Lopes Viana Nunes, Sofia; (2023) Establishing Ultra Scale-Down Tools for the Rational Design of Linked Bioprocesses: Flocculation and Centrifugation. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The processing of cell homogenates to recover intracellular products comprises one of the most challenging solid-liquid separation stages in bioprocessing. One promising option to meet this challenge is to use flocculation to aid cell debris removal by high-speed, continuous centrifuges. However, flocculation is complex, with multiple effects determining its performance, often requiring case-by-case adjustments. This thesis focuses on establishing an ultra scale-down (USD) platform for flocculation and centrifugation to mimic the full-scale processing performance and product yield and study the impact of operating parameters. A combination of experimental techniques and data-handling methods is used to achieve this target. This research develops the design of USD flocculation and its link with USD centrifugation. An initial validation was performed using homogenised baker’s yeast, which demonstrated equivalent supernatant clarity between USD (50 mL) and pilot scale (6.7 L). Multivariate data analysis was used with the USD platform, accelerating the extraction of meaningful process information. Furthermore, a method that calculates four indices (Gn, R, Snh and Rdc) from particle size distribution was developed for quantifying floc growth and strength, showing to characterise and select flocculation conditions. The USD platform was then applied to study the recovery of CV2025 w-transaminase expressed in E. coli. This USD platform for processing homogenised E. coli was characterised and validated using a strategy proposed in this thesis. The results of the USD approach mimicked enzyme yield, revealing that losses occurred during flocculation rather than in centrifugation. Flocculation studies using poly(ethyleneimine) demonstrated that a large Camp number (≥ 105 ) does not necessarily lead to strong flocs. Optimisation of the flocculation chemistry (e.g., flocculant, salt addition) is key for robust flocculation. USD centrifugation for flocs required the addition of a correction factor. This adjustment was successfully validated using flocs from homogenised E. coli.

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