Reaching the clinic as quickly as possible is only beneficial if you get there having already established an optimized, robust, and scalable production process. For simple to complex proteins expressed in E. coli, that means working with a CDMO with the capability to develop unique bacterial strains that afford high-quality proteins in high yields. 

Many Challenges to Bacterial Protein Expression

Since the advent of monoclonal antibodies, the focus of the industry has shifted to mammalian cell culture to produce these larger proteins. However, microbial fermentation offers many advantages for smaller proteins, antibody fragments, hormones, and other types of biotherapeutics. Working in bacterial systems is easier, and the results are often better for these biomolecules. In particular, E. coli is a popular bacterial host for recombinant protein production because it grows quickly, is relatively well understood, and can reach high cell densities at a reasonable cost.

It can be challenging, however, to achieve both high titers and high quality for some proteins. Every protein is unique and poses its own unique set of difficulties. It isn’t possible to use just a few different expression vectors or strains to find the optimum solution in every case. Indeed, a wide range of protein attributes — from the gene sequence that encodes a given protein’s primary structure to its secondary and tertiary structures, domains and signal peptide localization, potential disorder zones, solubility, stability, and hydrophobicity — can impact the yields of proteins expressed in bacteria. 

Many proteins can be difficult to express. Others have hydrophobic regions or multiple disulfide bonds that result in aggregation and the formation of insoluble inclusion bodies (IBs). IBs simplify purification, but typically require development of a unique refolding protocol, which adds costs to the development project. Additionally, the refolding strategy itself may be expensive and have low efficiency, resulting in loss of product. As such, IBs present a double-edged sword: they can help to increase titers, but this benefit can be offset by a suboptimal refolding strategy that itself makes it difficult to generate high titers of soluble recombinant proteins with the right structures and biological activities. However, in cases where refolding is intrinsically poor, increased titers can rescue a production process that would otherwise be impractical. Additionally, IBs and proteins that are enzymatically active or that interact with DNA can also be toxic to the host E. coli cells. 

Importance of Avoiding Risk

Without considering the multitude of factors that can impact protein expression in E. coli systems, there is always a risk that the target will not advance from the preclinical to the clinical stage. There are generally two causes of failure at this stage: the expression titers are not sufficient to allow for a viable production process, or the quality of the protein is unacceptable due to aggregation, misfolding, or degradation. In both cases, the production process affords too little of the biologically active protein per fermentation volume.

If misfolding occurs, the process may potentially be salvaged through downstream manipulations, including refolding. However, doing so adds additional steps to the purification process and can be very expensive; it is generally difficult to predict the costs and timelines required to develop a good refolding process. Regardless, the need to perform a refolding step at commercial scale substantially increases the overall cost of goods (COGs).

These issues are not always apparent at laboratory scale, and thus it is possible that a drug developer may be unaware of the significant risk of encountering such problems before they manifest themselves. Since it is essential to be able to quickly and smoothly go from preclinical to clinical development, it is paramount that these issues are avoided. It is therefore essential to develop — from the outset — both a production strain (cell line) and process that produce the protein in sufficient quantity in a biologically active form from small to large scale.

Reducing COGs 

Of course, development timelines and costs are not the only concern. It is also necessary to ensure that the process implemented for commercial manufacturing will have the lowest possible COGs. While it is possible to move through the early phases of development with a bacterial strain and production process that is barely good enough, making changes at later development stages is often prohibitively expensive in terms of both project delays and added costs. As a result, that poor strain and process will generally be used at commercial scale as well, leading to much higher COGs.

It is much more efficient to create an optimal strain immediately to save costs and hassle later on when (hopefully) moving into commercial manufacture. This approach is particularly paramount with biosimilars, for which reducing COGs is absolutely crucial. Being able to manufacture a protein at a smaller scale or in fewer batches can result in much lower production costs, which translates into measurable economic advantage vis-a-vis competitors. 

