Protein nucleation and crystallisation on novel 3-D templates

The crystallisation of biopharmaceuticals is poorly understood and is a rarely used commercial process for the primary separation and purification of proteins. This proposal describes a fundamental investigation into the nucleation and crystallisation of proteins via the development of novel solid templates with known surface chemistry and topography for their controlled heterogeneous nucleation and crystal growth. These templates will enhance the success rates for protein crystallisation/ nucleation, allowing an improved understanding of both processes as well as potentially forming the basis for the development of industrial bio-crystallisation processes. This proposal represents the first step towards a major new downstream opportunity for biopharmaceutical manufacturing, providing potentially major cost and productivity benefits in product separation and purification, as well as the major product stability and delivery advantages offered by crystalline products over traditional product/dosage forms. The ultimate success of this strategy would give the UK industry an international competitive advantage in biopharmaceutical processing.

The direct crystallisation of proteins from fermentation broths is an industrially attractive route for protein manufacture. This proposal describes an integrated and innovative research programme for the improved understanding of the effects of both surface chemistry and topography on heterogeneous protein nucleation and crystallisation via the use of novel templates. The main objectives of this study include: 1. The use of specific surface chemistry in combination with precise surface topographical features to allow novel surface templates to be created. 2. Use of these novel templates for protein crystallisation studies 3. An improved understanding of protein nucleation via novel detection methods. 4. Improved protein crystallisation fundamentals to enable the control and optimisation of bioprocessing. The methodologies to be employed include: 1. Sub 100 nm surface topographies will be templated onto surfaces by a PDMS stamping technique and via colloidal particle arrays. Other features will be fabricated via an anodisation approach. 2. A wide range of controllable surface chemistry's to be controlled via an established method; the self-assembled monolayers (SAMS). Surface characterisation of the templates will include wettability, FTIR, zeta potential, SEM, AFM, TEM. 3. A Quartz Crystal Microbalance capable of detecting depositions of nanogram levels protein onto the surfaces will monitor crystal nucleation, as well as measuring the viscoelastic properties of the protein layer. 4. The protein structure, morphology, habit and purity will be characterised for the crystals obtained. Our hypothesis is that these novel protein crystallisation templates will be superior to current nucleation media and methodologies. Coupled with an improved understanding of the fundamentals of protein nucleation and crystallization, these templates could directly, or indirectly, facilitate direct crystallisation in the reactor broth.