Human growth hormone separation

Although gradient elution is generally required for RPLC separations of proteins, isocratic elution can be successful in some instances. For example, isocratic elution has been used for the determination of purity of production batches of biosynthetic human growth hormone (HGH).42 The method was used to... 

McNerney, T. M., Watson, S. K., Sim, J. H., and Bridenbaugh, R. L. (1996). Separation of recombinant human growth hormone from Escherichia coli cell pellet extract by capillary zone electrophoresis./. Chromatogr. A 744, 223 — 229. 

Peptide mapping (the separation of the tryptic digest) of /i-lactoglobulin and human growth hormone (hGH). 

G. Teshima and E. Canovadavis, Separation of oxidized human growth hormone variants by reversed-phase HPLC-effect of mobile phase pH and organic modifier, J. Chromatogr., 625 201 (1992). 

R. C. Chloupek, W. S. Hancock, and L. R. Synder, Computer simulation as a tool for the rapid optimization of the HPLC separation of a tryptic digest of human growth hormone, J. Chromatogr., 594 65 (1992). 

COLUMN STABILITY. The absence of a porous support structure results in enhanced column stability at elevated temperature and pH even with micropellicular sorbents prepared from siliceous supports (14). This is illustrated by the chromatogram in Figure 5 which shows the separation of minor conformers of human growth hormone by using a moderately alkaline mobile phase (pH 8.5). Prior to obtaining the above chromatogram, the column was perfused with 4000 column volumes of the mobile phase at 80°C, yet no noticeable changes in retention behavior, separation efficiency and sample recovery had been observed with respect to initial column performance. 

Because it contains 191 amino acid residues, although in a single chain, human growth hormone presented a more difficult problem than insulin. Two separate segments of cDNA had to be constructed, each inserted and joined in a vector, to obtain the entire coding sequence for the protein. 

Capillary isoelectric focusing (CIEF) separates proteins based on differences in their isoelectric points. CIEF has been used to separate product-related impurities of recombinant proteins, mainly deamidated species, such as those of human growth hormone.71... 

The idea of using CIEF to separate bound from free antibody was realized for the first time by Shimura et al. [25], These authors quantified human growth hormone (hGH) using tetramethylrodamine/iodoacetamide-labeled anti-hGH Fab fragment. Excess fluorescendy labeled Fab was added to solutions with variable amounts of hGH and, upon incubation, Fab -Ag complex was separated from the excess Fab by CIEF. LIF detection was used, which provided a very low detection limit of 5 x 10 12 M of methionyl hGH. In this report different forms of Ag were detected simultaneously in one run, since complexes of labeled Fab with non-, mono-, and di-deaminated variants of met-hGH exhibited different isoelectric points and hence could be resolved by CIEF. 

As an illustration of HIC technique, the recombinant human growth hormone (hGH) and methionyl hGH (met-hGH) were well-separated by the HIC technique [14]. The optimized conditions were found to be IM ammonium phosphate dibasic, pH 8.0/propanol (99.5 0.5) and 0.1 M sodium phosphate dibasic, pH 8.0/propanol (97.5 2.5) for mobile phase A and B, respectively, with a descending gradient from 100% A to 100% B in 30 minutes at a column (TSK-phenyl 5PW, 75 x 7.5 mm) temperature of 30°C. Note that the addition of a small amount of propanol as organic modifiers significantly decreases elution time while maintaining resolution and efficiency. This HIC method allowed separation of several hGH variants from the main hGH peak while retaining their native structures. 

Fig. 1 Tryptic maps of the intact and (insets) degraded forms of recombinant human growth hormone (RHGH). The separation was achieved on a Nucleosil C-18 column (150 X 3.9 mm inner diameter) with a 120-min mobile-phase gradient from 10 mM potassium phosphate (pH 2.85) to 60% acetonitrile in the starting buffer. The flow rate was 1 mL/min and detection was at 214 nm. [Reprinted from J. Frenz, W.S. Hancock,...

Electrophoretic elution and "switch" monoclonal antibodies are combined in a new rapid recycle method an affinity-mediated membrane transport process reported by Dall-Bauman and Ivory (8). In this modeling paper, a "switch" monoclonal antibody incorporated into a supported liquid membrane is used to facilitate the transport of human growth hormone from a high-pH to a low-pH environment. Electrochemical effects, including Donnan equilibria between the membrane and external environments, and imposition of external electrical fields, significantly affected the flux of protein across the membrane. Experimental confirmation of the simulation results could introduce affinity-mediated transport as a powerful new biospecific separation method. 

Arcelloni, C. Fermo, I. Banfi, G. Pontiroli, A.E. Paroni, R. Capillary electrophoresis for protein analysis separation of human growth hormone and human insulin molecular forms. Anal.Biochem., 1993, 212, 160-167... 

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