BioPharm: What parts of the protein expression process are still rate-limiting, and how do you see these challenges being addressed?
What improvements do you expect to see in the future?
Squires: We anticipate that given the continued need for protein supply across the spectrum of applications, many of the existing
weak links in protein expression will be strengthened. One of the most pressing unmet needs is production of small amounts
of protein for research purposes. Often, dozens of proteins ranging from antigens for preclinical testing to related variants
of a target protein for some form of differential testing are required. While only a small amount of protein, a few mg, is
generally required for activity evaluation or crystallization for these types of studies, currently all of the cloning steps
needed to get even that small amount of protein are identical to those required to assemble a strain or cell line for large-scale
biopharmaceutical production. This is highly inefficient and a consistent bottleneck in the process. Clearly, there is a need
to develop off-the-shelf solutions to fill this gap. New, precise, and more adaptable tools allowing more rapid cloning and
characterization will need to be developed. These tools will necessarily include the development and adoption of more effective
high throughput processing methods. Improvements in genetic, analytical, and processing methods practiced in high throughput,
parallel modes will enable the researcher to access more tools and reach a wider range of solutions more rapidly.
Clearly, for these approaches to have an impact, methods to allow the researcher to identify productive clones at the earliest
possible stage must be developed. Better reporter systems that may enable screening, even at the colony or single cell level,
would be very valuable. Processing to obtain small amounts of protein would then benefit from the development of more efficient
purification tags. These may also be envisioned to include reliable expression reporter sequences, sequences that promote
proper protein folding, and ideally be efficiently and universally removable. Pfenex is exploring this segment, adapting its
expression platform strategy specifically for custom reagents. Development in this area would be of great benefit not only
to the core of researchers producing proteins for research purposes but the benefits would be amply felt in the area of target
protein expression as well.
Another route to solve the problem of small-scale supply would be to adopt a cell-free approach such as that employed by Sutro
Biopharma. Such an approach is enabled today by the relatively rapid, commercial availability of synthetic, gene-length DNA
sequences. Developing an off-the-shelf cell-free system would jump the cloning and cell growth steps in the current processes
and potentially deliver advantages of protein quality as well. An in vitro system would also be able to take advantage of the reporter and purification technologies mentioned above for in vivo systems.
As more and more dimensions to protein production are added, the researchers' ability to deliver will continue to improve.
The challenge will continue to evolve, however, as more and more types of hybrid or completely synthetic proteins are developed
that may or may not have some evolutionarily defined path to proper folding and thus activity.