It is highly advantageous to be able to develop bioprocesses early on in the product design lifecycle, where strategic options and alternatives can be considered cheaply and effectively. Resources are however often in very short supply at this point in the process, typically with very limited quantities of feedstock available and with minimal access to capital equipment. As such there is significant advantage in being able to develop chromatographic processes at very small scales that will only require small quantities of feedstock and can utilise common laboratory equipment. If the height of the packed bed is not maintained during scale up then it becomes difficult to predict chromatographic behaviour and efficiency. This seriously limits how small a chromatography system can be and still be representative of how a process scale chromatographic separation will behave. Ultra scale-down (USD) methodologies for chromatography take a different approach. A small scale device, which may or may not have geometric similarity to the process scale equipment, generates data which when combined with a specific methodology and mathematical model allows the prediction of the large scale equivalent. The work in this thesis sought to develop a USD methodology and model to accurately predict large scale chromatographic behaviour using very small scale devices. The development of a model separation was required to act as a source of realistic experimental data for the development of a USD methodology. This model separation required a suitable feedstock, a suitable USD device and a suitable sorbent on which to perform the separation. It was found that the most suitable feedstock of those tested was a FAb fragment containing periplasmic lysate produced in E. coli, due to the ease of pre-chromatographic processing and industrial relevance. Several designs of very small column were investigated and it was found that PRC pre-packed columns (Pall Life Sciences) had superior separation characteristics and were therefore selected as the USD device of choice. This was combined to produce a viable separation process using MEP HyperCel presented in the PRC pre-packed column format with FAb fragment containing periplasmic lysate as a feedstock. A linear pH gradient produced a clearly resolved two peak system with excellent FAb fragment purity that was deemed very suitable as a reference separation for the USD methodology development. The premise of the USD methodology was the deconvolution of each relevant peak within an elution profile into its four curve coefficients, namely height, width at half height, skew and peak location. These four curve coefficients for each peak in the small scale chromatogram could then be individually modelled using a transformation function into the large scale equivalents and then recreate a large scale chromatogram prediction from these values. This was also used to predict how the chromatograms will change with respect to altering the packed bed height and linear velocity of the loading and elution steps. The methodology that was developed was shown to be effective and was typically accurate to under 5% difference normalised root mean square when the predicted large scale chromatograms and real large scale data was compared. The methodology was further validated by testing with a range of different chromatographic systems and processes. These included changing the feedstock by reducing the FAb fragment titre by 50%, changing the chromatographic ligand to PPA and also a multi-variate change that altered the ligand, the sorbent backbone and the feedstock all at once. The transformation functions were found not to be generic and required system specific alterations. However, the methodology itself was shown to be very effective across a wide range of chromatographic conditions and systems and as such would be a good basis for ultra-scale down development in elution chromatography.

Biocatalysis continues to be a powerful tool for the efficient synthesis of optically pure pharmaceuticals that are difficult to access via conventional chemistry. The efficient application of biocatalysis requires the availability of suitable enzymes with high activity and stability under process conditions. However, the borderline stability of biocatalysts in many types of reaction media has often prevented or delayed their implementation for industrial-scale syntheses of fine chemicals and pharmaceuticals. Consequently, there is great interest in understanding the effects of solution conditions on protein stability, as well as in developing strategies to improve enzyme stability and activity in desired reaction media. The enzyme transketolase (TK; E.C. 2.2.1.1) from Escherichia coli is an important biocatalyst in stereo-specific carbon-carbon bond synethesis. The power of transketolase is further augmented when the bioconversion takes place in a multi-step biotransformation in which transketolase and transaminases are employed in series to create chiral amino alcohols from achiral substrates. These compounds are synthetically very useful in the production of a range of compounds with pharmaceutical application. Although many useful