Protein nucleation and crystallisation on novel 3-D templates

Lead Research Organisation: Imperial College London
Department Name: Chemical Engineering


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.

Technical Summary

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.


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Description Can crystallise proteins at lowest ever achieved concentrations, allowing high process efficiency and close alignment to solution concentrations provided by bioreactors

Can customise nano-template design for any potential target protein requiring separation and purification based on knowledge of the protein dimensions and its chemistry

Can selective separate protein mixtures including recombinant proteins

Can crystallise proteins not possible by any other technique

Can use flow crystallisation approaches in small tubes/capillaries for potential scale up to crystallisation based

Also screen for habit and polymorph discovery /control using nanonucleants

Have demonstrated success of this approach using a range of therapeutically relevant proteins including Insulin, MIS12,Thaumatin, Concanavalin A

Have crystallised 16 different proteins from 14kDa to >1MDa including some not previously crystallised.
Exploitation Route To use these approaches to crystallise industrially relevant proteins.
Sectors Chemicals,Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

Description BBSRC 2012 BRIC Doctoral Programme Application
Amount £90,000 (GBP)
Funding ID BB/K020994/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 09/2013 
End 10/2017
Description Enhancement of Protein Crystal Growth and Quality by Solution Flow through Glass Tubes
Amount £30,000 (GBP)
Funding ID BB/FOF/PF/13/08 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 01/2009 
End 03/2009
Description Protein Crystallisation of MAb's
Amount £90,000 (GBP)
Organisation University College London 
Sector Academic/University
Country United Kingdom
Start 09/2014 
End 10/2018
Description Collaboration with Industry 
Organisation Fujifilm
Department Fujifilm Diosynth Biotechnologies
Country United States 
Sector Private 
PI Contribution Established solution conditions for probable MAB's crystallisation Also determined second virial coefficients for these MAB's Established the aggregation propensity for these MAB's
Collaborator Contribution Provision of unique but research critical protein samples from Fuji BioSyn UK
Impact Support for 2 PhD studentships
Start Year 2008