Martina Micheletti

How can measurement, monitoring, modeling and control advance cell culture in industrial biotechnology?

This report highlights the potential of measurement, monitoring, modeling and control (M(3) C) methodologies in animal and human cell culture technology. In particular, state-of-the-art of M(3) C technologies and their industrial relevance of existing technology are addressed. It is a summary of an expert panel discussion between biotechnologists and biochemical engineers with both academic and industrial backgrounds. The latest ascents in M(3) C are discussed from a cell culture perspective for industrial process development and production needs. The report concludes with a set of recommendations for targeting M(3) C research toward the industrial interests. These include issues of importance for biotherapeutics production, miniaturization of measurement techniques and modeling methods.

A generic hierarchical screening method for the analysis of microscale refolds using an automated robotic platform

The refolding of protein derived from inclusion bodies is often characterized by low yields of active protein. The optimization of the refolding step is achieved empirically and consequently is time‐consuming slowing process development. An automated robotic platform has been used to develop a dilution refold process‐screening platform upon which a hierarchical set of assays rapidly determine optimal refolding conditions at the microscale. This hierarchy allows the simplest, cheapest, and most generic high‐throughput assays to first screen for a smaller subset of potentially high‐yielding conditions to take forward for analysis by slower, more expensive, or protein specific assays, thus saving resources whilst maximizing information output. An absorbance assay was used to initially screen out aggregating conditions, followed by an intrinsic fluorescence assay of the soluble protein to identify the presence of native‐like tertiary structure, which was then confirmed by an activity assay. Results show that fluorescence can be used in conjunction with absorbance to eliminate low‐yielding conditions, leaving a significantly reduced set of conditions from which the highest yielding ones can then be identified with slower and often more costly activity or RP‐HPLC assays, thus reducing bottlenecks in high‐throughput analysis. The microwell‐based automated process sequence with generic hierarchical assays was also used to study and minimize the effect on redox potential or misfolding, of oxygenation due to agitation, before demonstrating that the platform can be used to rapidly collect data and evaluate different refolding conditions to speed up the acquisition of process development data in a resource efficient manner.

Microscale methods to rapidly evaluate bioprocess options for increasing bioconversion yields: application to the ω-transaminase synthesis of chiral amines

This work aims to establish microscale methods to rapidly explore bioprocess options that might be used to enhance bioconversion reaction yields: either by shifting unfavourable reaction equilibria or by overcoming substrate and/or product inhibition. As a typical and industrially relevant example of the problems faced we have examined the asymmetric synthesis of (2S,3R)-2-amino-1,3,4-butanetriol from l-erythrulose using the ω-transaminase from Chromobacterium violaceum DSM30191 (CV2025 ω-TAm) and methylbenzylamine as the amino donor. The first process option involves the use of alternative amino donors. The second couples the CV2025 ω-TAm with alcohol dehydrogenase and glucose dehydrogenase for removal of the acetophenone (AP) by-product by in situ conversion to (R)-1-phenylethanol. The final approaches involve physical in-situ product removal methods. Reduced pressure conditions, attained using a 96-well vacuum manifold were used to selectively increase evaporation of the volatile AP while polymeric resins were also utilised for selective adsorption of AP from the bioconversion medium. For the particular reaction studied here the most promising bioprocess options were use of an alternative amino donor, such as isopropylamine, which enabled a 2.8-fold increase in reaction yield, or use of a second enzyme system which achieved a 3.3-fold increase in yield.

An automated microscale platform for evaluation and optimization of oxidative bioconversion processes

In this work an integrated robotic platform has been used for the development of a fully automated microscale process sequence comprising fermentation and bioconversion using E. coli TOP10 [pQR210] expressing cyclohexanone monooxygenase (CHMO). Ninety six‐Deep Square Well (96‐DSW) microtiter plates were used for microbial culture and enzyme‐catalyzed conversion, where plate preparation, reagent addition, and sampling were all carried out without manual intervention. The adoption of automated robotic procedures has enabled the rapid collection of kinetic data for whole process optimization at the microscale. This high‐throughput approach enabled a range of amino acid sources for media formulation and well fill volumes to be investigated highlighting when nutritional limitation and oxygen limitations took place. The automated process sequence has been applied to test six CHMO substrates including norcamphor and cycloheptanone all of which to the best of our knowledge have yet to be tested with E. coli TOP10 [pQR210]. Substrate specificity and product selectivity were effectively demonstrated and compared to both the natural substrate cyclohexanone and the model substrate bicyclo[3.2.0]hept‐2‐en‐6‐one used to demonstrate asymmetric synthesis. The results obtained using the developed process sequence could be reproduced at 75 L scale when a matched oxygen transfer coefficient kLa approach was used. The study demonstrates how automated microscale processing enables the rapid collection of kinetic quantitative data in a robust manner with clear implications for accelerating bioprocess development, optimization, and scale‐up.

