OBTAINING HIGH CELL DENSITY CULTURES FOR ECONOMIC PRODUCTION OF POLYBIND–Z
Commercialization of biopolyesters and biopolyester-derived products has been slow because of the excessive cost of production.
However, production of biodegradable polyesters as commodity products has become economically feasible due to a significant
amount of applied research and engineering on bacterial production strains and fermentation methods, allowing high cell density
E.coli fermentations up to 190 g/L dry cell weight (7, 9–11). Today, E. coli genetically engineered to overproduce biopolyesters as renewable commodity plastics have been reported to accumulate polyester
levels approaching 80% of the dry cell weight. Further, volumetric productivity for commodity plastics using commercial technologies
has reached levels above 3 g/Lxh.
Here, the authors describe a new platform technology that uses biodegradable biopolymer beads as the carrier for the presentation
of a functional PS–Z fusion protein, increasing the complexity of the product. Given that this platform technology incorporates
functional proteins presented on the surface, productivity assessment of the PolyBind–Z beads inherently includes functionality.
Initial fermentations produced low yields of PolyBind–Z beads (25%–30% of the dry cell weight) that displayed IgG binding
functionality in the range of 30 mg/g of drained beads. To increase E.coli cell density, productivity, and functionality of the PolyBind–Z beads, the fermentation conditions, as well as the IgG binding
fusion protein, were examined and modified.This resulted in a scalable industrial fermentation process for high-performance
PolyBind–Z. Using a fed-batch strategy and incorporation of a defined media lacking animal products, the volumetric productivity
of the PolyBind–Z beads has reached levels of approximately 50% when compared with commercial commodity bioplastics production
processes. Further, the specific IgG binding functionality (discussed below) of the PolyBind–Z beads was also significantly
improved to 100 mg/g of drained beads.
While significant advances have been achieved in the production of highly functional PolyBind–Z beads, the current production
process holds the promise to be further optimized. To increase the productivity of our system and approach levels obtained
by the commercial commodity bioplastics industry, additional fermentation optimization studies using quality-by-design are
CURRENT DOWNSTREAM PROCESSING: LYSIS AND REMOVAL OF IMPURITIES
Although this platform technology has allowed the development and production of tailor-made highly functional biobeads, a
substantial practical and financial hurdle must be overcome. As with many products generated that use microorganisms in their
production process, the intracellular polymer or protein must be extracted and purified from the host cell components by an
economically feasible process. Fortunately, there are a number of scaled extraction technologies currently used by both industries
that can be applied to the extraction of PolyBind–Z beads. These technologies include both mechanical disruption and chemical/enzymatic
extraction. However, in contrast to the commodity bioplastics industry, extraction and purification of the PolyBind–Z beads
requires retention of protein function. Inherent to the platform technology is a significant proportion of covalently linked
surface protein. As a result, the chemicals/solvents applied by the commodity bioplastics industry to extract and purify biopolyester,
such as chloroform, butyrolacetone, and sodium hypochlorite, are not viable with this platform; such chemicals would damage
the functional proteins displayed on the surface of the biopolymer beads. However, incorporating some of the advances in industrial
scale cell disruption and extraction techniques, we have developed a method that uses several different technologies to efficiently
disrupt E. coli cells and to release PolyBind–Z.
Once the PolyBind–Z beads are extracted from the E.coli, a stringent purification process is required. Given the resin purity required for the mAb production process, purification
of the PolyBind–Z beads to remove host cell proteins (HCPs), nucleic acids, and endotoxins is critical. Informed by already
designed and industrially-used protein inclusion body purification processes, we have developed scalable purification steps
to remove the majority of the HCPs, nucleic acids, and endotoxins (see Table I). The impurities and contaminants are continuously
monitored using sensitive biopharmaceutical standard methods as we further develop the purification process to reduce the
impurity levels associated with the beads to levels acceptable by the industry (12).
Table I: Current PolyBind–Z impurity profile.