2.1. Specification of Product Requirements
A critical starting point for selecting the right expression system is a clear specification for your product, bioprocess, and commercial requirements. This specification should be comprehensive and include details pertinent to questioning standard assumptions that vendors may not be forthcoming about.
When considering bioprocessing solutions, there can be pressure to immediately opt for a particular expression system based on specific technical details or past experience. However, decades of protein expression and multiple “omics” studies have shown that the factors limiting expression systems are typically different for each specific protein, including even variants of the same protein (Idiris et al., “Engineering of protein secretion in yeast: strategies and impact on protein production”. 2010). Consequently, the first expression system selected may not deliver the required output, especially when scale-up and commercial targets are factored in. To ensure objectivity in the assessment of the solution required, the key commercial and technical parameters should be laid out clearly:
2.2 Product Details
What are the specific details of the required biologic, and what are its Critical Quality Attributes (CQAs), e.g. purity, identity, homogeneity, potency, safety and stability?
2.3. Batch-to-Batch Consistency
To achieve regulatory approval for its intended use, the product must be consistently manufactured within the acceptable limits of product-related and process-related impurities. It must be stable for the required shelf-life, e.g. within limits for degradation and aggregation. It must be highly homogeneous with the correct structure and potency and be proven to be safe, including by immunogenicity and toxicity testing. Because many of these parameters are primarily affected by the production strain, (e.g. correct protein folding, post-translational modifications (PMTs), and host cell protein contaminants), selecting the correct expression platform has far-reaching implications for the entire bioprocessing operation and its economics. For example, the removal of even small amounts of incorrectly processed or modified product-related impurities introduced by a sub-optimal expression host, which are physiochemically challenging to separate from the desired product, can result in significantly lower product recoveries and additional DSP requirements, all of which increase the final CoGs.
2.4. Cost of Goods:
What is the likely competitive landscape, including potential new products entering the market during the anticipated lifespan of the bioprocess, and what does this create in terms of the benchmark required for cost of goods sold to be competitive and achieve the market share and margins needed for a successful product?
2.5. Intellectual Property:
In today’s commercial landscape, intellectual property extends beyond protecting the product by composition of matter patents and includes the potential to block rival manufacturing technologies from being used to produce off-patent protein therapeutics, e.g. biosimilars, thereby delivering significant competitive advantage.
2.6. Manufacturing Plant
What is the intended design for the manufacturing plant? Are the proposed fermentation titre and net yield after downstream processing such that they require an excessively large-scale plant with an unacceptably high level of capital expenditure? Will the proposed bioprocessing solution require high-cost specialist equipment or non-standard downstream processing matrices to overcome upstream issues related to a suboptimal production strain? Does this involve highly skilled operational staff, and are they available at the manufacturing plant’s location? If the plan is to use CDMOs to scale up a process developed elsewhere, do they have the available capacity, equipment, and experienced staff for the proposed project, and is this affordable for the planned development budget and manufacturing costs and available at the optimal time for your clinical program?
Clarity for Objectivity
Clarity on the above parameters creates a more objective grounding for the specification against which expression solutions can be fairly assessed. When genomic engineering-based solutions, such as those offered by CDMOs or synthetic biology companies, are being considered, the above specification allows the track record of first-generation off-the-shelf expression systems lacking full genome optimisation to be placed in proper context.
Irrespective of whether an existing expression host has a track record of manufacturing a particular product type, the important consideration for any new process is whether it will succeed for the new specification in the future manufacturing and market environment. This may mean competition from lower-cost rival product from a fully optimised bioprocess with a pricing advantage that traditional manufacturing methods cannot match. Full genome optimisation to make production strains bespoke to these products thereby creates opportunity for biobetters to enter markets and outcompeting less ambitious products.
Proper consideration of the points raised above will lead to a strong foundation for selecting the most appropriate technical solutions for the manufacturing process, including:
- The optimal expression host system and strain genotype.
- A stable plasmid system with a high copy number and selection system that is amenable for sustainable manufacture.
- A final expression construct, e.g. comprising a bespoke combination of promoters, leader sequences (for secreted products only), codon usage, terminators, and other regulatory elements.
- A scalable fermentation regime with appropriate media ingredients and supply.
- Affordable DSP steps offering maximum recoveries and meeting target product profiles, including for host cell protein and endotoxin risks.
Evaluate against the future commercial landscape not the present or past
In certain cases, the solutions offered by in-house teams or existing vendors may be adequate for your product. However, where this falls short, fully optimised solutions may be required to meet future market conditions significantly different from those that have prevailed in the past. The development risk in terms of financial cost is borne by the project owner, not the supplier. There is also an opportunity cost associated with the project should it fail, where other projects might have been successful. Failure can be fatal for the project with dire consequences for the product owner, especially when there is insufficient time and budget to try an alternative. Additionally, once the development of one manufacturing solution has started for regulatory purposes, it is incredibly disruptive and potentially costly to alter the approach. Late-stage changes may require new clinical trials, bridging studies and regulatory approval approaches. In other words, the costs and implications of making the incorrect selection of expression system might not be realised until it is too late.
The ceiling of genetic engineering
Underlying this is the reality that all the existing solutions, including synthetic biology, are based on genetic engineering approaches. Genetic engineering has inherent limitations for solving the more complex problems associated with bioprocess optimisation that the purveyors of existing solutions are unlikely to highlight and may not be made aware of. These limitations often relate to ceilings on fermentation titre, controlling post-translational modifications, downstream processing efficiency, and scalability. This is especially relevant when the ideal solution requires the optimisation of complex phenotypes controlled by multiple genes, or multiple phenotypes require simultaneous optimisation, and the number of permutations exceeds what can be practically achieved by traditional genetic engineering techniques.
The advent of full genome optimisation technology has removed such limitations for a significant portion of biologics. Therefore, when selecting manufacturing solutions, it needs to be considered whether rival products may deploy such technologies and how the competitive advantages of the resultant products are likely to change the future market landscape.
