Peter Arletti Dephillips

A microscale yeast cell disruption technique for integrated process development strategies

Miniaturizing protein purification processes at the microliter scale (microscale) holds the promise of accelerating process development by enabling multi‐parallel experimentation and automation. For intracellular proteins expressed in yeast, small‐scale cell breakage methods capable of disrupting the rigid cell wall are needed that can match the protein release and contaminant profile of full‐scale methods like homogenization, thereby enabling representative studies of subsequent downstream operations to be performed. In this study, a noncontact method known as adaptive focused acoustics (AFA) was optimized for the disruption of milligram quantities of yeast cells for the subsequent purification of recombinant human papillomavirus (HPV) virus‐like particles (VLPs). AFA operates by delivering highly focused, computer‐controlled acoustic radiation at frequencies significantly higher than those used in conventional sonication. With this method, the total soluble protein release was equivalent to that of laboratory‐scale homogenization, and cell disruption was evident by light microscopy. The recovery of VLPs through a microscale chromatographic purification following AFA treatment was within 10% of that obtained using homogenization, with equivalent product purity. The addition of a yeast lytic enzyme prior to cell disruption reduced processing time by nearly 3‐fold and further improved the comparability of the lysate to that of the laboratory‐scale homogenate. In addition, unlike conventional sonication methods, sample heating was minimized (≤8 °C increase), even using the maximum power settings required for yeast cell disruption. This disruption technique in combination with microscale chromatographic methods for protein purification enables a strategy for the rapid process development of intracellularly expressed proteins.

Design-for-Six-Sigma for Development of a Bioprocess Quality-by-Design Framework

An initial quality-by-design (QbD) framework was assembled for biopharmaceutical product, process, and analytical development using the design-for-six-sigma (DFSS) methodology. This technique was both streamlined and efficient, which permitted development of a QbD framework with minimized team leader and member resources. DFSS also highly emphasized voice-of-the-customer, information considered crucial to development and implementation of a bioprocess QbD framework appropriate for current development needs of the organization and its regulatory environment. The bioprocess QbD final design and implementation plan was comprised of seven teams, constructed from six QbD elements plus a communication/training team. Each elements detailed design was evaluated against internal and external established best practices, the QbD charter, and design inputs. Gaps were identified and risks mitigated to assure robustness of the proposed framework. Aggregated resources and timing were estimated to obtain vital implementation sponsorship. Where possible, existing governance and information technology efforts were leveraged to minimize additional bioprocess resources required. Finally, metrics were selected to track success of pilots and eventual implementation. LAY ABSTRACT: An initial quality-by-design (QbD) framework was assembled to guide biopharmaceutical product, process, and analytical development. QbD starts by defining the patient requirements which then are translated into required quality attributes for the product. The production process then is designed to consistently meet these quality requirements by identifying and understanding those parameters which influence them. A control strategy is developed that specifically relates each point of control to a desired quality measure. Overall, this approach results in a robust process, capable of reliably producing quality product. The bioprocess QbD framework was developed to guide implementation of the desired QbD strategy. It was comprised of seven teams, constructed from six QbD elements plus a communication/training team. Each elements detailed design was evaluated against internal and external established best practices, the charter, and design inputs. Gaps were identified and risks mitigated to assure robustness of the proposed framework. Aggregated resources and timing were estimated to obtain vital implementation sponsorship. Where possible, existing governance and information technology efforts were leveraged to minimize additional bioprocess resources required. Finally, metrics were selected to track success of pilots and eventual implementation.

893. Using QPCR and Automation to Assign Infectious Potencies to Adenovirus Based Vaccines and Vectors for Gene Therapy

The potencies of test articles generated during bioprocess development supporting the manufacture of Ad5 based HIV vaccine have been assigned since 1999 using a QPCR-based Potency Assay (QPA). We report here the simplification of the Ad5 QPA through (1) the introduction of a facile method for the harvest of DNA for QPCR quantitation and (2) the integration of automated liquid handling systems for performing semi-automated or completely automated QPA assays. We demonstrate semi-automated QPA operation using the Beckman Coulter Multimek for the addition of reagents, preparation of dilutions, and set-up of PCR reactions in 384 well formats, which greatly reduce reagent cost and analyst time involved with QPA assays. We show preliminary results indicating that a fully automated assay is possible using a more versatile liquid handling system such as the Tecan Freedom Evo. We also present the results of a PreValidation Exercise (PreVEx) for the semi-automated QPA assay we designate Triton Lysis with the Multimek (TLM) Ad5 QPA, which exhibits a remarkable precision. The PreVEx demonstrated that the TLM Ad5 QPA has a root variability of approximately 16.8% and a format dependent variability (1|[times]|3 assay format, with 4 infection replicates per assay) of approximately 5.8%, allowing samples differing in potency by 17.4% to be discriminated with 95% confidence. This precision equals or exceeds the precision associated with the previous Ad5 QPA.