reactions have been reported for TK, many of the substrates and products are unstable or insoluble at the pH or temperature range for which the enzyme has optimum activity in aqueous media. Understanding the activity and structural stability of transketolase under bioprocess conditions will Biocatalysis continues to be a powerful tool for the efficient synthesis of optically pure pharmaceuticals that are difficult to access via conventional chemistry. The efficient application of biocatalysis requires the availability of suitable enzymes with high activity and stability under process conditions. However, the borderline stability of biocatalysts in many types of reaction media has often prevented or delayed their implementation for industrial-scale syntheses of fine chemicals and pharmaceuticals. Consequently, there is great interest in understanding the effects of solution conditions on protein stability, as well as in developing strategies to improve enzyme stability and activity in desired reaction media. The enzyme transketolase (TK; E.C. 2.2.1.1) from Escherichia coli is an important biocatalyst in stereo-specific carbon-carbon bond synethesis. The power of transketolase is further augmented when the bioconversion takes place in a multi-step biotransformation in which transketolase and transaminases are employed in series to create chiral amino alcohols from achiral substrates. These compounds are synthetically very useful in the production of a range of compounds with pharmaceutical application. Although many useful reactions have been reported for TK, many of the substrates and products are unstable or insoluble at the pH or temperature range for which the enzyme has optimum activity in aqueous media. Understanding the activity and structural stability of transketolase under bioprocess conditions will improve our capacity to comprehend and ultimately to engineer it to make it work at a broader range of pHs and temperatures, and also in the presence of organic co-solvents. This will potentially help to reduce process development times and also increase the stability and solubility of substrates and products. To provide further insight into the underlying causes of TK deactivation in process conditions, the effects of temperature, pH and organic solvents on the structure, stability, aggregation and activity of Escherichia coli transketolase were characterized in Chapters 3 and 4. The results provided useful information for the engineering of TK enzymes with improved thermostability or extreme pH tolerance and in organic solvent mixtures. For thermostability and tolerance to low pH, mutations may be usefully targeted towards regions of protein sequence predicted to have a high propensity for aggregation. For the retention of biocatalytic activity at high pH or temperatures, stabilisation of the cofactor binding loops were found to be an attractive target. By contrast, the results in aqueous-solvent mixtures instead implied that the solvent dependence of catalytic activity cannot be simply explained by only one mechanism such as active-site binding or the replacement of water molecules, and that the effect of different solvents on protein structure penetration, denaturation and aggregation must also be considered. In the final Chapter, mutagenesis was targeted to the cofactor binding loops to further evaluate their impact on thermal stability. one mutant was found that successfully improved the stability of E. coli transketolase at elevated temperatures, giving a 3 fold specific activity increase at 60 oC compared to wild-type TK.

The effect of uncertainty in biopharmaceutical manufacturing can be a barrier to robust, scalable process design. The ideal is for a process in development to complete technology transfer to full scale manufacturing with no redevelopment costs or surprises. Essential to achieving this is a systematic method for analysing large complex datasets and extracting critical combinations of fluctuations that lead to product loss and scheduling delays. This thesis describes a dynamic database-driven decision-support tool to facilitate such efforts and identify robust optimal purification strategies to match the high productivity cell cultures whilst coping with uncertainties. The benefits of a databasedriven approach using MySQL (MySQL AB, Uppsala, Sweden) are harnessed to capture the process, business and risk features of multiple biopharmaceutical purification sequences in a multi-product facility and better manage the large datasets required for multiple processes, uncertainty analysis and optimisation. Principal component analysis combined with clustering algorithms are used to analyse the complex datasets from complete batch processes for biopharmaceuticals. The challenge of visualising the multidimensional nature of the dataset was addressed using hierarchical and k-means clustering as well as parallel co-ordinate plots to help identify process fingerprints and characteristics of clusters leading to facility fit issues. Industrially-relevant case studies are presented that focus on tech transfer challenges for therapeutic antibodies moving from early phase to late phase clinical trials.

