IE Case Study - rHuPH20 Bioreactor Optimization Applied Research

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Recombinant Human Hyaluronidase (rHuPH20) Bioreactor Optimization utilizing Amino Acid Analysis and Supplementation

Nov 2018

An important focus of BioProcess Development is to improve product yields and minimize cost of goods while maintaining product quality. 

Recombinant human hyaluronidase enzyme (rHuPH20) is expressed using a Chinese Hamster Ovary (CHO) cell line in a fed-batch bioreactor process. 

The current manufacturing process uses a chemically defined basal media and complex feeds consisting of carbohydrates, amino acids, lipids, growth factors, vitamins, etc. 

 To further improve bioreactor performance, as measured by an increase in Integral Viable Cell Density (IVCD) and productivity (enzyme titer), spent media analysis was used.   This analysis revealed that 3 amino acids in particular (asparagine, cysteine, and tyrosine) were either depleted or reduced during the early stages of the 14 day process. Using a Design of Experiment (DOE) approach, optimal concentrations of each of the 3 amino acids were assessed to increase cell growth and enzyme titer, while reducing waste product production (eg, lactate). Subsequent amino acid supplementation studies using a mini-bioreactor system at a fixed concentration for the 3 depleted amino acids led to an overall increase in the bioreactor IVCD by approximately 68% and enzyme titer by 90%, while decreasing cell specific lactate production by 45%.

The  work  demonstrated the effectiveness of spent media amino acid analysis to optimize amino acid supplementation of the cell culture feed media to improve the cellular growth and metabolic profile as well as increase productivity in a cell culture process.

Method: 

Cell culture intermediate retains obtained from a commercial production 300L Bioreactor were analyzed for amino acid concentrations. This informed the design of a screening study in 500 mL shake flasks varying the concentrations of asparagine, cysteine, and tyrosine. Based on the results of the screening study, 2 optimal conditions were identified and tested on a 250 mL mini-bioreactor along with a control condition. 

Measurements: Cell growth parameters (e.g. viable cell density) were measured using an automated cell counter, while cell metabolites (e.g. lactate) were quantified using a biochemical analyzer. Enzyme activity levels for rHuPH20 were quantified using a micro-turbidity method, and amino acid concentrations were quantified by Ultra Performance Liquid Chromatography (UPLC) analysis.

Results:  Spent media analysis  revealed that asparagine and cysteine are depleted by day 5-7, while tyrosine is reduced by approximately 40%.

In the initial screening study, a statistical trend of the impact of additional asparagine, cysteine, and tyrosine on integral viable cell density (IVCD), harvest cell viability, titer, specific productivity, and specific lactate production  demonstrated that cysteine had the most significant impact on overall bioreactor performance with modest gains from varying concentrations of asparagine and tyrosine.

Using a combination of asparagine, cysteine and tyrosine (condition C), IVCD increased by up to 68% while a 45% reduction in cell specific lactate production was observed. Along with increased cell growth, product yield significantly improved by up to 90% while maintaining specific productivity.

Conclusion: Bioreactor spent media analysis is a valuable tool and provides critical information on amino acid consumption that can be used to optimize individual components. Supplementation of depleted amino acids in the bioreactor manufacturing process significantly increases IVCD (68%) and productivity (90%), while decreasing specific lactate production (45%). Collectively, these improvements reduced the manufacturing cost (CoG).

Presenting Author, Main Author, Primary/Principal Investigator

Dan Nguyen

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– Sr. Process Development Associate, Halozyme, San Diego, California

https://www.eventscribe.net//2018/PharmSci360/fsPopup.asp?efp=UUFSQlZZVFM1OTQ2&PosterID=165894&rnd=0.1926764&mode=posterinfo

Patent - EP2674487A2 European Patent Office

Large-scale production of soluble hyaluronidase

Abstract

Provided are methods for preparing large-scale preparations of soluble hyaluronidases. The methods employ cells that contain a plurality of active copies of nucleic acid encoding the soluble hyaluronidase and a plurality of feedings and temperature changes, whereby the encoded soluble hyaluronidase is secreted into the cell culture medium.

https://patents.google.com/patent/EP2674487A2/en

A harvested cell culture fluid, comprising soluble rHuPH20 with an enzymatic activity that is greater than 5000 units/mL as harvested, wherein:

soluble rHuPH20 is a soluble form of human PH20 that is recombinantly expressed in Chinese Hamster Ovary (CHO) cells;

hyaluronidase enzymatic activity is measured in a hyaluronidase turbidometric assay and compared to a standard curve;

the harvested cell culture fluid is the fluid as separated from cells, cell debris and aggregates without further purification or concentration.

Recombinant human hyaluronidase (rHuPH20): an enabling platform for subcutaneous drug and fluid administration

Expert Opin Drug Deliv

. 2007 Jul;4(4):427-40. doi: 10.1517/17425247.4.4.427.

https://pubmed.ncbi.nlm.nih.gov/17683255/

Case Study - rHuPH20 Bioreactor Optimization