Microbial fermentation Process development
Microbial Fermentation Process Development
Our goal during upstream microbial fermentation process development is to develop high titer, scalable production processes that will express our customer’s target molecules efficiently with long term commercial production in sight.
The Process Development team can generate and qualify scale down-models and fully characterize process parameters in support of process validation.
We have modern facilities with access to state- of- the art equipment. With over 40 fermentors (1-100L) as well as ambr ™ systems (24x 250ml and 48x 15ml), FUJIFILM’s scientists develop and optimize batch and fed batch processes for microbially expressed products.
The Process Development team can generate and qualify scale down-models and fully characterize process parameters in support of process validation. Extensive use is made of DOE approaches, with the support of in-house statisticians.
Upstream Process Development
- Process Transfer-in and Development
- Process monitoring
- Recovery of Solids or Soluble Material
- Cell Disruption
- Supply & Toxicology Study Supply Batches
- Resin screening
- Intermediate stability UF/DF development
- Protein refolding
- Protein modifications
- Pegylation, hapten conjugation, enzymatic cleavage
- Set processing targets
- Process characterization
- Scale down models
Process Development – based on E. coli
E. coli is a tried and true expression platform for biologics. E. coli is usually chosen based on productivity. You can potentially get up to 20g/L , although titers of about 8 g/L are typically seen.
In our experience, E. coli is also preferred for some classes of molecules that interact with mammalian expression systems. These include growth factors, cytokines and interferons.
Advantages of E. coli expressions include:
- Fast processing: Plant time is short even if you are accounting for recovery steps. This can have an impact on cost but also minimizes the contact time with manufacturing systems.
- Flexibility: Microbial E. coli systems allow for processing with natural decoupling steps (cell paste for intracellular soluble and inclusion bodies). This increases the opportunities to assign manufacturing slots in a multi-product facility. It also allows for staggering of readiness activities which can decrease time to manufacture.
- Cost effectiveness: Materials can be more cost effective and are less blocked by proprietary issues, (i.e. cell culture feeds). In addition, most formulations are made from individual components that are well controlled and understood.
The challenges of working with E. coli
E. coli post-translational modifications are limited if not non-existent. Unlike chinese hamster ovary (CHO) cells that are able to produce humanized recombinant proteins with similar post-translational modifications, E. coli can offer very few chemical changes after translation.
Another challenge involved in E. coli process development is getting the product out of the cell. Lysis may not be enough and an additional processing step is often required. In our experience, high-pressure homogenization of the cells at about 800 bar solves the problem and break open the cells. This releases the product and ensures that the whole batch is homogenized and equal in composition, but it also creates a “soup of protein” to clean up. It also introduces heat into the culture that may induce the release proteases that in turn might degrade the therapeutic protein. Finally, it introduces one or more.
With inclusion bodies however, homogenization and IB washing is a very quick and efficient way to get a purity of 90% or higher in a simple step. The disadvantage being only that we need to use refolding techniques to recover the correctly folded proteins from the inclusion bodies.
Process Development – based on yeast
Yeast-based expression systems, using Pichia pastoris or Saccharomyces cerevisiae hosts for the expression of your recombinant therapeutic proteins, can sometimes provide an elegant solution in terms of obtaining expression levels at several grams per liter of soluble, active protein. High cell densities and good volumetric productivities can be achieved with more favorable batch times and a lower cost of goods (COGs) compared to mammalian expression systems.
The challenges of working with Yeast
Yeast based expression systems can have a propensity to express proteins with clipped N-terminals due to the presence of endogenous proteases. In our experience, this can be highly protein dependent and sometimes difficult to predict.
Saccharomyces sp. in particular can also produce non-native (hyper-mannosylated) glycoproteins.
For each separate case, careful consideration of the pros and cons of yeast-based expression is necessary – but the rewards can be considerable.
We bring experience and a long track record to your Yeast expressed program
FUJIFILM Diosynth Biotechnologies has extensive experience with yeast expression. Especially P. pastoris expression systems, and in particular with those based on the AOX1 promoter. These expression system provides an attractive option for the production of many proteins, and complements our world leading experience in the process development, scale up and commercialization of yeast-based processes for the production of biopharmaceuticals.
Our process development experience includes working with MutS, Mut+ strain, secreted and intracellular expressed proteins. We also have long experience of performing in-depth product characterization to assess product quality.