeast system for producing SARS-CoV-2 RBD-dimer immunogens, providing a promising platform and pipeline for the future continuous updating and production of SARS-CoV-2 vaccines.

Enhanced production of human interferon α2b in glycoengineered Pichia pastoris by robust control of methanol feeding and implications of various control strategies

Interferon α2b (IFNα2b), a prominent cytokine molecule, is bestowed with immunomodulatory, antiproliferative, and antiviral properties. Like any other recombinant protein produced in methylotrophic yeast Pichia pastoris, Human interferon α2b (huIFN α2b) production in P. pastoris depends on methanol utilization during the induction phase. This study investigates the impact of varying residual methanol concentrations on huIFNα2b productivity and assesses control strategies towards the tight control of residual methanol concentration in fed-batch experiments carried out in the fermentation calorimeter. Real-time monitoring of P. pastoris metabolism was facilitated by metabolic heat rate measurements and exhaust gas analyser. Optimal methanol concentration screening performed at different concentrations viz., 1.5, 3, 5, and 7 g/L, revealed the highest huIFNα2b productivity at 3 g/L (0.21 mg/g h). Methanol sensor provided input data for three different control strategies (on/off, proportional integral (PI), and model-based adaptive PI) to maintain optimal methanol levels (3 g/L). Model-based adaptive control resulted in the highest huIFNα2b yield, productivity (244.34 mg/L, 0.31 mg/g h), surpassing on/off (188.7 mg/L, 0.16 mg/g h) and PI (202.3 mg/L, 0.28 mg/g h) control strategies. The model-based adaptive PI control framework developed in this study could be employed for other heterologous protein production in P. pastoris.

A Delta-Omicron Bivalent Subunit Vaccine Elicited Antibody Responses in Mice against Both Ancestral and Variant Strains of SARS-CoV-2.

Continued mutation of the SARS-CoV-2 genome has led to multiple waves of COVID-19 infections, and new variants have continued to emerge and dominate. The emergence of Omicron and its subvariants has substantially increased the infectivity of SARS-CoV-2. RBD genes of the wild-type SARS-CoV-2 strain and the Delta, Omicron BA.1 and Omicron BA.2 variants were used to construct plasmids and express the proteins in glycoengineered Pichia pastoris. A stable 4 L-scale yeast fermentation and purification process was established to obtain high-purity RBD proteins with a complex glycoform N-glycosyl structure that was fucose-free. The RBD glycoproteins were combined with two adjuvants, Al(OH)3 and CpG, which mitigated the typical disadvantage of low immunogenicity associated with recombinant subunit vaccines. To improve the broad-spectrum antiviral activity of the candidate vaccine, Delta RBD proteins were mixed with BA.2 RBD proteins at a ratio of 1:1 and then combined with two adjuvants-Al(OH)3 and CpG-to prepare a bivalent vaccine. The bivalent vaccine effectively induced mice to produce pseudovirus-neutralizing antibodies against SARS-CoV-2 variants, Delta, Beta, and Omicron sublineages BA.1, BA.2, BA.5. The bivalent vaccine could neutralize the authentic wild-type SARS-CoV-2 strain, Delta, BA.1.1, BA.2.2, BA2.3, and BA.2.12.1 viruses, providing a new approach for improving population immunity and delivering broad-spectrum protection under the current epidemic conditions.

Deployment of metabolic heat rate based soft sensor for estimation and control of specific growth rate in glycoengineered Pichia pastoris for human interferon alpha 2b production

