Effect of Excipients on the Stability and Structure of Lyophilized Recombinant Human Growth Hormone

Abstract

We have investigated the effect of mannitol, sorbitol, methyl alpha-D-mannopyranoside, lactose, trehalose, and cellobiose on the stability and structure of the pharmaceutical protein recombinant human growth hormone (rhGH) in the lyophilized state. All excipients afforded significant protection of the protein against aggregation, particularly at levels to potentially satisfy water-binding sites on the protein in the dried state (i.e., 131:1 excipient-to-protein molar ratio). At higher excipient-to-protein ratios, X-ray diffraction studies showed that mannitol and sorbitol were prone to crystallization and afforded somewhat less stabilization than at lower ratios where the excipient remained in the amorphous, protein-containing phase. The secondary structure of rhGH was determined using Fourier transform infrared (FTIR) spectroscopy. rhGH exhibited a decrease in alpha-helix and increase in beta-sheet structures upon drying. Addition of excipient stabilized the secondary structure upon lyophilization to a varying extent depending on the formulation. Samples with a significant degree of structural conservation, as indicated by the alpha-helix content, generally exhibited reduced aggregation. In addition, prevention of protein-protein interactions (indicated by reduced beta-sheet formation) also tended to result in lower rates of aggregation. Therefore, in addition to preserving the protein structure, bulk additives that do not crystallize easily and remain amorphous in the solid state can be used to increase protein-protein distance and thus prevent aggregation.

Characterization of S-layer proteins of Lactobacillus by FTIR spectroscopy and differential scanning calorimetry

“…These are, however, well-established procedures since long ago. Empirical association was made between the position of Amide I band and/or the position and relative areas of component bands after deconvolution and the type and relative content of secondary structure in the protein [22,10,23]. The information obtained by infrared spectroscopy can also be enriched by complementary analysis by other methods.…”

“…The assignment of protein secondary structures to the principal Amide I frequencies was as previously described in literature [22,10,23,30].…”

CE‐ESI‐TOF/MS for human growth hormone analysis

“…Several methods have been developed to analyze these products. Among them, reversed-phase [5,6], ion exchange [6,7], and size-exclusion [6,8] LC have been described. An immunological method has also been developed [9,10] to detect rhGH abuse using a change in hGH isoform ratios [11].…”

Temperature scanning FTIR analysis of secondary structures of proteins embedded in amorphous sugar matrix

“…The resolution factor, K, and half-width of the unresolved bands were 2.8 and 30 cm À1 , respectively, which are in the range of previously published values. 9,[17][18][19][20] The overlapping bands in the deconvolved amide I band region were resolved using a PeakFit software program (SeaSolve Software Inc., Framingham, MA). The resolved bands were assigned to the secondary structural components, as previously described in the literature.…”

“…The resolved bands were assigned to the secondary structural components, as previously described in the literature. 9,[17][18][19][20][21][22] The contents of the secondary protein structures were then calculated from the areas of the resolved bands. The deviations in secondary structure contents in a set of replicates were less than 15% of the average content values.…”