Developing downstream bioprocessing for gene and cell therapy applications

Ghosh, Ronit
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Dordick, Jonathan
Royer, Catherine Ann
Cramer, Steven M.
Karande, Pankaj
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Chemical engineering
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Attribution-NonCommercial-NoDerivs 3.0 United States
This electronic version is a licensed copy owned by Rensselaer Polytechnic Institute (RPI), Troy, NY. Copyright of original work retained by author.
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With the recent progress in gene and cell-based regenerative therapies, the challenge of producing pure and concentrated stocks of complex biologicals, in particular, gene therapy vectors and functional cells, has gained significant importance. As various gene and cell therapy-based treatments progress from pre-clinical to clinical and finally to the commercialization phase, the high demand for these new biologics necessitates developing robust, fast, and scalable downstream purification techniques that will guarantee optimum product yield, purity and viability. With this in mind, chromatographic purification process was developed for lentiviral and adeno-associated viral vectors, which are the two major vectors currently used in gene therapy-based clinical trials. Later, a novel separation process was also designed for the isolation of cells for cell therapy applications. Lentiviral vectors are mainly used as gene therapy agents in cancer immunotherapy and for the treatment of neurodegenerative diseases. However, the purification process suffers from low yield mainly due to the poor stability of lentiviral vectors and low product titer in the feed material, resulting in limited scalability and high manufacturing cost. To address this, we first performed a detailed study to assess the stability of LVV under different fluid phase conditions and identified optimal conditions for vector stability. While the vector was observed to be stable at pH 6.5-7.5, high salt concentration (> 500 mM NaCl) was found to be detrimental to vector stability. The selection of buffer species also impacted vector stability, with LVV exhibiting increased stability in phosphate buffer in the pH range of 6.5-7.5. The stability study helped set the operating condition boundaries of pH and salt for the subsequent evaluation of chromatographic resins. The first phase of process development involved the high-throughput batch screening of chromatographic resins in bind-and-elute mode in a 96-well slurry plate format to identify the best performing candidates. These candidates were then used in the second phase of a high throughput process development screen to determine wash and elution conditions by varying pH and salt concentration within the acceptable operating ranges based on LVV stability. The best-performing adsorbents were further tested in a miniature column format and ranked based on LVV yield and impurity clearance. Product and impurity retention maps obtained from linear gradient experiments were then used to define a bind-and-elute step process for top anion-exchange resins, including Amino Sepharose FF, Poros 50D, and Capto Q ImpRes. The bind-and-elute step process with these resin candidates resulted in LVV yields greater than 70% while also achieving greater than 90% residual protein clearance and more than 50% hcDNA clearance. A second downstream purification step was also developed using Capto Core 700, a resin with combines size exclusion and multimodal adsorptive properties, in the flow through mode. A preliminary two-column purification of LVV was performed by combining the flow-through Capto Core 700 step with the AEX bind and elute step, resulting in greater than 70% overall LVV yield, with nearly 99.9% host cell protein removal. Work was also carried out on the affinity capture of adeno-associated viral vectors. Since AAV mediated gene therapy treatments are delivered in vivo, the downstream purification of these vectors needs to meet higher purity standards. As a result, affinity chromatography was selected as the capture step over other modes of chromatography. A set of three variants of an AAV9 serotype with varying capsid ratios was evaluated using pH gradient experiments on AAV9 specific affinity resins (Poros Capture Select AAV9 and AviPure AAV9) and a more “universal” AAV affinity resin (Poros Capture Select AAVX). Comparable elution recoveries of 60-70% were obtained under all conditions examined. In addition, it was also observed that the elution pH was higher for the AAVX resin (pH 4.2) as compared to the AAV9 specific resins (pH 3). In order to improve resin lifetime, the efficacy of several cleaning in place (CIP) protocols were evaluated for these resins. The results indicated that a strategy using two guanidine hydrochloride sanitization steps per cycle was successful in extending the resin lifetime. The final phase of the thesis involved the identification of affinity peptides for cell separations and their evaluation in affinity cell capture was evaluated using magnetic beads. Phage display was used to screen and identify affinity peptides that target and bind to either dermal fibroblasts or endothelial cells (HUVECs) with high affinity and specificity. A cell-based binding assay was developed and used to determine the binding strengths and specificities of the affinity peptides to the two cell types. Two peptide candidates, B8 and F12 (out of 120 tested), showed high specific binding to HUVECs and fibroblasts, respectively. Parallel flow cytometry experiments were also performed to confirm the binding strength and specificity of the two lead peptide candidates. A proof of concept cell capture study was then carried out. The results indicated that both the B8 and F12 functionalized Dynabeads® were indeed successful in capturing the fibroblast and endothelial cells, respectively. This workflow for the identification and screening of affinity peptides for cell separations can be readily extended to other therapeutically relevant cells of clinical importance. The work presented in this thesis will help advance the state of the art of downstream bioprocessing for these important new classes of gene and cell therapy based biological products.
August 2021
School of Engineering
Dept. of Chemical and Biological Engineering
Rensselaer Polytechnic Institute, Troy, NY
Rensselaer Theses and Dissertations Online Collection
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