Optical sensor enabled rocking T‐flasks as novel upstream bioprocessing tools

During the past decade, novel disposable cell culture vessels (generally referred to as Process Scouting Devices or PSDs) have become increasingly popular for laboratory scale studies and seed culture generation. However, the lack of engineering characterization and online monitoring tools for PSDs makes it difficult to elucidate their oxygen transfer capabilities. In this study, a mass transfer characterization (kLa) of sensor enabled static and rocking T‐flasks is presented and compared with other non‐instrumented PSDs such as CultiFlask 50®, spinner flasks, and SuperSpinner D 1000®. We have also developed a mass transfer empirical correlation that accounts for the contribution of convection and diffusion to the volumetric mass transfer coefficient (kLa) in rocking T‐flasks. We also carried out a scale‐down study at matched kLa between a rocking T75‐flask and a 10 L (2 L filling volume) wave bioreactor (Cultibag®) and we observed similar DO and pH profiles as well as maximum cell density and protein titer. However, in this scale‐down study, we also observed a negative correlation between cell growth and protein productivity between the rocking T‐flask and the wave bioreactor. We hypothesize that this negative correlation can be due to hydrodynamic stress difference between the rocking T‐flask and the Cultibag. As both cell culture devices share key similarities such as type of agitation (i.e., rocking), oxygen transfer capabilities (i.e., kLa) and disposability, we argue that rocking T‐flasks can be readily integrated with wave bioreactors, making the transition from research‐scale to manufacturing‐scale a seamless process. Biotechnol. Bioeng. 2012;109: 2295–2305.

Mode decomposition and Lagrangian structures of the flow dynamics in orbitally shaken bioreactors

In this study, two mode decomposition techniques were applied and compared to assess the flow dynamics in an orbital shaken bioreactor (OSB) of cylindrical geometry and flat bottom: proper orthogonal decomposition and dynamic mode decomposition. Particle Image Velocimetry (PIV) experiments were carried out for different operating conditions including fluid height, h, and shaker rotational speed, N. A detailed flow analysis is provided for conditions when the fluid and vessel motions are in-phase (Fr = 0.23) and out-of-phase (Fr = 0.47). PIV measurements in vertical and horizontal planes were combined to reconstruct low order models of the full 3D flow and to determine its Finite-Time Lyapunov Exponent (FTLE) within OSBs. The combined results from the mode decomposition and the FTLE fields provide a useful insight into the flow dynamics and Lagrangian coherent structures in OSBs and offer a valuable tool to optimise bioprocess design in terms of mixing and cell suspension.

Engineering characterisation of single-use bioreactor technology for mammalian cell culture applications

The thesis describes an experimental investigation of the fluid dynamics within novel single-use bioreactors (SUBs), including stirred, rocked and pneumatically driven mixing systems. Biological studies to ascertain the impact of hydrodynamic conditions within these systems, on the growth and protein productivity of a mammalian cell line, are also presented. Two-dimensional velocity measurements within different SU technology were acquired with the use of a whole flow field laser-based technique, Particle Image Velocimetry (PIV). Fluid dynamic characteristics including velocity, turbulence, turbulent kinetic energy and vorticity were determined from time-resolved and phase-resolved velocity measurements. Commercial bioreactor systems were modified, if needed, in order to perform experiments within bioreactors commonly used for cell culture experiments, in preference to using vessel mimics. The fluid flow characteristics in both the impeller region and bulk fluid of a single-impeller stirred bioreactor were investigated, facilitating an enhanced understanding of the spatial distribution of velocity and turbulence throughout the vessel. PIV was also used to study the flow in a dual-impeller stirred bioreactor, providing a rare examination of the interaction between the flow fields generated by two impellers. The whole flow field velocity and turbulence characteristics measured within a rocked bag and pneumatically driven vessel, allow a unique insight into the flow pattern and turbulence distribution within two novel cell culture systems. Cell viability, size, growth, protein productivity and metabolites concentration were monitored under different cell culture operating conditions. Cell culture experiments, combined with the hydrodynamic information acquired using PIV, offer an insight into the physiological response of the cells to highly disparate flow conditions. This information helped to understand how the hydrodynamics induced by novel commercially used mixing systems, can impact upon a mammalian cell line. Having implications for an augmented capacity for cross-compatibility, in addition to enhanced strategies for scale translation and optimal bioreactor design.

Impact of hydrodynamics on iPSC-derived cardiomyocyte differentiation processes

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.