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Evaluation of butyrate-induced production of a mannose-6-phosphorylated therapeutic enzyme using parallel bioreactors.

ABSTRACT: Bioreactor process changes can have a profound effect on the yield and quality of biotechnology products. Mannose-6-phosphate (M6P) glycan content and the enzymatic catalytic kinetic parameters are critical quality attributes (CQAs) of many therapeutic enzymes used to treat lysosomal storage diseases (LSDs). Here, we have evaluated the effect of adding butyrate to bioreactor production cultures of human recombinant β-glucuronidase produced from CHO-K1 cells, with an emphasis on CQAs. The β-glucuronidase produced in parallel bioreactors was quantified by capillary electrophoresis, the catalytic kinetic parameters were measured using steady-state analysis, and mannose-6-phosphorylation status was assessed using an M6P-specific single-chain antibody fragment. Using this approach, we found that butyrate treatment increased β-glucuronidase production up to approximately threefold without significantly affecting the catalytic properties of the enzyme. However, M6P content in β-glucuronidase was inversely correlated with the increased enzyme production induced by butyrate treatment. This assessment demonstrated that although butyrate dramatically increased β-glucuronidase production in bioreactors, it adversely impacted the mannose-6-phosphorylation of this LSD therapeutic enzyme. This strategy may have utility in evaluating manufacturing process changes to improve therapeutic enzyme yields and CQAs.

SUBMITTER: Madhavarao CN

PROVIDER: S-EPMC10723619 | biostudies-literature | 2014 Mar-Apr

REPOSITORIES: biostudies-literature

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Evaluation of butyrate-induced production of a mannose-6-phosphorylated therapeutic enzyme using parallel bioreactors.

Madhavarao Chikkathur N CN Agarabi Cyrus D CD Wong Lily L Müller-Loennies Sven S Braulke Thomas T Khan Mansoor M Anderson Howard H Johnson Gibbes R GR

Biotechnology and applied biochemistry 20140220 2

Bioreactor process changes can have a profound effect on the yield and quality of biotechnology products. Mannose-6-phosphate (M6P) glycan content and the enzymatic catalytic kinetic parameters are critical quality attributes (CQAs) of many therapeutic enzymes used to treat lysosomal storage diseases (LSDs). Here, we have evaluated the effect of adding butyrate to bioreactor production cultures of human recombinant β-glucuronidase produced from CHO-K1 cells, with an emphasis on CQAs. The β-glucu ...[more]

PMID: 24033810

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Project description:Since 1997, the highly pathogenic influenza H5N1 virus has spread from Hong Kong. According to the WHO bulletin report, the H5N1 virus is a zoonotic disease threat that has infected more than 850 humans, causing over 450 deaths. In addition, an outbreak of another new and highly pathogenic influenza virus (H7N9) occurred in 2013 in China. These highly pathogenic influenza viruses could potentially cause a worldwide pandemic. it is crucial to develop a rapid production platform to meet this surge demand against any possible influenza pandemic. A potential solution for this problem is the use of cell-based bioreactors for rapid vaccine production. These novel bioreactors, used for cell-based vaccine production, possess various advantages. For example, they enable a short production time, allow for the handling highly pathogenic influenza in closed environments, and can be easily scaled up. In this study, two novel disposable cell-based bioreactors, BelloCell and TideCell, were used to produce H5N1 clade II and H7N9 candidate vaccine viruses (CVVs). Madin-Darby canine kidney (MDCK) cells were used for the production of these influenza CVVs. A novel bench-scale bioreactor named BelloCell bioreactor was used in the study. All culturing conditions were tested and scaled to 10 L industrial-scale bioreactor known as TideCell002. The performances of between BelloCell and TideCell were similar in cell growth, the average MDCK cell doubling time was slightly decreased to 25 hours. The systems yielded approximately 39.2 and 18.0 μg/ml of HA protein with the 10-liter TideCell002 from the H5N1 clade II and H7N9 CVVs, respectively. The results of this study not only highlight the overall effectiveness of these bioreactors but also illustrate the potential of maintaining the same outcome when scaled up to industrial production, which has many implications for faster vaccine production. Although additional studies are required for process optimization, the results of this study are promising and show that oscillating bioreactors may be a suitable platform for pandemic influenza virus production.

Project description:BackgroundMicrobial-driven solubilization of lignocellulosic material is a natural mechanism that is exploited in anaerobic digesters (ADs) to produce biogas and other valuable bioproducts. Glycoside hydrolases (GHs) are the main enzymes that bacterial and archaeal populations use to break down complex polysaccharides in these reactors. Methodologies for rapidly screening the physical presence and types of GHs can provide information about their functional activities as well as the taxonomical diversity within AD systems but are largely unavailable. Targeted proteomic methods could potentially be used to provide snapshots of the GHs expressed by microbial consortia in ADs, giving valuable insights into the functional lignocellulolytic degradation diversity of a community. Such observations would be essential to evaluate the hydrolytic performance of a reactor or potential issues with it.ResultsAs a proof of concept, we performed an in silico selection and evaluation of groups of tryptic peptides from five important GH families derived from a dataset of 1401 metagenome-assembled genomes (MAGs) in anaerobic digesters. Following empirical rules of peptide-based targeted proteomics, we selected groups of shared peptides among proteins within a GH family while at the same time being unique compared to all other background proteins. In particular, we were able to identify a tractable unique set of peptides that were sufficient to monitor the range of GH families. While a few thousand peptides would be needed for comprehensive characterization of the main GH families, we found that at least 50% of the proteins in these families (such as the key families) could be tracked with only 200 peptides. The unique peptides selected for groups of GHs were found to be sufficient for distinguishing enzyme specificity or microbial taxonomy. These in silico results demonstrate the presence of specific unique GH peptides even in a highly diverse and complex microbiome and reveal the potential for development of targeted metaproteomic approaches in ADs or lignocellulolytic microbiomes. Such an approach could be valuable for estimating molecular-level enzymatic capabilities and responses of microbial communities to different substrates or conditions, which is a critical need in either building or utilizing constructed communities or defined cultures for bio-production.ConclusionsThis in silico study demonstrates the peptide selection strategy for quantifying relevant groups of GH proteins in a complex anaerobic microbiome and encourages the development of targeted metaproteomic approaches in fermenters. The results revealed that targeted metaproteomics could be a feasible approach for the screening of cellulolytic enzyme capacities for a range of anaerobic microbiome fermenters and thus could assist in bioreactor evaluation and optimization.

Dataset Information

Evaluation of butyrate-induced production of a mannose-6-phosphorylated therapeutic enzyme using parallel bioreactors.

Publications

Evaluation of butyrate-induced production of a mannose-6-phosphorylated therapeutic enzyme using parallel bioreactors.

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