Conformational effects in the reversed-phase chromatographic behavior of recombinant human growth hormone (rhGH) and N-methionyl recombinant human growth hormone (Met-hGH)

Abstract

This paper examines the retention behavior of recombinant DNA-derived human growth hormone (rhGH) in reversed-phase chromatography and its separation from the closely related N-methionyl variant (Met-hGH). It is first shown that retention for rhGH decreases with increasing column temperature when 1-propanol (1-PrOH) is used as organic modifier. On the other hand, retention increases with temperature when acetonitrile (CH3CN) is employed. The differences in behavior for the two organic modifiers could be related to conformational changes in the protein as determined by solution and adsorption intrinsic fluorescence spectroscopy. Specifically, desorption and elution of rhGH using 1-PrOH could be correlated with a solvent-induced conformational change, with retention decreasing with increasing temperature due to the increasing ease of structural alteration. On the other hand for CH3CN the increase in retention correlated with temperature rise was related to a partial structural change yielding a more hydrophobic species. In this case, a surface-driven process is suggested. The work then turned to the separation of rhGH and Met-hGH where it was found for both organic modifiers optimum separation occurred at 45 degrees C and pH 6.5. Separate studies revealed that during the conformational change Met-hGH appeared more hydrophobic than rhGH since protein-protein aggregation was observed at a lower 1-PrOH concentration. It is suggested that this hydrophobic difference, which was optimized under the conditions cited above, resulted in the separation. The study demonstrates the importance of conformational changes in retention behavior and separation of protein samples.

Human Growth Hormone Adsorption Kinetics and Conformation on Self-Assembled Monolayers

“…The observed maximum at 338 nm for hGH in solution might be the result of the polarity which arises from an H-bond of the tryptophan with an aspartic acid residue. 29 As can be seen from both the λ m shifts and the quenching constants, relatively large changes are observed for adsorption onto the OTS and DDS surfaces. These were also the surfaces for which hGH showed the highest affinity.…”

Adsorption Kinetics, Conformation, and Mobility of the Growth Hormone and Lysozyme on Solid Surfaces, Studied with TIRF

“…The concentration and intensity are both a function of the distance from the interface into the cell. The observed fluorescence signal, S, can therefore be written as [3] in which f is an instrumental factor characterized by the efficiency in which the fluorescence is detected.…”

“…The fluorescence of TRP emanates from molecules in solution which are excited by the evanescent wave and can be derived by integration of Eq. [3]. The size of an adsorbed protein is two orders of magnitude less than the penetration depth and therefore the protein concentration profile can be considered as a quantity equal to the surface concentration, Γ p , positioned at z = 0.…”

Hydrophobic interaction chromatography selectivity changes among three stable proteins: conformation does not play a major role

“…The potential for conformational changes and yield losses induced by HIC media can further complicate process development. Protein conformational changes are known to occur when exposed to the strong hydrophobic surfaces of reversed phase chromatography (RPC) (Benedek et al, 1984;Cohen et al, 1985;Lu et al, 1986;Gooding and Regnier, 1990;Oroszlan et al, 1992;Fernandez, 1999, 2001;). Losses of enzyme activity (Kato et al, , 1986Josic et al, 1986) and low chromatographic recovery (Ichinose et al, 1985;Fausnaugh et al, 1984a,b;Kato et al, 2002) have also been reported for some HIC media, even though they are the much less hydrophobic than RPC resins.…”