Session 2

Ian Gough

McMaster University

Selective protein quantification on continuous chromatography equipment with limited absorbance sensing: A partial least squares and statistical wavelength selection solution

Monoclonal antibodies are a class of protein that provide targeted and effective treatments for a wide range of diseases. Continuous chromatography is emerging as a vital component of the monoclonal antibody manufacturing process. The long-term operation of continuous chromatography processes relies on monitoring process performance with real-time selective protein concentration quantification, such as estimating the concentrations of monoclonal antibodies and host cell protein impurities. Partial least squares (PLS) models fit with spectroscopic UV-Vis absorbance data have demonstrated the ability to selectively quantify proteins [1]. However, the practical adoption of such models has been hindered as standard chromatography equipment is only capable of measuring UV-Vis absorbance at a few user-defined wavelengths, while existing demonstrations rely on complete spectral data. To address this limitation, we propose a method for selecting wavelengths that can selectively quantify proteins using continuous chromatography equipment. First, we generate spectral absorbance data from 200 – 900 nm with a plate reader and a set of independent protein mixtures composed of bovine serum albumin and lysozyme per a D-optimal experimental design. We show the statistical t-test and an absorbance magnitude test are important to refine the spectral data by identifying wavelengths that do not follow the Bouguer-Beer-Lambert law. We then rank the wavelengths within the refined region, and we show the choice of wavelength selection algorithm is important among sequential forward search, variable importance to projection scores, and the least absolute shrinkage and selection operator. Next, we generate in-line absorbance data with the set of protein mixtures on the Sartorius BioSMB PD. We prove the set of in-line absorbance measurements at the selected wavelengths produces more accurate selective protein quantification than a set of measurements at 230 nm, 260 nm, and 280 nm, by a factor of four in terms of root mean squared prediction error. We demonstrate the importance of verifying the selected wavelengths on the continuous chromatography equipment. Finally, we show the choice of wavelengths is dependent on the measurement path length and protein concentration ranges. Overall, we provide a process analytical technology that chromatography practitioners can utilize to generate more information about their process state. While the development and use of soft sensors is well established in the biopharmaceutical industry with accessory analytical equipment, the method proposed here helps alleviate the analytical bottleneck that hinders practitioners from developing more advanced monitoring and control technologies with standard chromatography equipment. [1] Brestrich, Nina, et al. Biotechnol Bioeng 111.7 (2014): 1365-1373

Roy Byung-Chul Kim

University of Toronto

Engineering anaerobic fungal-bacteria consortia for medium chain fatty acid production from lignocellulosic biomass

Consolidated bioprocessing of lignocellulosic biomass (LCB) for chemical and fuel production has emerged as a sustainable solution to replace conventional petroleum-derived products. However, this promising bio-based approach has been limited by the high costs associated with biomass pretreatment and aerobic microbial processes. Anaerobic microbiomes that directly breakdown solid substrates in the absence of oxygen could enable cost-effective conversion of LCB to chemicals and fuels. In particular, anaerobic rumen fungi have been shown to efficiently convert LCB to simple fermentation products, which could be further upgraded to valuable chemicals through co-culturing with specialized anaerobic bacteria. Here, we established synthetic microbial consortia consisting of anaerobic fungi and anaerobic chain elongating bacteria that efficiently convert LCB to valuable medium chain fatty acids (MCCs). We initially leveraged an automated high-throughput anaerobic cultivation platform to collect detailed growth and fermentation kinetic profiles of different chain elongating bacteria. In total, five chain elongating species were screened in four different medias containing one of six different carbon sources. Subsequently, anaerobic fungal-bacterial co-cultures were cultivated on cellulose fiber or sorghum, which produced butyrate and hexanoate as primary end products via lactate-based chain elongation. Our results demonstrate the ability to use synthetic consortia of anaerobic fungi and bacteria as a platform for MCFA production. High-throughput screening with fungal strains and synthetic consortia will be further conducted to reveal key metabolic interaction and to further optimize LCB to MCFA conversion.

Aishik Chakraborty

University Of Western Ontario

Nanosilicate-incorporated hydrogel scaffold for drug delivery

Hydrogels derived from natural polymers are three-dimensional (3D), water-retaining scaffolds that can serve as drug delivery vehicles. They are suitable for various biomedical applications because of their (a) easily tunable physicochemical functionalities, (b) biocompatibility, and (c) biodegradability. However, hydrogels prepared from natural polymers are mechanically weak and degrade quickly. Different nanoparticles have been used to enhance the mechanical strength of natural polymeric hydrogels. Recently, two-dimensional (2D) nanosilicates have emerged as reinforcing agents because of their biocompatibility and highly tunable physicochemical properties. Here, we have incorporated nanosilicates in a naturally derived gelatin-based polymeric matrix to formulate mechanically tough scaffolds. Furthermore, the hydrogels were prepared by cryogelation, where the crosslinking process is carried out at sub-zero temperatures. At such freezing temperatures, ice crystals form, and when thawed, the ice crystals melt to leave behind large pores in the polymeric matrix. These macroporous hydrogels are expected to demonstrate improved nutrient transfer through the polymeric network and allow cell infiltration. Our study shows that the designed scaffold is (i) mechanically tough, (ii) macroporous, (iii) cytocompatible, and (iv) capable of delivering a model small molecule drug, metformin. Finally, we hypothesize that this nanosilicate-incorporated scaffold will be suitable for diverse biomedical applications, including wound regeneration.

Madhuja Chakraborty

University of Waterlooo

Optimization of the Autographa californica multiple nucleopolyhedrovirus genome by CRISPR-Cas9

The baculovirus expression vector system (BEVS) has proven to be a promising platform for producing recombinant proteins, vaccines, virus-like particles, and/or other biologics. However, with co-production of recombinant baculovirus and other viral proteins, the system could benefit with the removal of non-essential genes, which, by our definition, are genes that do not decrease protein yield. To this end, our lab has developed CRISPR-Cas9 assay to determine the essentiality of genes in the vector genome. We identified 92 AcMNPV genes that are active in the late and very late phases of the infection cycle based on the presence of a 5'-TAAG-3' promoter motif and literature review to be targeted. Briefly, a sgRNA plasmid having a targeting sequence under the SfU6 promoter was constructed and used in the transfection of a Cas9 expressing Sf9 cell line seeded on tissue culture-treated multi-well plates. 16-24 hours post transfection, the medium is replaced with fresh Sf-900TM III medium containing p6.9mAG rBEV. This baculovirus vector contained a green fluorescent protein gene under the control of the p6.9 promoter. Approximately 48 hours post infection, the cell pellet and supernatant were harvested for analysis by flow cytometry and end-point dilution assay, respectively. The assay confirmed that targeting essential genes like pk-1 and lef1 decreased both fluorescence intensity and infectious virus titer (IVT), whereas targeting AcOrf-19 which is predicted to be non-essential resulted in little change in fluorescence intensity and IVT. However, targeting pkip which is known to be essential from the literature did not decrease fluorescence intensity and IVT.