Even for novel medicines, having a cost-efficient production process is important because there could be competition with other branded therapeutics, price controls may exist in some markets, there might be future competition from biosimilars, and lower COGs benefit patients. They of course also strengthen profitability.

Relatively Simple, but Still Requires Expertise

While protein expression in E. coli is simpler than protein expression in mammalian cells, development of the optimum expression system and process conditions for a specific protein requires deep knowledge of E. coli biology and recombinant protein expression. 

There are many aspects to identifying the optimal E. coli expression system beyond selecting the right strain. The vector system, including promoters; the codons; the stabilizing and solubilizing tags, for both large proteins and smaller peptides; and whether co-expression of molecular chaperones, folding modulators, or fusion partner proteins is needed — all have a direct impact on the titer and quality of the expressed protein. As mentioned above, process parameters (e.g., media, temperature) also play an important role.

Therefore, the bacterial strain, vector, and other aspects of the expression system, as well as the process conditions, must be carefully selected to overcome the challenges associated with difficult-to-express proteins in order to achieve high-quality proteins in high titers at commercial scale. What is most crucial is to have the toolbox of expression elements that allows for the development of an expression vector and strain that facilitates good yields. Absent such a toolbox, most companies rely on a limited set of elements that can only generate a small range of possible variation, or — even less ideal — rely on a single set of parameters that worked well for one protein. However, each protein has unique characteristics, and forcing a square peg into a round hole will not result in an optimal process.

Even possession of such a toolbox of expression elements does not guarantee robust process development without the know-how to combine them optimally for each and every protein. The predictive ability of the expression elements is limited, and as such Vectron’s strategy is a mix of random tests of different expression vectors to determine the best expression strategy for each protein (e.g., strong promoters, tight promoters, low copy number, translocation to the periplasm, tags), combined with more rational engineering (testing of many more elements) once the fundamental strategy has been established. 

Finding the Right Partner

Time flies fast in preclinical development, and many companies have specific dates for when they want to move into clinical development. It can be frustrating to experience production issues at the preclinical stage that delay progress. Those delays can get magnified if a drug developer must switch from one CDMO to another because the original service provider did not have the expertise needed to identify a robust strain and process that could be taken forward. Switching also increases the cost of a project. 

It is much better to partner with a company that is best equipped to solve all of the issues immediately and develop an optimum E. coli expression system and process at the earliest possible development stage.

Vectron Biosolutions Can Help

Vectron Biosolutions is such a CDMO. We have developed technologies that enable us to fine-tune and optimize many aspects of expression vectors, even for hard-to-produce proteins.¹ The technology works well with any protein that can be produced in E. coli, including antibody fragments, enzymes, hormones, and so on. In many cases, we can develop strains and processes that yield protein titers of more than 10 g/L — in one case reaching titers as high as 60 g/L.      

The key to our success is a truly unprecedented toolbox of expression elements — a theoretical vector space of over 5 million potential variants — that allows us to determine the unique combination that enables high yields for each protein. Rather than focus on single systems or solutions that were successful in past projects, we develop a novel, optimized system for each protein from the bottom up. Our technology creates tailor-made, bespoke expression vectors for each and every protein that we work with through an iterative process that is analogous to what occurs in nature. 

In addition, Vectron is developing a technology that enables the secretion of proteins outside of the cell, which is not a typical attribute. Once optimized in E. coli, the production platform will not only be superior for proteins that could already be produced in E. coli but will also enable the manufacture of proteins that would typically be produced in yeast because of the secretion possibilities.

Our approach is also innovative because of our proprietary ultra-high-throughput screening method. Rather than needing to develop a screening method for each protein, which can take months, our universal screen focuses on titers, not activities, and thus works independently of the protein. 

With this technology, we bring value to customers by not just increasing titers, which allows for smaller batch sizes or fewer batches and therefore reduced COGs, but also by improving quality and reducing time to market through development of a robust production strain during preclinical development.

References

1. Aune, Trond Erick Vee. “Unique Vector Expression Technologies for Boosting Protein Production in E. coli.” Pharma’s Almanac. 30 Mar. 2022.