Lack of appropriate process models, reliable online sensors, and process variability in bioprocess systems are poising challenges in real-time monitoring and control of critical process parameters (CPPs). This present investigation deals with the development of a non-invasive soft sensor by utilizing metabolic heat rate as input signal for online estimation of specific growth rate (μest) during the induction phase of glycoengineered Pichia pastoris for human interferon-alpha 2b (huIFNα2b) production. Feedforward strategy employing a predetermined exponential feeding of methanol during the induction phase was dealt at defined setpoint values (μSP). Standard PID controller with predetermined gain values regulated methanol feeding in accordance with the deviation from the μSP value. An adaptive PID (gain scheduling) significantly minimized the deviation of μ from its μSP value, reduced the amplitude of oscillation and achieved long-term controller stability. Robust control of methanol feeding by adaptive PID resulted in a 1.5 and 2.2-fold increase in productivity of huIFNα2b compared to standard PID and feedforward controls respectively. Moreover, adaptive PID control facilitated narrow range control of μ for longer durations (> 20 h) with a low average tracking error (< 6%) enumerating its scope of application in therapeutic protein production in near future.

Kappa-RBD produced by glycoengineered Pichia pastoris elicited high neutralizing antibody titers against pseudoviruses of SARS-CoV-2 variants.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) kappa (B.1.617.1) variant represented the main variant of concern (VOC) for the epidemic in India in May 2021. We have previously established a technology platform for rapidly preparing SARS-CoV-2 receptor-binding domain (RBD) candidate vaccines based on glycoengineered Pichia pastoris. Our previous study revealed that the wild-type RBD (WT-RBD) formulated with aluminum hydroxide and CpG 2006 adjuvant effectively induces neutralizing antibodies in BALB/c mice. In the present study, a glycoengineered P. pastoris expression system was used to prepare recombinant kappa-RBD candidate vaccine. Kappa-RBD formulated with CpG and alum induced BALB/c mice to produce a potent antigen-specific antibody response and neutralizing antibody titers against pseudoviruses of SARS-CoV-2 kappa, delta, lambda, beta, and omicron variants and WT. Therefore, the recombinant kappa-RBD vaccine has sufficient potency to be a promising COVID-19 vaccine candidate.

Production of Recombinant Human Ceruloplasmin: Improvements and Perspectives.

The ferroxidase ceruloplasmin (CP) plays a crucial role in iron homeostasis in vertebrates together with the iron exporter ferroportin. Mutations in the CP gene give rise to aceruloplasminemia, a rare neurodegenerative disease for which no cure is available. Many aspects of the (patho)physiology of CP are still unclear and would benefit from the availability of recombinant protein for structural and functional studies. Furthermore, recombinant CP could be evaluated for enzyme replacement therapy for the treatment of aceruloplasminemia. We report the production and preliminary characterization of high-quality recombinant human CP in glycoengineered Pichia pastoris SuperMan5. A modified yeast strain lacking the endogenous ferroxidase has been generated and employed as host for heterologous expression of the secreted isoform of human CP. Highly pure biologically active protein has been obtained by an improved two-step purification procedure. Glycan analysis indicates that predominant glycoforms HexNAc2Hex8 and HexNAc2Hex11 are found at Asn119, Asn378, and Asn743, three of the canonical four N-glycosylation sites of human CP. The availability of high-quality recombinant human CP represents a significant advancement in the field of CP biology. However, productivity needs to be increased and further careful glycoengineering of the SM5 strain is mandatory in order to evaluate the possible therapeutic use of the recombinant protein for enzyme replacement therapy of aceruloplasminemia patients.

A Vaccine Based on the Receptor-Binding Domain of the Spike Protein Expressed in Glycoengineered Pichia pastoris Targeting SARS-CoV-2 Stimulates Neutralizing and Protective Antibody Responses

In 2020 and 2021, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a novel coronavirus, caused a global pandemic. Vaccines are expected to reduce the pressure of prevention and control, and have become the most effective strategy to solve the pandemic crisis. SARS-CoV-2 infects the host by binding to the cellular receptor angiotensin converting enzyme 2 (ACE2) via the receptor-binding domain (RBD) of the surface spike (S) glycoprotein. In this study, a candidate vaccine based on a RBD recombinant subunit was prepared by means of a novel glycoengineered yeast Pichia pastoris expression system with characteristics of glycosylation modification similar to those of mammalian cells. The candidate vaccine effectively stimulated mice to produce high-titer anti-RBD specific antibody. Furthermore, the specific antibody titer and virus-neutralizing antibody (NAb) titer induced by the vaccine were increased significantly by the combination of the double adjuvants Al(OH)3 and CpG. Our results showed that the virus-NAb lasted for more than six months in mice. To summarize, we have obtained a SARS-CoV-2 vaccine based on the RBD of the S glycoprotein expressed in glycoengineered Pichia pastoris, which stimulates neutralizing and protective antibody responses. A technical route for fucose-free complex-type N-glycosylation modified recombinant subunit vaccine preparation has been established.

