Single-use technologies have transformed conventional biopharmaceutical manufacturing, and their adoption is increasing rapidly for emerging applications like antibody–drug conjugates and cell and gene therapy products. These disruptive technologies have also had a significant impact during the coronavirus disease 2019 pandemic, helping to advance process development to enable the manufacturing of new monoclonal antibody therapies and vaccines. Single-use systems provide closed plug-and-play solutions and enable process intensification and continuous processing. Several challenges remain, providing opportunities to advance single-use sensors and their integration with single-use systems, to develop novel plastic materials, and to standardize design for interchangeability. Because the industry is changing rapidly, a holistic analysis of the current single-use technologies is required, with a summary of the latest advancements in materials science and the implementation of these technologies in end-to-end bioprocesses.

Cardiomyocytes (CMs), derived from pluripotent stem cells (PSCs), have the potential to be used in cardiac repair. Addition of physical cues, such as electrical and mechanical stimulations, have proven to significantly effect morphology, density, cardiogenesis, maturity and functionality of differentiated CMs. This work combines rigorous fluid dynamics investigation and flow frequency analysis with iPSC differentiation experiments to identify and quantify the flow characteristics leading to a significant increase of differentiation yield. This is towards a better understanding of the physical relationship between frequency modulation and embryoid bodies suspension, and the development of dimensionless correlations applicable at larger scales. Laser Doppler Anemometry and Fast Fourier Transform analysis were used to identify characteristic flow frequencies under different agitation modes. Intermittent agitation resulted in a pattern of low intensity frequencies at reactor scale that could be controlled by varying three identified time components: rotational speed, interval and dwell times. A proof of concept biological study was undertaken, tuning the hydrodynamic environment through variation of dwell time based on the engineering study findings and a significant improvement in CM yield was obtained. This work introduces the concept of fine-tuning the physical hydrodynamic cues within a three-dimensional flow system to improve cardiomyocyte differentiation of iPSC.

Protein Glycan Coupling Technology (PGCT) uses purposely modified bacterial cells to produce recombinant glycoconjugate vaccines. This vaccine platform holds great potential in this context, namely due to its modular nature, the simplified production process in comparison to traditional chemical conjugation methods, and its amenability to scaled-up operations. As a result, a considerable reduction in production time and cost is expected, making PGCT-made vaccines a suitable vaccine technology for low-middle income countries, where vaccine coverage remains predominantly low and inconsistent. This work aims to develop an integrated whole-process automated platform for the screening of PGCT-made glycoconjugate vaccine candidates. The successful translation of a bench scale process for glycoconjugate production to a microscale automated setting was achieved. This was integrated with a numerical computational software that allowed hands-free operation and a platform adaptable to biological variation over the course of a production process. Platform robustness was proven with both technical and biological replicates and subsequently the platform was used to screen for the most favourable conditions for production of a pneumococcal serotype 4 vaccine candidate. This work establishes an effective automated platform that enabled the identification of the most suitable E. coli strain and genetic constructs to be used in ongoing early phase research and be further brought into preclinical trials.

Intermittent agitation strategies have been increasingly used for a range of process development applications, i.e., to modulate physical cues, to improve stem cell differentiation yields, and to control hydrodynamic shear stresses in microcarrier suspension; however, there is a distinct lack of characterization. Both continuous and intermittent agitation modes in relation to suspension and mixing dynamics within a DASGIP bioreactor were characterized. Suspension dynamics were found to be affected by microcarrier porosity and the degree of settling was found to be more pronounced at the top of the bioreactor. Mixing time characterization showed a marked improvement in mixing efficiency for intermittent agitation, with an overall dependence on the timing of tracer insertion.

This work aims to characterize the mixing and suspension dynamics occurring within two commercially available DASGIP bioreactor configurations, equipped with a twoblade paddle impeller with large impeller to tank diameter ratio, D/T = 0.97. Both continuous and intermittent agitation modes were employed to determine the impact that agitation strategy has upon mass transfer and microcarrier settling/suspension. This paper builds upon the flow dynamics data presented in Part 1 for a flat bottom DASGIP bioreactor and shows how intermittent agitation can break-up regions of slow mixing observed during continuous agitation, therefore substantially increasing the mixing efficiency of the system. Similarly, it was found that microcarrier characteristics might significantly affect the level of suspension when the impeller is in dwell status when intermittent agitation modes are used.