Heterogeneous crystallisation of therapeutic proteins for downstream processing and structure determination
Creators: name: Ahmed, Sabiyah Jannat
Publisher: name: Imperial College London
Year: 2018

description: This thesis explores the crystallisation of 8 proteins; lysozyme, superoxide dismutase, monoclonal antibody anti-CD20, native state human serum albumin and 4 engineered human serum albumins HSA-0098, HSA- 0354, HSA-0575 and HSA-0311. The application of mesoporous silica nanotemplates for protein crystallisation for both downstream therapeutic protein manufacture and for protein structure determination perspectives are studied in this thesis. Mesoporous silicas were synthesised with controlled pore diameters and narrow pore size distributions to validate the relationship between protein hydrodynamic diameter and nucleant surface porosity previously proposed by Shah et. al. Results confirm that such a correlation influences the propensity of crystallisation demonstrated by all proteins studied here. The thesis reports on the crystallisation of the enzyme superoxide dismutase in concentrations x10 lower than that previously reported in literature, for the enzymes family, and at a x68 lower concentration for the specific enzyme complex CuZnSOD for concentrations as low as 0.2mg/ml. Such low concentrations for protein crystallisation are rarely reported, and indeed are occurring at the lowest levels of supersaturation concentrations. The success of crystallisation was due to the use of mesoporous silica nanotemplates with specific pore size distributions which correlated with CuZnSOD’s hydrodynamic diameter in solution. High quality diffraction crystals with resolution of 2.2Å were produced in solution conditions that failed to grow crystals without silica nanotemplates. A native state polymorph of therapeutic human serum albumins was successfully crystallised to produce diffraction quality crystals with a resolution of 2.8Å. Data was collected for structure determination and successfully solved to reveal a novel polymorphic structure which has never been reported before. The protein revealed a P 21 21 21 space-group unique to human serum albumins. The structure shows a high degree of symmetry with superimposable dimers which sets it apart from the other known structural forms. In addition to crystallisation for structure determination, the crystallisation conditions proved to be very robust and highly reproducible which allowed the construction of a phase diagram to map out the limits of crystallisation within the bounds of the reagent conditions. Further experiments advantageous for process development such as batch to batch reproducibility, product crystal yield were also reported. Attempts to crystallise engineered forms of Human Serum Albumins (HSA) showed varying levels of success. Out of four different engineered samples, one was demonstrated to be crystallisable to diffraction quality of 2.8Å with its resolved structure revealing two octanoate ligands which bind in domain II and IIIA. Other HSA forms showed crystallisation of low crystal quality in the form of spherulites. Nevertheless, these are the first crystals of this kind reported. To understand the interaction occurring between mesoporous nanotemplates and proteins, novel Inverse Liquid Chromatography (ILC) experiments resulted in confirmation that crystallisation does indeed occur within the porous structure as opposed to its surface which has been a common question with this type of porous material mediated crystallisation. The ILC results are two-fold, firstly it demonstrates a novel way to observe actual porosity in a porous material in solution conditions using colloidal gold nanoparticles which are similar to conditions to the crystallisation experiments. Secondly, the resulting pore size distribution can be mapped out before and after protein adsorption which demonstrates unequivocally the protein uptake into the pores. Thus, verifying a key step in the protein crystallisation mechanism of these mesoporous nanotemplates.