Recombinant H7 hemagglutinin expressed in glycoengineered Pichia pastoris forms nanoparticles that protect mice from challenge with H7N9 influenza virus

Cases of H7N9 human infection caused by an avian-origin H7N9 virus emerged in eastern China in 2013, leading to the urgent requirement of developing an effective vaccine to reduce its pandemic potential. In this report, the full-length recombinant H7 protein (rH7) of A/Hangzhou/1/2013 (H7N9) virus was expressed by a glycoengineered Pichia pastoris system. The rH7 protein underwent complex glycosylation modifications and polymerized to nanoparticles of 30–50 nm in diameter. Recombinant H7 (1.9 µg) elicited a > 1:40 hemagglutination inhibition titer, and 3.75 µg rH7 protected 100% of the mice in the mice challenge model with 10-fold 50% lethal dose of the A/Shanghai/2/2013 (H7N9) rat lung-adapted strain. In conclusion, rH7 produced by the glycoengineered P. pastoris can be used for vaccination against the H7N9 virus, and provides an effective platform for the rapid production of future influenza vaccines.

Biocalorimetric monitoring of glycoengineered P. pastoris cultivation for the production of recombinant huIFNα2b: A quantitative study based on mixed feeding strategies.

Real-time monitoring of glycoengineered Pichia pastoris by employing process analytical technology (PAT) tools is vital for gaining deeper insights into the therapeutic protein production process. The present study focuses on influence of mixed feed carbon substrates during the induction phases of glycoengineered P. pastoris cultivation, for recombinant human interferon α2b (huIFNα2b) production by employing calorimetric (biological heat rate, q B ) and respirometric (oxygen uptake rate and carbon dioxide evolution rate) measurements. Mixed feed stream of carbon substrates (methanol + glycerol, methanol + sorbitol) at a predetermined "C-molar ratios" were added during the induction phases. Methanol- and sorbitol-based mixed feeding approach resulted in an improved huIFNα2b titer of 288 mg/L by channeling of methanol predominantly towards an optimal functioning of AOX expression system. A stand-off between biomass yield coefficient, degree of reduction of methanol and its cosubstrate (glycerol and sorbitol) determines the fraction of carbon energy channeled toward biomass and protein production, under strict aerobic conditions. Calorespirometric monitoring and assessment of thermal yields enables a reliable prediction of process variables, leading to futuristic efficient PAT-based feed rate control.

Control of specific growth rate for the enhanced production of human interferon α2b in glycoengineered Pichia pastoris: process analytical technology guided approach

AbstractBACKGROUNDProcess variability in bioprocess systems involving genetically engineered strains is a common bottleneck and a real‐time insight of the on‐going process is crucial to achieve the desired product and its quality. In this study, a process analytical technology (PAT) platform was developed for the monitoring and control of specific growth rate during methanol induction phase (μmet) of glycoengineered Pichia pastoris fermentation for human interferon alpha 2b (huIFNα2b) production.RESULTSThe PAT guided approach involves real‐time monitoring of capacitance (ΔC) facilitating online estimation of specific growth rate (μest), which serves as a process input during controller application. Fed‐batch experiments using pulsed‐feeding of methanol at different dosage (20 g and 30 g) did not significantly influence μmet. Exponential methanol feeding was achieved using a modified proportional, integral and derivative (PID) controller for different predefined specific growth rate set point (μsp) values, namely 0.015, 0.03, 0.04 and 0.06 h−1. Exponential feeding strategy during the induction phase resulted in two crucial outcomes: (i) controlled methanol feeding rate (regulated by the developed PID controller) balanced the methanol consumption rate (qs, met) of the P. pastoris; (ii) significant improvement in huIFNα2b titer (1483 mg L−1) and specific productivity(&gt;0.4 mg g−1. h) was achieved by the robust control of μmet at optimal (0.04 h−1) value. The purified huIFNα2b was found to exhibit antiproliferative effect against human breast cancer cell lines.CONCLUSIONSEfficient control of μmet at a very low narrow range was achieved with a long‐term controller stability (&gt;10 h) and the highest titer reported to date for huIFNα2b (at optimal μmet) in the yeast expression platform. © 2019 Society of Chemical Industry

