Mixed polyelectrolyte brush modified adsorbents for the separation of proteins

Theeranan, Thantawat (2020). Mixed polyelectrolyte brush modified adsorbents for the separation of proteins. University of Birmingham. Ph.D.

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Abstract

Bioprocess chromatography plays key roles in the manufacture of biotherapeutic proteins and guarantees the safe removal of critical impurities, but in its present guise future sustainability is questionable. The past decade has seen a conspicuous rise in the application of ‘smart’ or ‘stimuli responsive’ polymers in downstream processing, and especially in adsorption chromatography. This interest is largely driven by the realization that smart polymer based separation systems might afford lean green solutions to endemic defects of conventional chromatographic systems. Temperature and pH responsive varieties are the most obvious candidates for bioprocess application. Effective use of temperature sensitive chromatography media materials in bioprocessing requires the development of specialized equipment. No such requirement affects the application of chromatography media embellished with pH responsive polymers, which have been used extensively, for example in the manufacture of high capacity tentacular ion exchange resins featuring single types of polyelectrolyte (PEL) chains.By sequentially tethering two weak oppositely charged PEL chains, poly(2-vinylpyridine) (P2VP) and poly(methacrylic acid) (PMAA), side-by-side within the pores of beaded meso-macroporous chromatography matrices (Tresyl activated Toyopearl HW65 and Praesto 45) mixed PEL brush adsorbents displaying the 'Chameleon-like' ability to reversibly transform between anion exchange (AEX), and cation exchange (CEX) binding modes, have been created in this study. This involved: (i) prior activation with tresyl chloride (for the Praesto 45 starting material only); (ii) patterning the pores of tresyl-activated media with two amine-terminated weak PELs, i.e. poly(2-vinylpyridine), P2VP (DP = 126) and poly(t-butyl methacrylate), PtBMA (DP = 204) or poly(methyl methacrylate), PMMA (DP = 98) used sequentially for creating mixed PEL adsorbents (grafting PtBMA or PMMA first, P2VP second), and singly for homo PEL supports; (iii) blocking unreacted tresyl functions; and (iv) deprotecting PtBMA and PMMA modified media to remove tertiary butyl protecting groups, thereby liberating the poly(methacrylic acid), PMAA chains.
PEL adsorbents and intermediates in their manufacture were subjected to a battery of physico-chemical and functional characterisation tests, which included: (i) FTIR (for composition and loading of each PEL type); (ii) gravimetry (for polymer loadings); (iii) fluorescence imaging with fluorophore-tagged proteins (to assess uniformity of PEL brush distribution within supports); (iv) zeta potential measurements (to measure points of zero charge and examine pH switching behaviour); and (v) functional protein binding and elution tests using model acidic, neutral and basic proteins.
Loading calibration studies with each PEL chain type provided a simple framework for creating of mixed PEL fimbriated supports with different ‘PMAA:P2VP’ contents and inter-chain spacings in two sequential grafting steps. Successful manufacture of ‘functional’ mixed PEL supports was gauged from the following observations: (i) Smooth reversible charge switching of mixed PEL brush layers on modified Toyopearl supports demonstrated from measurements of zeta potential as the pH was swung back and forth across the point of zero charge; (ii) The point of zero charge of mixed PEL adsorbents strongly correlated with brush composition; (iii) mixed PEL supports displayed ‘chameleon-like’ pH switchable AEX-CEX protein binding-desorption behaviour; (iv) Protein binding and elution performance of mixed PEL brush supports correlated with composition and individual PEL loadings, the balance of AEX-CEX character being clearly determined by the relative populations of the two oppositely charged PELs rooted ‘side-by-side’ in the same surface, and the loading of each type correlates with the support binding and elution performance; (v) The mixed PEL supports also displayed similar and in some cases superior pH mediated elution behaviour to homo PEL supports (e.g. when shifted by 1 or 2 pH units); and (vi) predictable pH mediated chromatographic separation of a model mixture of acidic, basic and neutral proteins under low-salt conditions.Attempts to improve the low operational binding capacity of mixed PEL porous adsorbents for lysozyme, involving utilisation of a shorter chained of PMAA, and a different base matrix (Praesto 45), met with some success, but necessary further increases in binding capacity will likely require a ‘grafting from’ manufacturing approach.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

Supervisor(s):