We can identify many challenges that must be met if we are to have an effective and profitable biotech industry. Increasing process productivity and reducing cost-of-goods are easy to identify as ongoing improvement targets, but of equal importance is the need to reduce the time lines for development of new treatments.
As an industry we have to become faster at moving projects into the clinic and then better at avoiding work repetition or recycling certain aspects of development because of incorrect process choices. Choices and decisions made early in development profoundly impact cost and time lines, and ultimately reduce value for all stakeholders.
Nowhere is the drive to achieve increased production titers more evident than in the monoclonal antibody field. New systems have been introduced that deliver grams per liter of product, and some companies are now reportedly reaching ten grams per liter. It will take some time for these performance levels to translate into profit and cost benefits, but the direction is clear. Antibodies, however, have one huge process advantage in that the purification strategies required are largely conserved from one product to the next.
But does the same benefit apply to biologics that fall outside the monoclonal antibody family? For non-mAbs, the world does look different. The products themselves have huge variety: enzymes, cytokines, interferons, growth factors, vaccines, various formats of fusion proteins, antibody fragments, and more. The target therapies and hence product characteristics vary enormously, leading to different purity, cost-of-goods, and volume requirements. In this market sector microbial, rather than cell culture, manufacturing is at the forefront introducing a different set of processing paradigms.
Due to these factors, processing strategies for products tend to be customized, both upstream and downstream. A wide range of process options have to be considered when commencing a development program covering expression vectors, including host strains, fermentation conditions, whether the product will be accumulated intracellularly as inclusion bodies or in a soluble form or secreted (periplasmic or extracellular), and the different downstream approaches that follow.
As our experience with microbial biologics has grown, so has our belief in the need to take an holistic approach to process development. As soon as we move away from monoclonal antibodies, product variability and thus the need for customized solutions becomes more clear. It isn’t ever simply a case of maximizing fermentation titer and everything else will be fine. Striving for high grams per liter is vital but it is not the full story.
Construction of the production strain and establishment of the fermentation production process are critical early points within process development. It is important to make the right choice of production system in order to avoid problems during clinical development.
Development of pAVEway™
The choice of expression vector, host organism, strain construction, and fermentation and purification strategies has to be viewed, and addressed, in an integrated approach and aligned with the specific characteristics of the product. At Avecia (www.avecia.com), we sought to deliver an approach that:
• Minimized the time needed to establish a process suitable for development, starting from a gene sequence
• Enabled a rapid comparison of process options
• Established the basis for a high-productivity process early in development
• Provided a sound basis for future scale up and validation
• Offered versatility to meet the demands of different products
• Used well-characterized systems to underpin regulatory confidence
The pAVEway platform is based on a set of unique protein-expression plasmids that have been developed by Avecia. Using a novel configuration of operators, promoters, and repressors, we have created a range of vectors that provide tightly controlled production of the target proteins while allowing high expression levels.
However, these vectors are also complemented by a range of selected and proven host strains with well-developed and -characterized high-titer fermentation strategies. By choosing the appropriate combination of plasmid, host strain, and fermentation conditions, high titers have been demonstrated for soluble, insoluble, and secreted proteins.
Our key belief is that no single system or approach will meet all demands. A development program has to be able to consider and explore most major options, and pAVEway allows us to narrow the scope of such work.
E. coli is the undisputed workhorse of our industry, well accepted by the regulators and capable of delivering high productivity processes. However, it is worth bearing in mind that not all E. coli vectors and host strains are the same. Selectivity here is also an essential prerequisite to successful development.
Many preexisting systems are limited in their ability to perform at large-scale because of “leakiness”, or high basal expression of the target protein in the absence of inducer. This can impact the overall performance, particularly in situations where the product has a negative impact on the host organism.
With pAVEway, the basal expression level can be fully suppressed so that no target protein is produced before induction or after prolonged incubation in the absence of inducer addition. This allows fermentations to achieve high cell densities with cells maintaining an optimum protein synthetic capacity resulting in high volumetric titer even for proteins that would normally impair the growth of the host.
The second key characteristic of pAVEway is its ability to modulate expression levels in an almost near-linear manner by varying the concentration of inducer added to the culture (Figure 1). Maximum expression rate is not always the best solution. Expression kinetics can thus be tightly regulated to match protein synthetic capacity, secretion capacity, or the protein-folding capabilities and capacity of the host cell, enabling, for example, the soluble accumulation of proteins of high folding complexity.
The application of pAVEway to many different proteins is a vital feature for any microbial platform. While E. coli is undoubtedly the first choice for microbial biologics, there are clearly occasions when alternative hosts can bring additional benefits.
There are situations with complex products where yeasts such as Pichia can bring tangible benefit. With some such products now late in development is it unlikely to be long before the regulatory track record starts to build impressively. Other bacteria such as Pseudomonas may also have utility, and there is evidence that some proteins expressing as inclusion bodies in E. coli can be made more effectively in Pseudomonas in a soluble state.
This is an area needing further study and better, more robust like-for-like comparisons, but we have shown that the pAVEway system is equally applicable in this host. Data achieved with enzymes and growth factors has shown delivery of yields in excess of ten grams per liter of active protein.