Novel glycosylated human interferon alpha 2b expressed in glycoengineered Pichia pastoris and its biological activity: N-linked glycoengineering approach

Human interferon alpha 2b (IFN α2b) is a type I interferon exhibiting antiviral, anti-proliferative and immunomodulatory activities. The clinical outcome of the approved recombinant human IFN α2b drugs in the market suffers from short plasma half-life, rapid clearance and other side effects. Human IFN α2b expression in mammalian cell lines results in significant heterogeneity in glycan moieties, inconsistent product quality and high production cost. Potential scope exists for the design and development of a successful expression platform for enhanced human IFN α2b production with improved pharmacokinetic property. Glycoengineering strategy was employed to construct IFN α2b with potential N-glycosylation site to evade the drawbacks of approved recombinant human IFN α2b drugs. Heterogeneity of glycosylation and hypermannosylation in the wild-type strains of Pichia pastoris was circumvented by employing glycoengineered strain (SuperMan5) to produce glycosylated IFN α2b with human type N-glycans. Recombinant SuperMan5 strain expressed human type N-glycosylated IFN α2b with greater homogeneity elucidated by glycan analysis (MALDI-TOF/MS). The purified glycosylated IFN α2b was biologically active, inhibiting the viral replication of HCV and HEV at 85% and 66%, respectively. Pharmacokinetic studies in Wistar rats revealed 1.3 fold increase in plasma half-life for glycosylated IFN α2b compared to standard IFN α2b produced by E. coli.

High level extracellular production of recombinant human interferon alpha 2b in glycoengineered Pichia pastoris: culture medium optimization, high cell density cultivation and biological characterization.

The present study was aimed at design of experiments (DoE)- and artificial intelligence-based culture medium optimization for high level extracellular production of a novel recombinant human interferon alpha 2b (huIFNα2b) in glycoengineered Pichia pastoris and its characterization. The artificial neural network-genetic algorithm model exhibited improved huIFNα2b production and better predictability compared to response surface methodology. The optimized medium exhibited a fivefold increase in huIFNα2b titre compared to the complex medium. A maximum titre of huIFNα2b (436mgl-1 ) was achieved using the optimized medium in the bioreactor. Real-time capacitance data from dielectric spectroscopy were utilized to model the growth kinetics with unstructured models. Biological characterization by antiproliferative assay proved that the purified recombinant huIFNα2b was biologically active, exhibiting growth inhibition on breast cancer cell line. Culture medium optimization resulted in enhanced production of huIFNα2b in glycoengineered P. pastoris at both shake flask and bioreactor level. The purified huIFNα2b was found to be N-glycosylated and biologically active. DoE-based medium optimization strategy significantly improved huIFNα2b production. The antiproliferative activity of huIFNα2b substantiates its potential scope for application in cancer therapy.

Engineering O-glycosylation in modified N-linked oligosaccharide (Man12GlcNAc2∼Man16GlcNAc2) Pichia pastoris strains.

