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The olfactory mucosa is a source of cell types that are associated with neural regeneration such as olfactory ensheathing cells (OECs), mesenchymal stromal cells (MSCs), and neural stem cells (NSCs). Although cells from this region have been used for autologous transplantation onto damaged spinal cord injury, there is a gap for an allogeneic cell therapy approach. The aim of this thesis was to establish key bioprocessing parameters for the expansion of c-MycERTAM-derived populations of human late-adherent olfactory mucosa cells (hOMCs). First, growth kinetics, identity and potency were characterised in both monolayer and suspension culture platforms. Profiles of cell growth kinetics were obtained for seeding densities ranging from 3,000 cells/cm² to 12,000 cells/cm² by manual counting and confluence measurements. PA5 hOMCs achieved between 35-41 population doublings in culture when seeded at 6,000 cells/cm² in long term culture. Moreover, removal of 4-hydroxytamoxifen (4-OHT) did not have an effect on cell growth kinetics. Replicative senescence was observed due to undesired silencing of c-MycERTAM. Next, alternative media compositions were examined as a means of optimising the process and transitioning to xeno-free culture. hOMCs showed a high dependence on fetal bovine serum (FBS) supplementation, and substitution with commercially available supplements did not sustain growth in serum-free conditions. However, human platelet lysate (hPL) was found to be a comparable to FBS when used to supplement basal media at concentrations of 2% and 5% (v/v). In this xeno-free media, hOMCs showed comparable cell growth kinetics to FBS supplemented media. Additionally, identity and potency marker expression in hOMCs was comparable between the FBS and the xeno-free hPL conditions. Finally, to progress towards scalable production of hOMCs, microcarrier screening and subsequent cell expansion on microcarriers in spinner flasks was undertaken. PA5 hOMCs were successfully expanded as a suspension culture on microcarriers at 80-mL scale in spinner flasks. A total of 8.40 ± 0.54 ×10⁶ viable cells were harvested when grown in medium supplemented with 5% hPL, and a similar number of 6.70 ± 1.05 ×10⁶ cells when grown in 10% FBS using Plastic microcarriers. PA5 hOMCs expanded on microcarriers conserved markers of identity post-harvest. In conclusion, by addressing bioprocessing fundamentals, advances towards scalable production of hOMCs using xeno-free culture reagents have been achieved.

The biopharmaceutical industry is becoming increasingly reliant on recombinant proteins as therapeutic agents. The work presented here details the development of rapid mammalian expression systems and novel capture methods for use in early recombinant protein development. A key aim was to investigate expression of recombinant proteins via cost effective production methods and to compare the resultant products at small scales of manufacture. A model single chain variable fragment Fc conjugate (scFv-Fc) targeted against a clinically relevant glycoprotein, the carcinoembryonic antigen (CEA), was expressed and characterised using both transient and stable-based expression of transgenic DNA. Transient gene expression in suspension HEK293 cells produced a maximum scFv-Fc level of 72μg/mL, which was used for initial protein characterisation. A stable pool of transfected CHO cells was also generated using a ubiquitous chromatin opening element (UCOE)-based vector achieving increased protein expression and culture periods when combined with a fed-batch regimen. Characterisation of the resulting proteins showed that stability and effector function was maintained across transient and stable production methods at all scales, indicating that preliminary data generated from transient expression in HEK293F cells could be generalised to predict that of protein stably expressed in CHO cell populations. A significant bottleneck in harvesting and purifying proteins from cell containing feed streams is the requirement for solid-liquid separation prior to capture. Therefore, a technique was proposed for direct capture of recombinant protein from unclarified feed streams that can integrate directly into the bioreactor harvest line. The process was demonstrated using immobilised metal affinity chromatography (IMAC) capture of recombinant CEA with a polyhistidine (His6) tag from a bioreactor culture. This provides a basis for direct primary capture of recombinant proteins from unclarified mammalian cell feed streams that could be generalized to other capture methods and proteins.

