Hans Holmegaard Sørensen

Variety in Growth Hormone Determinations due to Use of Different Immunoassays and to the Interference of Growth Hormone-Binding Protein

More than 30 years after their introduction, growth hormone (GH) immunoassays showed the poorest inter-laboratory agreement of the various hormone assays evaluated in 1998 by the UK National External Quality Assessment Scheme, in which different laboratories using different assays reported that analyses of identical samples differed two- to threefold in value. There is therefore an urgent requirement and desire within the scientific community, particularly within centres diagnosing and treating GH deficiency and acromegaly, to resolve this problem and to develop a GH assay(s) that measures solely all of the relevant components of circulating GH immunoreactivity. The main confounders in the estimation of GH levels (now that the use of GH standards other than that recommended by the World Health Organization has largely been eliminated) are GH heterogeneity, anti-GH antiserum binding site specificity and interference from circulating high-affinity GH-binding protein (GHBP). The effects of these factors are closely related. The present study investigates these factors, focussing on the influence of GHBP and antibody binding site specificity on various assays for GH. The findings lead the authors to suggest that a solution to the problem may be to develop a GH assay that measures specifically and solely all serum 22 kDa GH, as this is the major circulating fraction and carries the dominant GH bioactivity.

Impact of GHBP interference on estimates of GH and GH pharmacokinetics

Objective Circulating GH‐binding protein (GHBP) may interfere with GH measurements in immunoassays by competing with the antibodies for ligands. The concentrations of circulating GHBP in humans are closely related to the amount of body fat, and subject to large interindividual differences.

Identification of aprotinin degradation products by the use of high-performance capillary electrophoresis, high-pressure liquid chromatography and mass spectrometry

A preparation of bovine aprotinin, bovine pancreatic trypsin inhibitor, was subjected to high-performance capillary electrophoresis (HPCE) analysis and the purity was calculated to be approximately 80%. The two dominating contaminants were integrated to approximately 7% each as compared to the intact molecule. Characterization by high-pressure liquid chromatographic (HPLC) and mass spectrometric analysis was carried out on digests of the reduced and alkylated molecules. The contaminants were identified as truncated aprotinin, missing one and two amino acids, respectively, at the C-terminus. No such structures were identified in similar amounts in preparations of recombinant aprotinin by HPLC or HPCE.

Recovery of polypeptides after reversed-phase high-performance liquid chromatography☆

Mass recovery of individual polypeptides may be estimated under various practical conditions. With the purpose of obtaining rapid and reliable standard procedures for recovery measurements, we have compared five individual methods utilizing a silica-based stationary phase [Nucleosil C18 (7 microns)/ammonium sulphate-perchlorate-acetonitrile, pH 3.0] and a resin-based stationary phase (TSK Phenyl 5 PW RP/ammonium phosphate-acetonitrile, pH 7.0). The recoveries of insulin (6 kilodaltons), human growth hormone (22 kilodaltons) and human serum albumin (68 kilodaltons) estimated under five different experimental conditions were found to be concordant. Variations in column load, flow-rate, gradient shape and column dwell time and addition of cyclame did not increase the (reduced) recovery of serum albumin and growth hormone.

Alternative mobile phases for the reversed-phase high-performance liquid chromatography of peptides and proteins.