Ultra scale-down (USD) approach is a powerful tool to predict large-scale process performance by using very small amounts of material. A new USD membrane tool for mimicking large scale crossflow filtration (CFF) would allow preliminary evaluation to be conducted at the early stages of process development to give an indication of the processability of the biomaterials, and provide crucial data for identifying optimal operating parameters with lower development cost. This thesis reports the development of an ultra scale-down shear device, method for USD mimicking, method for large scale performance prediction as well as method of verification based on an industrial relevant and complex feed material, E. coli lysate containing antibody fragment (Fab’). The Pellicon 2 labscale system (Millipore Corporation, Bedford, MA) is used as the benchmark for the mimic evaluation which can readily be scaled to small pilot or industrial scale. Large scale CFF was attempted to be scaled down with a rotating disc filter in recycle mode previously. However, the volume of feed material required cannot be reduced effectively, and the resulting loading ratio of the feed volume to the membrane area was large, which led to long experimental time. In addition, the operating conditions such as transmembrane pressure were hard to maintain due to the small size of membrane and the inaccurate monitor of the permeate flow, hence the accurate operation at USD cannot be achieved. A new mimicking method has overcome these difficulties. Adopted from the pulsed sample injection technique by Ghosh and Cui (2000), the rotating disc filter has been modified by building in inserts to allow the flexibility of the chamber volume, so that a large volume reduction can be achieved and only 1.5 mL of processing material is required for each diafiltration experiment. The USD mimicking method uses the modified rotating disc filter operated in dead-end mode, processing material is prefilled in the shear chamber and only buffer is pumped in through inlet port. The new set-up is much simpler and accurate control of operating conditions has been achieved. As the establishment of shear rate correlation between USD and labscale is essential, a specific correlation between shear rate on membrane surface and disc rotating speed for USD has been generated by CFD simulations with high accuracy and validated by Laser Doppler Velocimetry. The correlation between wall shear rate on the membrane and the inlet flow rate in labscale cassette is based on the developed analytical models, which can be readily modified and used for membrane cartridge systems of different designs and scales. Flux model regarding TMP and shear rate has been established using USD mimicking method based on E. coli lysate. Recognising the difference of system configuration between USD and large scale, e.g., the existing of screen on both side of the membrane in cassette, a new flux prediction method is developed firstly to accurately calibrate the system resistance in large scale CFF with water flux tests, and secondly to predict large scale performance accurately with USD model inputs. The important benefit is that the method allows accurate predictions with a simple procedure, so it can be widely applied. The transmission prediction method has also been developed based on mass balance technique. It has been found that the transmembrane pressure and shear rate do not show significant impact on the transmission of antibody fragment, but the ionic strength of the solution has strong influence on the observed transmission. Predicted transmission data agrees well with the experimental results of a labscale diafiltration.

This thesis focused on the development of ultra scale-down (USD) approaches to predict dewatering levels of high cell density yeast cell suspensions in centrifuges. Two yeast cell and feed types were used: Baker’s yeast suspensions and P.pastoris fermentation cultures. P.pastoris was grown to high cell densities equivalent to that obtained in industry (2 100 g/L DCW). Two methodologies were successfully developed and verified at scale (p-value 2 0.05). A small-scale (~ 15mL) USD methodology was described to mimic dewatering levels of two pilot-scale centrifuges with intermittent solids discharge, a tubular-bowl CarrpowerfugeTM P6 centrifuge and a disc-stack CSA-1 machine, in ready available laboratory centrifuge tubes. This was followed by a separate USD device, designed to recreate dewatering conditions in a continuous solids discharge pilot-scale centrifuge, in particular a scroll decanter centrifuge (SDC), at very small scale (2 mL). The basis of both USD approaches was to use a feed concentration that would mimic the maximum final height of wet solids experienced in the pilot-scale machine; and to use the same range of equivalent residence times (Gt) practised at pilot-scale. The USD device simulated two different dewatering operations that occurred in a SDC: compression and decanting. The validation of the use of this USD device was successfully undertaken with high cell density Baker’s yeast suspensions (p-value 2 0.05). Finally, the first developed USD methodology was further used on a case study where the impact of the choice of P.pastoris cell strain during fermentation was assessed on centrifugation performance. This work therefore presented simple and novel methodologies that were performed in the laboratory and that required small quantities of feedstock material (~ 1 5ml) hence reducing the need for repeated pilot-scale runs during early stages of bioprocess development.

