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Flexible Microbial Manufacturing. BioPharmaceutical Microbial Fermentation. The BioPharm industry has good selection of bacterial and yeast recombinant expression platforms. Companies have made signinficant investments in production strains to meet their in house requirements.
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BioPharmaceutical Microbial Fermentation • The BioPharm industry has good selection of bacterial and yeast recombinant expression platforms. • Companies have made signinficant investments in production strains to meet their in house requirements. • Merck purchased the glyco-modified Pichia pastoris system from Glycofi. • Amgen uses an E. coli platform that is designed specifically to produce inclusion bodies. • Very scalable technology • Scaling-up to 10,000s L is well documented in Industry • Scaling-down to mLs with similar productivity at the 100 L -1,000 L scale is in it’s infancy. • Established fermentation control strategies exist, though they tend to be protein specific. • Microbial physiology and metabolism is understood, but we need to better understand and enhance recombinant protein production metabolism.
Microbial Fermentors • Stainless steel pressure vessel • High oxygen transfer capabilities • High heat transfer capabilities • On-line sensors • Standard probes: pH, temp, dissolved oxygen, level, pressure, anti-foam. • Specialty probes: OD, NIR (Acetate, protein, ethanol, methanol, glucose), redox • Off-gas mass spectrometer: OUR, CER and RQ, methanol, ethanol. • Very flexible systems
Applikon Micro-Bioreactors DASGIP Parallel Bioreactor Bioengineering Bioreactor Flexible Microbial Fermentation • Current SS fermentors are very flexible and can handle just about any microbial fermentation. • Several companies have developed scaled-down microbial fermentation systems that are capable of high throughput screening of clones and media optimization and fermentation optimization studies. • The microbioreactor systems are well suited for screening clones using a defined fermentation process. • Fermentation optimization is typically left to stirred tanks…DASGIP and larger…
How Does One Go From the Desired Microorganism to High Output Capabilities? • Once a recombinant strain produces “enough” product to satisfy Phase I, the objective is ONLY Phase I studies. • The highest producing strain(s) are 1% or less of the total number clones. • Fermentation optimization can increase productivity 2 to 3 fold. • Strain optimization can increase productivity 10 to 20 fold. • Strain development is the most critical step. • What is the ideal production strain? • Do we create a whole new production organism…a new species…???
High Throughput Process Development - HTPPD • What is HTPPD? • A process that is ready to produce Phase I material in….weeks??....day???....hours???? • The current model is months. • Fermentation is just one step in the process…but it can significantly impact downstream processing. • Purification is dance. The greatest challenge is purifying closely related degradation products. • Do not forget about all of those analytical methods that are needed for in-process, release testing and characterization…Often times can take the longest amount of time to put everything in place.
Miniaturizing The Fermentor • The fermentation group can never have enough tanks. • HTP robotic system exist for cell culture for clone and media selection. We need one for microbial production systems. • The objective is to find the right combination of media and operating conditions the maximizes product quantity and quality…the most important attribute! • Fermentor miniaturization is down to the 5 -10 mL volume, with pH and DO control and some addition…but still a long ways from being identical in performance to 1 L fermentor. • What volume or how many cells do we need to be able to match cell productivity at the 100, 1000 and 10,000 L scale.
Product Recovery and Capture • For a secreted product, recovery and capture is relatively straight forward…clarify and chromatography. • Some proteins are best produced intracellularly. • Chemical methods of cell disruption are amenable to microtiter plates. • Mechanical disruption requires very high pressures…can this be miniaturized for HTP screening of disruption conditions. • HTP protein folding optimization has occurred in microtiter plates. • What about HTP ultra high pressure protein refolding.
HTP Protein Purification • The UNL BPDF has 6 BioCad systems. We can generate more samples than we have the capacity to analyze. • We use 96 well plates to screen resins followed by dynamic studies. • Our favorite analytical tool is our ESI-TOF which has mass accuracy down to 2 ppm. The best process development tool we have. • Can we miniaturize all three into a single system that can evaluate 100s to 1000s of chromatographic separation…using scalable resins or technology.
Release Testing • Multiple instruments are required for the 10 to 15 different release tests for the BDS. • There has not been a demand for miniaturization of the release testing…yet. • Each of the methods must be qualified and validated. • Cell based assays are the most labor intensive and difficult of the assays. • Do we need to take another look at what information we really need to safely release a product given the analytical technology available today.
