Another great day of talks, the sessions this year have been excellent and I am not the only one who thinks so. Many of the sessions have been completely full. Today I chose the following three talks to highlight.
James Brooks, Ph.D., R&D Manager, BD Biosciences-Advanced Bioprocessing, gave a talk titled “Chemically Defined Media Optimization: Challenges and Solutions.”
Dr. Brooks provided an interesting background as to where we have been with media and where we are at now. In addition he provided two interesting case studies from media optimization projects at BD Biosciences including, steps taken, challenges faced and solutions discovered. I will summarize one of the case studies below.
Dr. Brooks presented his thoughts on a new or re-emerging paradigm of optimizing media for a particular cell line, clone and process to meet the increased need for reduction in process times and to increase speed to market.
In the talk he identified the following reasons for optimizing media:
- No universal medium – each cell line/clone has different nutritional requirements and genomic diversity
- Removal of components – continued desire to remove animal derived and hydrolysate components
- Enhanced culture performance – product yield and product quality
Case Study 1
In the first case study presented by Dr. Brooks, the goal was to develop a chemically defined medium and feed strategy to enhance protein production two-fold. The cell line was CHO K1. BD Biosciences then performed the following steps for optimization.
- Chemically defined library media screen to select media to identify media with at least 150% or more compared to control production.
- Chemically defined library media scale up of selected media, looking at growth production and viability. They selected two to carry forward.
- Bioreactor optimization and feed evaluation. With the addition of a feed strategy they were able to increase production six-fold. Based on these results they selected one media to move forward.
- Conducted “CHO Flow” Viability Evaluation to check for apoptosis.
- Bioreactor performance confirmation with a benchtop bioreactor where they met goal of two-fold increase without feed and when feeds were added achieved six-fold increase in production.
- mAb quality analysis – glycosylation profile and confirmation that quality was maintained.
Veronique Chotteau, Ph.D., Principal Investigator, KTH – Royal Institute of Technology, Sweden, gave a talk titled “Perfusion of IgG Producing Chinese Hamster Ovary Cells by Alternating Tangential Flow Filter at Very High Cell Density.”
In the talk Dr. Chotteau discussed a study in which the goal was to develop perfusion in a disposable WAVE bioreactor and a stirred tank bioreactor to evaluation ATF and TFF and test the limits of the system with respect to cell density. The study used CHO cells producing IgG and the cell densities achieved were quite remarkable. These high cell density cultures have many applications in bioprocessing.
The WAVE bioreactor was tested in perfusion mode with both ATF and TFF and very high cell density was achieved in both. Max cell density of 132 x 106 cells/ml was reached using ATF. However, WAVE with ATF was challenged at very high cell densities due to pressure limitation to push highly viscous fluid. Max cell density of 200-230 x 106 cells/ml was reached using TFF. Cell viability was very good in both with viability equal to or greater than 90%, mostly around 95%. There was comparable cell growth between ATF and TFF.
Next the study was conducted in a stirred tank bioreactor with working volume of 1 liter, comparing ATF and TFF. Cell density was stabilized at 20 x 106 cells/ml by daily cell bleeds.
One interesting finding of the study was that there was smaller cell diameter at such high cell density and when distance between cells becomes too small i.e. 2 micrometers, cells shrink. They found that 250 x 106 cells/ml was the limit for 16 micrometer diameter cells.
- Very high cell density of 100 x 106 cells/ml were stable and maintained
- 200 x 106 cells/ml in stirred tank with ATF or in WAVE with TFF were successfully achieved. WAVE with ATF was limited by high viscosity
- Applicable limit for cell density in suspension depends on cell diameter and equipment
- Not clear on the impact of cell shrinking so perhaps best to avoid shrinking cells
Applications for use of high cell density cultures include:
- Seed bioreactor
- Production bioreactor
- Rapid non-optimized production of protein for exploratory research
- Cell expansion for cell banking
Mireli Fino, Vice President, Manufacturing Operations, Protein Sciences Corporation, gave a talk titled “Flublok: Developing the World’s First Recombinant, Highly Purified, Egg-Free Influenza Vaccine
Ms. Fino gave a very interesting talk about the Flublok technology and also the journey to receiving approval. Protein Sciences uses a Baculovirus Expression Vector System (BEVS) to produce Flublok. They have a pilot facility in Meriden, Connecticut, which runs a 500 liter bioreactor and can produce 250,000 doses of Flublok per year. They also have a large scale manufacturing facility in Pearl River, New York where they use 2,000 liter bioreactor that can produce 2-5 million doses/year of Flublok. Their technology enables them to manufacture 50 million doses of pandemic flu vaccine within six months.
BEVS begins with engineering the baculovirus with the gene of interest using a powerful promoter that generates high yields. Insect cells are then grown in fermenters and infected with engineered virus. A serum-free media is used for culture. Then protein is purified and formulated with PBS into influenza vaccine.
During the talk the following advantages were given for use of BEVS:
- Safe eukaryotic cells
- Fast and flexible manufacturing
- High expression level particularly with large proteins
- Scale proven up to 20,000 liters