The use of a high content of acetic acid as mobile phase additive for the reversed-phase high-performance liquid chromatography (RP-HPLC) of several proteins and extracts of biological tissues was evaluated for a divinylbenzene (DVB)-based stationary phase, and the separations obtained with acetic acid gradients in acetonitrile, isopropanol or water were compared with classical polypeptide RP-HPLC on silica C4 with trifluoroacetic acid (TFA)-acetonitrile. The separation patterns for recombinant derived interleukin-1 beta (IL-1 beta) on the C4 column eluted with TFA-acetonitrile and the DVB column eluted with acetic acid-acetonitrile were similar, but only the polymeric column was able to separate the components present in an iodinated IL-1 beta preparation. Neither eluent had any harmful effect on the biological activity of IL-1 beta isolated after RP-HPLC. Several standard proteins could be separated when the polymeric column was eluted with acetic acid gradients in acetonitrile, isopropanol or water and, although the separation efficiency with acetic acid in water was lower than that in combination with classical organic modifiers, insulin, glucagon and human growth hormone (hGH) were eluted as sharp, symmetrical peaks. The recoveries of insulin and hGH were comparable for all three mobile phases (80-90%). The separation patterns obtained from a crude acetic acid extract of a normal and a diabetic, human pancreas analysed using acetic acid gradients with or without organic modifiers were found to be similar and comparable to those obtained on a silica C4 column eluted with an acetonitrile gradient in TFA. The principal differences resulted from the use of different UV wavelengths (215 nm for TFA-acetonitrile, 280 nm for acetic acid). Acetic acid extracts of recombinant derived hGH-producing Escherichia coli were separated on the DVB column eluted with an acetic acid gradient in water. Although the starting material was a highly complex mixture, the hGH isolated after this single-step purification was surprisingly pure (as judged by sodium dodecyl sulphate-polyacrylamide gel electrophoresis). Consequently several (pure) polypeptides and complex biological samples were separated on a polymeric stationary phase eluted with acetic acid gradients in water without the use of organic modifiers.

A practical approach to high performance capillary electrophoresis with biosynthetic human growth hormone as a model protein.

Biosynthetic human Growth Hormone (B-hGH) is a protein comprising 191 amino acids. The molecular weight is 22,125 and the isoelectric point is close to pH 5. Due to the ready availability of closely related analogues B-hGH was used as a model protein thus allowing for the demonstration and evaluation of the high resolution capability of high performance capillary electrophoresis (HPCE). The same apparatus was used throughout the experiments and an optimum signal-to-noise ratio was found at 200 nm. Linearity was observed between peak area, retention time and the hGH concentration or sample introduction time. Baseline separation of hGH, desamido hGH and didesamido hGH was obtained. Examples showing analysis with 1 million theoretical plates per meter, high speed separation, simultaneous analysis of multiple samples, sample stacking, hGH tryptic digest, and hGH lysate are reported. The use of electrophoretic velocities instead of apparent velocities for peak identification is illustrated.

High-performance liquid chromatographic separation of membrane proteins isolated from erythrocyte ghosts

Membrane proteins extracted from erythrocyte ghosts with sodium dodecyl sulphate (SDS), 3-(3-cholamidopropyl)-dimethylamminopropane sulfonate (CHAPS) or octylglucoside have been analyzed in various reversed-phase high-performance liquid chromatographic systems. Only SDS was able to solubilize considerable amounts of membrane proteins with mol.wt. greater than 15,000 daltons, but these membrane proteins were recovered in poor yield from a silica-based C4 column eluted with an acetonitrile gradient in trifluoroacetic acid (TFA). A resin-based phenyl column eluted with a similar TFA-acetonitrile gradient was found to be a better choice with respect to the recovery of membrane proteins with mol.wt. greater than 15,000 daltons, and when this column was eluted with an acetic acid gradient with increasing amounts of acetonitrile, erythrocyte ghost membrane proteins solubilized in SDS (mol.wt. 10,000-200,000 daltons) were separated in six major and several minor components with satisfactory recovery.

Biosynthetic Human Growth Hormone Identical to Authentic Material

The major component of human growth hormone (hGH) is a protein with 191 amino acid residues, and a molecular weight of approximately 22,000 D (22K-hGH). A minor component which constitutes about 5% of the more abundant form has a molecular weight of approximately 20,000 D. The minor form is derived from the major by deletion of 15 amino acid residues (32–46 of the 22K-isomer). Both molecules are single stranded, and two disulfide bridges stabilizes the structure. The N-terminus as well as the C-terminus is phenylalanine (Chawla et al., 1983).