Lignin is an essential component of the cell wall of various types of plants and represents an abundant and renewable natural resource. Both thermo-chemical and biological pre-treatment can be applied to break down the strong ether bonds and phenylpropanoid polymer subunits present in lignin. These liberate a range of phenolic compounds which represent potential substrates for bioconversion by ω-transaminases (ω-TAm). In this work the utility of the CV2025 ω-TAm from Chromobacterium violaceum DSM30191 is explored for selective amination of lignin breakdown intermediates into value-added products. Eight potential ω-TAm substrates were initially screened using (S)-α-methylbenzylamine (MBA) as the amino donor. Vanillin was identified as the best potential substrate which is converted into vanillylamine, an intermediate in the preparation of pelargonic acid vanillylamide used as a hyperemia inducing active substance in wound dressings. At low vanillin and MBA concentrations (<10mM) and with an excess of the amine donor (1:4 mol/mol) 100% w/w conversion of vanillin into vanillylamine was observed within 60 min. At vanillin concentrations above 10 mM, inhibition was observed, decreasing the rate and yield of the bioconversion. A kinetic model of the bioconversion was subsequently established, based on the ping-pong bi-bi mechanism, which indicated that the reaction product, vanillylamine and by-product (acetophenone) were also inhibitory. Mutant libraries prepared by saturation mutagenesis at ten amino acid residues located in the active site of the CV2025 TAm enzyme were next evaluated in order to enhance the activity and increase the substrate range. A microwell-based high-throughput screening approach (300 µL scale) identified one particular mutant, R416Q, that increased both the rate of the vanillin bioconversion and those involving substrates (acetovanillone and p-hydroxyacetophenone) that would lead to formation of chiral amine products. For the vanillin bioconversion, the R416Q mutant displayed reduced inhibition from both vanillylamine product and the acetophenone by-product. Finally, fed-batch operation in larger scale, pH-controlled, stirred bioreactors (15 mL) was examined in order to overcome vanillin substrate inhibition. With the wild-type ω-TAm fed-batch bioconversions (feeding high concentrations of both vanillin and MBA) enabled a doubling of the final product concentration obtained. Introduction of the R416Q mutant ω-TAm with the fed-batch process enabled a further 3-fold increase in the final product concentration. Through this combination of enzyme engineering and bioprocess engineering technologies a 93.8% w/w substrate conversion was achieved at a final product concentration of 17.3 g.L-1 (112.6 mM) after three consecutive batches within 12 hr. These results demonstrate the potential for bioconversion of lignin breakdown products into value-added chemicals but illustrate the need for enzymes with improved substrate range and reduced sensitivity to product inhibition.

Sterile filtration is considered as a final step in processing pharmaceutical grade plasmid DNA. During the development of filtration process, fundamental understanding on the mechanism of fouling and the DNA degradation is critical to improve filtration performance. This study focuses on the fouling and degradation of large plasmid DNA (> 20 kb) in sterilising grade filters. Scanning electron microscopy (SEM) was applied to investigate the mechanism of fouling of pGEc47 plasmid DNA (56 kb) in sterile filter. The investigation contributes to the fundamental understanding of the behaviour of large plasmid molecules during filtration through 0.22 µm membrane. The SEM images suggested that the fouling was due to entrapment of plasmids on the surface of membrane that created meshes of plasmids DNA. The severe form of the superposition of DNA meshes blocked the entrance of pores and restricted the passage of other incoming plasmid molecules. The observations of cross sections of the membrane showed that after filtration with 200 µg of plasmid, the blockage of internal pores was detected. Quantitative analysis of the progression of fouling using digital image processing technique suggested that the transition of fouling occurred. During filtration of 50 µg plasmid, the blockage was due to superposition of DNA molecules. However, after filtration of 200 µg plasmid, complete blockage of the pores was observed. In order to understand the blockage of membrane by plasmid DNA, the mechanism of fouling of pQR150 (20 kb) and pGEc47 (56 kb) plasmids during constant pressure filtration inside 0.22 µm PVDF membrane is experimentally investigated. The decline of filtrate flux as function of time is analysed using the framework of classical and combined blocking models (Bolton et al., 2006). The results for both plasmids indicate a transition between fouling mechanisms. Initially, during early part of the filtration, the intermediate blocking model provided the best fit of the experimental results suggesting that fouling of the membrane was mainly caused by deposition of particles onto its surface. Afterwards, the result trends were best captured by the standard blocking model indicating that internal fouling of membrane was the dominant fouling mechanism. A study of the transmission (Cf /C0) of both plasmids shows a significant reduction of plasmid transmission which coincides with the transition of the fouling mechanism from intermediate to standard blocking. The study elucidates the applicability of filtration blocking model to explain the fouling behaviour of large plasmid DNA during sterile filtration. The loss of plasmid is also related to the degradation of supercoiled (SC) plasmid to other topologies such as open circular and linear DNA. The degradation is correlated to fluid stresses in bioprocessing equipments that may contribute to the breakage of the phosphodiester bond between bases in the double-helical structure of DNA. The computational fluid dynamics (CFD) simulation was carried out to estimate the magnitude of elongational strain rate inside 0.22 µm polyvinylidene fluoride (PVDF) membrane which is a critical for plasmid DNA degradation. The results were compared with the critical elongational strain rate for DNA breakage and the experimental data that correlates with the loss of 20 kb plasmid DNA. Two approaches have been developed to determine the strain rates inside the membrane, which are the macro- and micro-scale models. During the filtration of plasmid at 5 and 8 psi transmembrane pressures, the macro-scale model estimated the average elongational strain rates up to 1x103 s-1. Furthermore, the micro-scale model detected wide range of local elongational strain rates up to 1x105 s-1. However, local elongational strain rates below ~4x103 s-1 were detected in most of regions of the membrane, which is below the critical elongational strain rate for DNA breakage.