Yeast have been engineered for the production of therapeutic glycoproteins with humanized N-linked oligosaccharides. Both N- and O-linked oligosaccharides engineered yeast have been attractive prospects, since yeast-specific O-mannosylated proteins were reported to induce an aberrant immune response and alter pharmacokinetics in vivo. In the present study, we genetically manipulated O-glycosylation by disrupting O-mannosyltransferase PMT1 and PMT5 in a low-mannose type N-linked oligosaccharide (Man12GlcNAc2∼Man16GlcNAc2) engineered Pichia pastoris strain to produce therapeutic glycoproteins. The O-mannosyltransferase PMT1 mutant produces anti-Her-2 antibodies with reduced O-linked oligosaccharides and protein degradation, but this strain exhibited growth defects. However, the deletion of O-mannosyltransferase PMT5 individually has a minimal effect on O-glycosylation, degradation of the anti-Her-2 antibody, and strain growth. Thus, by disrupting O-mannosyltransferase PMT1 in an N-glycosylation engineered Pichia pastoris strain, we generated an effective glycoengineered Pichia pastoris strain to effectively produce therapeutic glycoproteins with both engineered N- and O-linked oligosaccharides.

Production of a recombinant peroxidase in different glyco-engineered Pichia pastoris strains: a morphological and physiological comparison

BackgroundThe methylotrophic yeast Pichia pastoris is a common host for the production of recombinant proteins. However, hypermannosylation hinders the use of recombinant proteins from yeast in most biopharmaceutical applications. Glyco-engineered yeast strains produce more homogeneously glycosylated proteins, but can be physiologically impaired and show tendencies for cellular agglomeration, hence are hard to cultivate. Further, comprehensive data regarding growth, physiology and recombinant protein production in the controlled environment of a bioreactor are scarce.ResultsA Man5GlcNAc2 glycosylating and a Man8–10GlcNAc2 glycosylating strain showed similar morphological traits during methanol induced shake-flask cultivations to produce the recombinant model protein HRP C1A. Both glyco-engineered strains displayed larger single and budding cells than a wild type strain as well as strong cellular agglomeration. The cores of these agglomerates appeared to be less viable. Despite agglomeration, the Man5GlcNAc2 glycosylating strain showed superior growth, physiology and HRP C1A productivity compared to the Man8–10GlcNAc2 glycosylating strain in shake-flasks and in the bioreactor. Conducting dynamic methanol pulsing revealed that HRP C1A productivity of the Man5GlcNAc2 glycosylating strain is best at a temperature of 30 °C.ConclusionThis study provides the first comprehensive evaluation of growth, physiology and recombinant protein production of a Man5GlcNAc2 glycosylating strain in the controlled environment of a bioreactor. Furthermore, it is evident that cellular agglomeration is likely triggered by a reduced glycan length of cell surface glycans, but does not necessarily lead to lower metabolic activity and recombinant protein production. Man5GlcNAc2 glycosylated HRP C1A production is feasible, yields active protein similar to the wild type strain, but thermal stability of HRP C1A is negatively affected by reduced glycosylation.

Recombinant human interferon alpha-2β: Cloning, expression and PAT-process analytical technology enabled production in glycoengineered Pichia pastoris

Recombinant human interferon alpha-2β: Cloning, expression and PAT-process analytical technology enabled production in glycoengineered Pichia pastoris

Production of Functional Anti-Ebola Antibodies in Pichia pastoris.

The 2013-2016 Ebola outbreak highlighted the limited treatment options and lack of rapid response strategies for emerging pathogen outbreaks. Here, we propose an efficient development cycle using glycoengineered Pichia pastoris to produce monoclonal antibody cocktails against pathogens. To enable rapid genetic engineering of P.pastoris, we introduced a genomic landing pad for reliable recombinase-mediated DNA integration. We then created strains expressing each of the three monoclonal antibodies that comprise the ZMapp cocktail, and demonstrated that the secreted antibodies bind to the Ebola virus glycoprotein by immunofluorescence assay. We anticipate that this approach could accelerate the production of therapeutics against future pathogen outbreaks.

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.

Engineering the Pichia pastoris N-Glycosylation Pathway Using the GlycoSwitch Technology.

