Upstream Bioprocess Optimization and Biofuels Development
We have developed high throughput technology for upstream biologics production [Datta et al. Biotechnol. J. 9, 386-395 (2014)]. Our cell-based microarray platform was used to assess the effect of culture conditions on Chinese hamster ovary (CHO) cells. Specifically, growth, transgene expression and metabolism of a GS/methionine sulphoximine (MSX) CHO cell line, which produces a therapeutic monoclonal antibody, was examined using a microarray system in conjunction with a conventional shake flask platform in a non-proprietary medium. The microarray system consists of 60-nL spots of cells encapsulated in alginate and separated in groups via an 8-well chamber system attached to the chip. The non-proprietary medium developed allows cell growth, production, and normal glycosylation of recombinant antibody and metabolism of the recombinant CHO cells in both the microarray and shake flask platforms. In addition, 10.3 mM glutamate addition to the defined base medium results in lactate metabolism shift in the recombinant GS/MSX CHO cells in the shake flask platform. Ultimately, the results demonstrate that the HT microarray platform has the potential to be utilized for evaluating the impact of media additives on cellular processes, such as cell growth, metabolism, and productivity.
We have expanded our upstream optimization through work funded by NIIMBL (National Institute for Innovation in Manufacturing Biopharmaceuticals). This work is focused on high-yield production of lentiviruses for various biomanufacturing applications, e.g., CAR-T therapy. Stay tuned for more updates on this research.
Room Temperature Ionic Liquids in Biomass Conversion
The conversion of lignocellulosic biomass from agricultural waste, wood or energy crops into liquid biofuels is necessary to meet the increasing worldwide energy demand. Highly efficient pretreatment of lignocellulosic biomass for subsequent enzymatic hydrolysis and fermentation is perhaps the most significant limiting step on reduced process cost. Over the past several years, we have studied selective lignin extraction of lignocellulosic biomass using room temperature ionic liquids (RTILs). This approach enables efficient extraction of pristine lignin (that may be used as a valuable feedstock for lignochemicals production), while the crystal structure of cellulose and the hydrogen bonding network of lignocellulosic biomass subcomponents is disrupted (Figure BF-1) [Lee et al. Biotechnol. Bioeng. 102, 1368-1376 (2009)].
We have demonstrated that 1-ethyl-3-methylimidazolium acetate ([Emim] [OAc]) is capable of rendering maple wood flour and corn stover biomass highly susceptible to cellulase action via extraction of a substantial fraction of the native lignin along with substantial loss of native cellulose crystallinity (Figure BF-2A). Upon removal of over 60% of the native lignin, >90% of the cellulose was converted enzymatically into reducing sugars suitable for subsequent fermentation. A clear correlation existed between lignin removal and reduced cellulose crystallinity (Figure BF-2B). The residual RTIL could then be recycled multiple times with no loss in effectiveness. Further optimization enabled 50% (w/w) biomass loading to undergo substantial loss in biomass crystallinity with nearly complete cellulose degradation (Figure BF-3). Finally, through analysis of the physicochemical properties of the RTIL, we uncovered a quantitative solvent parameter that can be used to fine-tune RTIL composition for optimal pretreatment of lignocellulosic biomass (Figure BF-2) [Doherty et al. Green Chem. 12, 1967-1975 (2010)]. Specifically, we determined that the ability to disrupt the crystal structure of cellulose can be predicted by measuring the Kamlet-Taft β parameter of RTILs. In addition, RTILs can be reused up to 10 cycles of pretreatment without being regenerated.
Figure BF-1. Interaction of ILs with cellulose and lignin. (A) The semi-crystalline structure of cellulose is a result of the hydrogen-bonding network among the polysaccharide chains. (B) Incubation with ILs disrupts cellulose crystalline structure because of the IL's ability to accept hydrogen bonds (blue spheres indicate the cation and green spheres indicate the anion). (C) Following pretreatment amorphous cellulose can be depolymerized by cellulases. D: Interaction between lignin and [Emim] cation by π- π interactions [Lee et al. Biotechnol. Bioeng. 102, 1368-1376 (2009)].
Figure BF-2. Correlation of Kamlet-Taft β parameter extrapolated to 90˚C with: (A) glucose yield; (B) extracted lignin; (C) xylose yield; and (D) CrI for a series of RTILs ([EMIM]OAc, [BMIM]OAc, and [BMIM]MeSO4) at different water concentrations (0%, 5%, and 10%, w/w) [Doherty et al. Green Chem. 12, 1967-1975 (2010)].
Figure BF-3. (A) Effect from incubation time on digestibility of pretreated maple wood flour with [EMIM]OAc at 90˚C, and (B) effect of pretreatment temperature on digestibility of pretreated maple wood flour. Both graphs were made from data reported by Lee et al. [Lee et al. Biotechnol. Bioeng. 102, 1368-1376 (2009)]. (C) SEM images of untreated maple wood flour (A and B), maple wood flour pretreated in [EMIM]OAc (C), [BMIM]OAc (D), and [BMIM]MeSO4 (E) and with 10% (w/w) added water in [EMIM]OAC (F), [BMIM]OAc [BMIM]MeSO4 (H) [Doherty et al. Green Chem. 12, 1967-1975 (2010)].
Steven Cramer – Rensselaer Polytechnic Institute
Michael Betenbaugh – Johns Hopkins University