Enzymes are used in home and personal care applications for the removal of dirt and grease from surfaces. Of these, laundry cleaning utilizes the most enzymes. Technological innovations in the application of enzymes may provide solutions to challenging substrate-surface problems. Poor stability in detergents and high non-specific binding to cloth fabric suggests there is a need for improved lipase technologies. Metagenome analysis of soil bacteria is a successful approach to identifying novel enzymes with improved properties. Novel lipases were found to have improved performance over standard commercial lipases, as well as reduced non-specific binding. Literature suggested that immobilization of lipases on hydrophobic surfaces results in hyperactivation and improved thermostability. Lipases immobilized on a highly hydrophobic surface of fungal hydrophobins resulted in hyperactivation towards large mono- and triglycerides. A matrix of plant polysaccharides, proteins and lipids immobilized on cloth fabric was characterized. Various enzymes active towards components of the cell wall matrix were suggested for its degradation. Subtilases hydrolyzed chlorophyll binding proteins, releasing most chlorophyll. Pectin lyase released chlorophyll, supporting a mechanism whereby polysaccharide matrix loosening enables the release of chlorophyll. Chlorophyllase was ineffective in reducing the redeposition of chlorophyll on cloth fabric. The washed plant matrix was found to contain residual hemicellulose, therefore xylanases and accessory hemicellulases were hypothesized and tested as effective solutions. Calcium ions hinder the protease wash removal of proteinaceous matrices such as the immobilized plant matrix. The recent move to concentrated liquid detergents leaves little formulation space for metal ion chelators, therefore there is a need for improved protease technologies. Calcium ions were found to significantly reduce the susceptibility of bovine serum albumin and milk proteins to proteolytic degradation. Alternative protease specificities were hypothesized to provide a solution to this problem. A calcium-dependent metalloprotease (thermolysin) outperformed standard commercial subtilase in an environment high in calcium ions.

A novel dual resin based, substrate feed and product removal (SFPR) strategy has been investigated to overcome the substrate and product inhibition in an industrially important Baeyer-Villiger monooxygenase catalysed bioconversion in order to enhance the productivity of the bioconversion process. The bioconversion of the ketone substrate, bicyclo[3.2.0]hept-2-en-6-one, to the lactone products, (1R,5S)-3-oxabicyclo[3.3.0]oct-6-en-2-one and (1S,5R)-2-oxabicyclo[3.3.0]oct-6-en-3-one, catalysed by a recombinant whole cell biocatalyst, Escherichia coli TOP10 [pQR239], expressing cyclohexanone monooxygenase from Acinetobacter calcoaceticus, was used as the model reaction to prove the feasibility of the novel dual resin SFPR concept. Before the application of the dual resin SFPR strategy to the Baeyer-Villiger bioconversion, adsorption of the ketone and lactone onto non-specific resins was investigated. Several resins were initially characterised at the bench scale by determining adsorption isotherms for the ketone and lactone compounds. Thereafter adsorption isotherms were generated via a high throughput resin screening (HTRS) method using both 96 wells and 24 wells microplate platforms. Comparison of the adsorption isotherm data between the bench scale and the two HTRS platforms, together with results of resin mixing in wells of the 96 wells and 24 wells microplate platforms, as investigated by high speed imaging experiments, shows that the 24 well microplate platform was the most suitable to investigate adsorption kinetics of the ketone and lactone on the resins. Based on the adsorption studies, resins Dowex® Optipore L493 and Amberlite® XAD7 were chosen to be separately used for substrate feeding in the dual resin SFPR strategy. Dowex® Optipore L493 was chosen for its high capacity of 0.21 g/gadsorbent for ketone, whereas Amberlite® XAD7 was chosen for its high selectivity of ketone over lactone compared to any other resin. Amberlite® IRC50 was chosen for lactone removal in the dual resin SFPR strategy because of its high selectivity of lactone over ketone than any other resin. To demonstrate the feasibility of the dual resin SFPR strategy, the Baeyer-Villiger bioconversion was performed in shake flasks and compared to bioconversions without the use of resins and with the use of a single resin based SFPR strategy. At an initial ketone concentration of 3g/l, both resin based strategies performed significantly better than the bioconversion performed without resins. The dual resin SFPR strategy, carried out with both types of resins free in suspension without spatial