Bioengineered Protein A Polymer Beads for High-Affinity Antibody Purification - The authors discuss an alternative to traditional Protein A resins. - BioPharm International


Bioengineered Protein A Polymer Beads for High-Affinity Antibody Purification
The authors discuss an alternative to traditional Protein A resins.

BioPharm International
Volume 24, Issue 5, pp. 36-42


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 underway.


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.

Table I: Current PolyBind–Z impurity profile.
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).

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