Ask the Expert Enabling Custom Solutions for Downstream Processing of Future Therapies: An Adenoassociated Virus Case Study

with Orjana Terova

Orjana Terova is a purification product manager in the bioproduction division of Thermo Fisher Scientific. In a BPI webinar on 9 December 2016, she discussed the company’s custom resin program for purification of biological products. Thermo Fisher Scientific has dedicated a pilot-plant facility for this program.

Terova’s Presentation
Speed is the main development driver in downstream processing, but quality and efficiency are always critical. Purification processes need the highest resolution, capacity, salt tolerance, and operation speed possible. Consistency and reproducibility are important, as are cost minimization and mitigation of supply risk.

Adenoassociated viruses (AAVs) have emerged as the vectors of choice for many gene therapies because they can mediate long-term, tissue-specific gene expression with low immunogenicity. The biopharmaceutical industry can purify viral vectors on a scale for clinical supply, Terova said, but it needs industrialized platform technologies to maximize productivity and enable efficient, simple, and inexpensive purification of bioactive viral vectors for large-scale commercial manufacturing. Most processes include multiple chromatographic steps: e.g., ion exchange, hydrophobic interaction, and heparin or metal affinity. These are expensive processes with long development time and cumulative yield losses.

CaptureSelect technology produces high-binding ligands for affinity chromatography with antibody-based specificity independent of feedstock. (In an example, similar selectivity was shown for cell supernatant and plasma.) Ligands are the variable domain of heavy-chain–only antibody fragments. A single antibody domain provides full functionality for antigen-specific recognition in high-affinity binding. With compact structure, the molecules can withstand typical chromatography conditions.

CaptureSelect and POROS products are adapted in US and EU commercial purification processes. With CaptureSelect technology, high purity and yield can be obtained in a single chromatography step. This simplifies processes and shortens development timelines. Using POROS CaptureSelect AAV8 and AAV9 affinity resins, customers have increased yields 20–60% and reduced costs by a factor of six compared with other alternative methods.

Affinity ligand development begins with discovery of binding fragments through immunization and library construction. High-throughput screening follows to identify single clones that bind to a protein of interest with desired specificity. Further screening evaluates selectivity, elution conditions, stability, specificity, and binding affinity. Then prototype ligands are produced by an animal-origin–free yeast expression system for evaluation in small-scale affinity chromatography. Results are discussed with the client to identify the best ligands, which are then produced at larger scale for evaluation at the customer’s site. Finally, to make a resin suitable for clinical and commercial manufacturing, a robust ligand-manufacturing process is set up, prototype resins and a product-specific immunoassay are developed, validation batches are prepared, and data are compiled for a regulatory support package.

Ligands are immobilized on a POROS backbone. Terova described three attributes differentiating it from other chromatography resins: POROS rigid and incompressible polystyrene–divinylbenzene structure for robust physical and chemical stability, as well as a linear and predictable pressure flow curve; a large pore structure for convective flow; and a small average particle size of 50 µm for resolution independent of scale.

POROS and CaptureSelect technologies were combined to develop two affinity resins for purification of AAV subclasses 8 and 9. “We have obtained capacities of >10¹⁴ for POROS CaptureSelect AAV9 affinity resin and >10¹³ for POROS CaptureSelect AAV8 resin,” Terova reported. Capacity studies with academic collaborators and customer feedback confirm those numbers and show high specificity. Both resins provide one-step purification from crude material with high purity and yield, frequently followed by a concentration step. That simplifies AAV purification process with fewer unit operations, reducing cost of goods and enabling speed to market.

A collaborative study with Généthon compared purification of an AAV9 viral vector using multiple ion-exchange steps with POROS CaptureSelect AAV9 resin. Although equivalent purity was obtained using both methods, using the POROS resin reduced processing steps and increased yield (>80% recovery). No residual proteins were detected. In this case, the combination of POROS and CaptureSelect offered an industrialized purification platform for AAV large-scale production.

More Online
Find more information about POROS online at www.thermofisher.com/poros. Find more information about CaptureSelect products online at www.thermofisher.com/captureselect. The full presentation of this webcast — along with slides and a Q&A session can be found here.