separation, also showed improvement compared to results obtained with the single resin SFPR strategy. The dual resin SFPR strategy was also performed with the spatial separation of the two resins by housing one of the resins in a porous bag. This allowed observation of the majority of lactone product adsorbed onto the Amberlite® IRC50 resin as expected based on adsorption studies. Carrying out the Baeyer-Villiger bioconversion with the implementation of the dual resin SFPR strategy in shake flasks saw an increase of productivity compared to the Baeyer-Villiger bioconversions carried out without resins by as much as 132% and in comparison with the single resin SFPR strategy by as much 10%, thus demonstrating a ‘proof of concept’ of the novel dual resin SFPR bioconversion strategy. After demonstrating a ‘proof of concept’ for the dual resin SFPR strategy, its application in a miniature stirred tank bioreactor was investigated to open the way for scale up studies. Two configurations were investigated, namely the conventional reactor system where both resins were added directly into the bioreactor, and the recycle reactor system where a column housed one of the two types of resins. Using resins with low adsorption capacities and the need of an extra resin type in a dual resin SFPR strategy, makes the recycle reactor configuration a more attractive system, however it was the conventional reactor configuration that performed better than the recycle reactor system. The L493-IRC50 combination in the conventional reactor configuration achieves a 21% greater productivity than in the recycle reactor. The dual resin SFPR strategy using the L493-IRC50 combination performed better than any other resin based SFPR strategy when carried out with both resins in the reactor. It reached a productivity of 0.85g/l/h after 2.5 hours of reaction, 5% higher than the productivity achieved with the single resin strategy in the conventional reactor configuration. The novel dual resin based SFPR strategy and the HTRS method developed in this work has the potential to be applied in any bioconversion that needs to overcome substrate and product inhibition.

While the potential of pluripotent cells and their role in the future of regenerative medicine has rapidly evolved since the derivation of human embryonic stem cells in 1998, ethical issues surrounding the derivation and clinical use of pluripotent cells still remain. Somatic cell reprogramming offers an ethically preferred, potentially patient specific method for deriving pluripotent cells, but this technology is based on integration of genes that have been linked to oncogenesis and therefore limit clinical usefulness. This project, started in late 2005, has explored new methods towards achieving somatic cell reprogramming with the specific goal of reprogramming human somatic cells without altering genomic DNA. Through the use of cytoplasm from pluripotent cells, total RNA from pluripotent cells, and specific mRNAs coding for known reprogramming factors, attempts to reprogram somatic cells were made, along with the goal to better understand the process of reprogramming and the associated gene expression changes that catalyse it. To gauge reactivation of the embryonic genome, an Oct4-GFP fibroblast reporter line was successfully established. A protocol for the isolation of membrane encapsulated, nuclear DNA free pluripotent cell cytoplasm, or cytoplasts, was developed. Following fusion of cytoplasts to somatic reporter cells resulted in temporary OCT4 activation, but no pluripotent cells were isolated. Subsequently, an electroporation protocol was developed and optimised to transfect total RNA and total mRNA from pluripotent cells into somatic cells, in place of cytoplasts. This method successfully showed temporary upregulation of key pluripotency genes, but not full reversion to a pluripotent state. In 2006, it was shown that only four factors (OCT4, SOX2, cMYC, and KLF4) are required for somatic cell reprogramming, but the issue of DNA manipulation remained. Synthetically produced mRNA coding for the key reprogramming factors was then made and transfected into human fibroblast cells. It was found that transfected mRNA can successfully upregulate specific genes of interest, including pluripotency factors, in a more controlled and predictable manner than DNA. Although mRNA only causes upregulation for 3-4 days, in some cases lasting changes on endogenous expression of pluripotency genes were detected, including OCT4. This work shows that mRNA transfection can be a useful tool for temporary upregulation of specific gene expression and, with further optimisation, may provide a method for catalysing somatic cell reprogramming without genetic alteration. Additionally, mRNA has potential as an important tool for differentiation, transdifferentiation, and pluripotency studies.