MICROBIAL SURVEILLANCE AND POPULATION ECOLOGY CONSIDERATIONS FOR BIOPRODUCTION OF BIOPHARMACEUTICALS

: (Alternative Format Thesis)

Abstract

Individual microbes serve as economic cell factories for the production of non-mammalian biopharmaceuticals such as therapeutic recombinant proteins and monoclonal antibodies. These individual microbes can dynamically alter their cellular processes through molecular interactions among the members of their population or based on chemical and physical environmental conditions. Frequently, the focus of microbiological testing during bioprocessing is limited to biomass growth measurements and contamination control to meet the release criteria set out by regulatory agencies using conventional microbiological methods specified by pharmacopeial programs. This Ph.D. thesis aimed to evaluate the measurement, quality assessment, and monitoring strategies for microbial populations (microbial cells /biomolecules used to produce biopharmaceuticals) within non-mammalian whole-cell biomanufacturing industrial operations.

An in-depth literature review of the engineering design of whole-cell non-mammalian production platforms and the implications related to the aim of this thesis for measuring and monitoring microbial contaminants during bioprocessing and wastewater systems is discussed (Chapters 1 and 2). This review shows a need to implement advanced technology and standardized testing methodologies to create effective microbial surveillance programs—including the measurement of microbial cells or biomolecules of interest, monitoring, and microbial activity quality risk assessment for devising a clearance strategy.

Given the facility and platform design variation, tailoring the clearance strategy improves the microbial surveillance program for cleaning dry surfaces within the production facility. There is a link between methodology challenges for monitoring surface-associated dry biofilm and product contamination incidents in industrial settings. A practical microbiological study design (Chapter 3) for testing surface bacteria's survival pattern paired with a first-order loss model for statistical analysis revealed that higher initial population density and recycling of resources from "zombie-like" cells might similarly support growth by providing access to cell lysates or the contents of heat-killed cells.

In Chapter 4, this thesis explores the challenges with detection and removal of biopharmaceuticals that are persistent in aquatic environments. Alternative detection, identification, and removal methods with applications for clearance determination in various matrices, including wastewater, replacing in vivo testing to improve bioprocesses' sustainability (Chapter 4). Employing biochemical procedures such as protein separation based on size and relative molecular weights followed by immunoblotting techniques within complex industrial mixtures was adequate for sensitive detection and identification of selected microbial protein by-products. A lab-scale ozone study also demonstrated the potential for degrading the same microbial contaminants for contamination control as an alternative sustainable heat/chemical treatment technology.

This thesis concluded that a paradigm shift is needed to reassess the current microbiological/biochemical protocols to allow the incorporation of multi-disciplinary approaches involving advanced technology, such as microfluidic chips, for rapid and reliable in-line microbial surveillance programs. Additional studies investigating the "in-between" metabolic stages for a given prokaryotic population at solid-air interfaces could improve cleaning clearance strategies. Furthermore, standardization of bioassays for monitoring in-process and wastewater matrices leads to the possibility of implementing sustainable technology options such as ozone systems and automation of bioprocess to enhance the overall health and safety of workers and the environment aligned with UN Sustainable Development Goals (e.g., SDGs 3,6,13,14).

Date of Award	26 Jun 2024
Original language	English
Awarding Institution	University of Bath
Supervisor	Jannis Wenk (Supervisor) & Barbara Kasprzyk-Hordern (Supervisor)