Pichia pastoris is an important host for recombinant protein production. As a protein production platform, further development for therapeutic glycoproteins has been hindered by the high-mannose-type N-glycosylation common to yeast and fungi. Such N-glycans can complicate downstream processing, might be immunogenic or cause the rapid clearance of the glycoprotein from circulation. In recent years, much effort has gone to engineering the N-glycosylation pathway of Pichia pastoris to mimic the human N-glycosylation pathway. This can be of pivotal importance to generate the appropriate glycoforms of therapeutically relevant glycoproteins or to gain a better understanding of structure-function relationships.This chapter describes the methodology to create such glyco-engineered Pichia pastoris strains using the GlycoSwitch(®). This strategy consists of the disruption of an endogenous glycosyltransferase and the heterologous expression of a glycosidase or glycosyltransferase targeted to the Endoplasmic Reticulum or the Golgi of the host. For each step in the process, we describe the transformation procedure, small-scale screening and we also describe how to perform DNA-Sequencer-Aided Fluorophore-Assisted Capillary Electrophoresis (DSA-FACE) to select for clones with the appropriate N-glycosylation profile. The steps described in this chapter can be followed in an iterative fashion in order to generate clones of Pichia pastoris expressing heterologous proteins with humanized N-glycans.

Real-time monitoring of glycerol and methanol to enhance antibody production in industrial Pichia pastoris bioprocesses

Monoclonal antibody production in glycoengineered Pichia pastoris was optimized for high cell density fed-batch fermentations. Results show that antibody productivity was 1.72-fold higher when maintaining oxygen-limited conditions compared to methanol-limited conditions during the protein production phase of the bioprocess. Under oxygen-limited conditions with a 30mmolL−1h−1 of oxygen uptake rate, an optimum methanol dosage level of 10gL−1 was established by comparing antibody titers under different methanol dosages during protein production. Real-time on-line NIR monitoring of glycerol concentration, methanol concentration, and relative cell density provided valuable data for process characterization and optimization. Real-time profiles of glycerol concentration and relative cell density documented run-to-run consistency during the batch and fed-batch phases by analyses of the glycerol consumption and cell growth kinetics. The volumetric methanol consumption rate was characterized for the induction process following the end of the glycerol fed-batch phase. Under oxygen-limited conditions, the volumetric methanol consumption rate increased over a 60-h period and this increase was independent of the methanol concentration over a range of 2.5–30gL−1 when the oxygen utilization rate was maintained at 30mmolL−1h−1. Utility of the on-line NIR monitor is demonstrated over the one-year period of this investigation by accurately tracking biomass, glycerol concentration, and methanol concentration without user required recalibration as well as by documenting abnormal events during high cell density bioprocesses.

Inactivation of a GAL4-like transcription factor improves cell fitness and product yield in glycoengineered Pichia pastoris strains.

With a completely reengineered and humanized glycosylation pathway, glycoengineered Pichia pastoris has emerged as a promising production host for the manufacture of therapeutic glycoproteins. However, the extensive genetic modifications have also negatively affected the overall fitness levels of the glycoengineered host cells. To make glycoengineered Pichia strains more compatible with a scalable industrial fermentation process, we sought to identify genetic solutions to broadly improve cell robustness during fermentation. In this study, we report that mutations within the Pichia pastoris ATT1 (PpATT1) gene (a homolog of the Saccharomyces cerevisiae GAL4 [ScGAL4] transcriptional activator) dramatically increased the cellular fitness levels of glycoengineered Pichia strains. We demonstrate that deletion of the PpATT1 gene enabled glycoengineered Pichia strains to improve their thermal tolerance levels, reduce their cell lysis defects, and greatly improve fermentation robustness. The extension of the duration of fermentation enabled the PpATT1-modified glycoengineered Pichia strains to increase their product yields significantly without any sacrifice in product quality. Because the ATT1 gene could be deleted from any Pichia strains, including empty hosts and protein-expressing production strains alike, we suggest that the findings described in this study are broadly applicable to any Pichia strains used for the production of therapeutic proteins, including monoclonal antibodies, Fc fusions, peptides, hormones, and growth factors.