Collagen-like block copolymers with tunable design : production in yeast and functional characterisation

Animal-derived collagen and gelatin have been extensively used in the past decades for several pharmaceutical and biomedical applications. However, there is need for collagen-based materials with predictable and tailorable properties. The aim of this thesis is the design and microbial production of gel forming non-hydroxylated collagen-like proteins. Recombinant protein expression and protein engineering are used to develop collagen-like polymers with defined composition, structure, and tunable physical-chemical properties. The possibility of using these proteins as controlled release systems is also explored, as well as the set-up of efficient and scalable production procedures using P. pastorisas a microbial factory. In chapter 2 we describe the genetic design, recombinant production and preliminary characterisation of a new class of ABA triblock copolymers forming thermosensitive gels with highly controllable and predictable properties. Gel formation is obtained by combining proline-rich collagen-inspired (Pro-Gly-Pro)9 end-blocks (T), which have triple helix-forming ability, with highly hydrophilic random coil blocks (Pn or Rn) defining the distance between the trimer forming end-blocks. We report the secreted production in yeast at several g/l of two such non-hydroxylated ~42 kDa triblock copolymers, TP4T and TR4T. The dynamic elasticity (storage modulus) of the gels from these collagen-inspired triblock copolymers was comparable to animal gelatin with a similar content of triple helices. In favourable contrast to traditional gelatin, the dynamic elasticity of the new material, in which only one single (well-defined) type of cross links is formed, is independent of the thermal history of the gel. The novel hydrogels have a ~37 °C melting temperature. However, the thermostability of the hydrogels formed by these polymers can be tailored by changing the number of (Pro-Gly-Pro) repeats. The concept allows to produce custom-made precision gels for biomedical applications. In chapter 3 it was shown that small, but tailored changes in the length of the mid-block of the collagen-inspired triblock copolymers results in significant changes in the viscoelastic properties of the hydrogels. We compared 4 different triblock copolymers, differing only in their mid-block size or mid-block amino sequence. The shorter versions, i.e. TP4T and TR4T, had mid-blocks made of ~400 amino acids, and their longer counterparts, i.e. TP8T and TR8T, ~800 amino acids. These results obtained indicate that the elastic properties of the network are not only a function of concentration and temperature but also of polymer length. The experimental results were well described by an analytical model that was based on classical gel theory and accounted for the particular molecular structure of the gels, and the presence of loops and dangling ends. These results suggest that, by controlling the structure of the present type of hydrogel-forming polymers through genetic engineering their physical-chemical properties can be predicted, and tailored in order to match a specific application In chapter 4 we explored the potential of hydrogels from collagen-inspired triblock co-polymers as drug delivery systems. We studied the erosion and protein release kinetics of two of these hydrogel-forming polymers, i.e. TR4T and TR8T, differing only in their mid-block length (mid-block molecular weights ~37 kDa and ~73 kDa). By varying polymer length and concentration, the elastic properties of the hydrogels as well as their mesh size, swelling and erosion behaviour can be tuned. We show that the hydrogel networks are highly dense and that the decrease of gel volume is mainly the result of surface erosion, which in turn depends on both temperature and initial polymer concentration. In addition, we show that the release kinetics of an entrapped protein is governed by a combined mechanism of erosion and diffusion. The prevalence of one or the other is strongly dependent on polymer concentration. Most importantly, the encapsulated protein was quantitatively released demonstrating that these hydrogels offer great potential as drug delivery systems. The development of efficient large-scale production processes can be a critical factor in whether or not a relevant pharmaceutical material is available in sufficient amounts to be used for application studies and eventually enter human clinical trials and the marketplace. In chapter 5 we describe the development of a pilot-scale process for the fermentation and purification of five collagen-inspired triblock copolymers (TP4T, TR4T, TP8T, TR8T and TP12T) with molecular weights ranging from ~42 kDa to ~114 kDa. P. pastoris strains were grown in a 140 liter bioreactor using a three-phase fermentation process. The fermentation culture reached high cell densities, and all proteins were efficiently expressed and secreted into the fermentation medium at a concentration of ~700-800 mg/l of cell free broth. The downstream processing principles elaborated previously at lab-scale were successfully adapted to the larger scale and resulted in 80-95 % recovery. The purified proteins were intact and showed a similar performance to those obtained using lab-scale procedures. The good productivity and efficient downstream processing (DSP) shown in this study provides a promising perspective towards a potential further scale-up to industrial production of these proteins. In chapter 6 some of the results obtained in the thesis are highlighted and suggestions for further research are given. The contents of this thesis provide a good starting point for future development of this novel class of hydrogel forming collagen-like proteins.