Process Development

Microbial 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.
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 ACTIVITIES INCLUDE:

  • Process Transfer-in and Development
  • Process monitoring
  • Recovery of Solids or Soluble Material
  • Cell Disruption
  • Supply & Toxicology Study Supply Batches

DOWNSTREAM DEVELOPMENT ACTIVITIES INCLUDE:

  • Resin screening
  • Intermediate stability
  • UF/DF development
  • Protein refolding
  • Protein modifications
  • Pegylation, hapten conjugation, enzymatic cleavage
  • Set processing targets
  • Process characterization
  • Scale down models

E. coli Process Development
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 ~8 g/L is a more typical titer. In our experience, E. coli is also preferred for some classes of molecules that could interact with mammalian expression systems. These include growth factors, cytokines and interferons.
ADVANTAGES 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 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.
  • Materials can be more cost effective and are less blocked by proprietary issues, (i.e., cell culture feeds). Most formulations are made from individual components that are controlled and understood.

What are particular challenges of working with E. coli?
E. coli post-translational modifications are limited if non-existent. Another one of the challenges with E. coli lies with getting product out of the cell. In our experience we have found that we may have have to homogenise to break open the cell (i.e., typical pressures ~800 bar). This has the disadvantage as it’s another processing step, it makes a “soup of protein” to clean up, puts heat into the culture, and may release proteases that might degrade the therapeutic protein.
If we express as inclusion bodies however, then homogenization and IB washing is a very quick and efficient way to get 90% plus purity in a simple step. Clearly however, the disadvantage with this is we need a refold post this step. At FUJIFILM Diosynth we have extensive experience and solutions to address these challenges.
What do we bring to your E. coli program?
One of our biggest differentiators as an E. coli CDMO is our pAVEway ™ expression platform. It is a tried and tested expression platform with over 100+ pAVEway studies completed. We have achieved significant titers in fully defined growth media, therefore robust and consistent. We also offer our customers with a well developed fermentation system via the ambr250, Caliper, and TECAN. This system has been proven to scale up to our cGMP Manufacturing facilities. In some instances, E. coli manufacturing could have larger barrier-to-entry with substantial utility and equipment requirements.
Yeast Process Development 
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 cost of goods (COGs) compared to mammalian expression systems.
What are particular 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 but this can be highly protein dependent and sometimes difficult to predict. Saccharomyces sp. in particular can also produce non-native (hyper-mannosylated) glycoproteins. FUJIFILM Diosynth Biotechnologies has extensive experience in performing in depth product characterization to assess product quality. Careful consideration of the pros and cons is necessary but the rewards can be considerable.
What do we bring to your Yeast expressed program?
Our process development experience includes working with MutS, Mut+ strain, secreted and intracellular expressed proteins.
FUJIFILM Diosynth Biotechnologies has extensive experience with P. pastoris expression systems, 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.