Synthetic Biology is ushering in a new era where reengineered genomes can enhance the capacity of host cells to produce biologic and chemical products. Standardisation is a key component of synthetic biology as it enables effective implementation (Müller and Arndt, 2012). This project has successfully generated synthetic biology standards for the quantitative polymerase chain reaction (qPCR), a highly specific and sensitive analytical platform, in order to increase its robustness for monitoring of host cell processes in an industrial setting. This project has also increased the assay throughput to allow for at-line analysis, in accordance with initiatives such as Process Analytical Technology (PAT) (Gnoth et al., 2007). Analysis was conducted on three commonly used host cell chassis and industrial contamination was also simulated by the addition of plasmid proxies. All assays were optimised by primer design and screening to ensure accuracy. End point PCR (e -pPCR) and quantitative PCR (qPCR) was conducted in the presence and absence of cellular material disrupted by a mild sonication procedure. We found that, whilst cellular material reduces assay sensitivity for a genomic locus, the presence of contaminating species can be accurately quantified. We also employed LRE-qPCR, which uses the CAL1 standard for quantification. LRE-qPCR matched the accuracy of a conventional standard curve qPCR method and we propose it as a Synthetic Biology standard. We next developed a modified standard curve method that streamlined methodology and bypassed errors inherent to the gold standard methodology to, for the first time, enable quantification of multiple targets from a single standard curve. The CyCal curve is a standard curve constructed from the CAL1 standard combined with the Cy0 data analysis. The approach was validated against 6 bioprocess targets and it was found that CyCal was able to replicate the accuracy of the gold standard approach. We then used CyCal to accurately determine how host cell plasmid copy number (PCN) evolves during fermentation. The combination of rapid sample preparation and a universal standard means that CyCal is capable of becoming the basis of an at-line qPCR assay when conducted on modern ultra-rapid qPCR thermocycler technology.

The controlled production of recombinant proteins and their high purity recovery are key goals of biochemical engineering, and can be met by concerted design of cellular hosts using synthetic biology tools. This thesis addresses five process areas with biological solutions. DNA is a major impurity in mammalian process streams, and can be removed by adding nuclease to the feedstream. Here, a HeLa cell line which encodes a recombinant nuclease is successfully adapted to serum-free growth and its activity is characterised. Optimising protein induction within E. coli is a time consuming process; here a synthetic gene network which is predicted to continually produce a recombinant protein once activated is assessed and the host cell line, culture environment, and analytical tools developed. Previously work with a biotherapeutic protein had failed to produce a dimer in a high yield or pure form from E. coli inclusion bodies. A cysteine residue was substituted into the sequence for dimerisation, and a bench scale production process yielding 108 mg of 95% pure dimer was developed. The monomeric form was chemically crosslinked to produce an enhanced stability dimer. DNA increases homogenate viscosity; here, an E. coli strain expressing a SRP-trafficked nuclease is constructed and transformed with a plasmid encoding an SEC-trafficked antibody fragment (Fab'); this strain autohydrolyses DNA, but maintained a high Fab' yield. This contrasts with a previously constructed SEC-trafficked nuclease, which reduced the yield and increased cell lysis. Proteomic analysis showed that utilising two translocation pathways depleted periplasmic proteins and a deterministic model suggested a maximum of 6% of the total periplasm volume could be occupied by recombinant proteins, above which lysis occurred. In order to test this threshold, a promoter-engineered variant of the Fab' production strain was constructed. Bench-scale fermentations showed improved product retention, cell viability, and validated the 6% hypothesis.

The aim of this project is to establish Roseobacter marine bacteria and Trypanosoma brucei (T. brucei) protozoa as synthetic biology chassis. This work addresses the gap within synthetic biology resulting from the limited choice of host cells available for use in practice. This was done by developing synthetic bacterial and trypanosomal genetic regulatory networks (GRNs) which function as an oscillator as well as by developing the necessary protocols and set-ups to allow for the analysis of GRN dynamics within the host. Roseobacter clade bacteria are naturally found in diverse oceanic habitats and have an important ecological role in balancing global carbon levels. This makes Roseobacter an ideal chassis for future efforts to apply synthetic biology to bioremediation and geo-engineering challenges. The aim of this investigation was to establish straight-forward molecular biology procedures in Roseobacter bacteria followed by characterisation and modelling of an E. coli oscillator in Roseobacter. Results showed that Roseobacter synthetic biology is non-trivial. Protozoa are exploited as host cells for industrial production of biotherapeutics due to fast doubling times and host proteins' mammalian-like post-translational glycosylation. As an established model organism for studying protozoa, T. brucei provided a test case for establishing synthetic biology in this phylum for the first time. T. brucei is highly divergent from eukaryotes commonly used in synthetic biology and possesses a sophisticated genomic machinery to evade host immune systems. The establishment of standard synthetic biology approaches in mathematical modelling and gene network design in T. brucei will underpin application of synthetic biology to enhance the industrial capability of the protozoa as a chassis and to probe its pathobiology. This investigation involved design and assembly of a Goodwin oscillator, followed by characterisation and modelling of the network and the development of a novel experimental set-up for live-cell imaging of single motile trypanosomes. Results showed that T. brucei is a promising novel synthetic biology chassis.