Comparison of reversed-phase and anion-exchange high-performance liquid chromatography for the analysis of human...

Separation of human growth hormones (hGH) has been studied by three chromatographic methods: gel filtration chromatography, reversed-phase high-performance liquid chromatography (HPLC) and anion-exchange HPLC. Six growth hormone preparations were used to characterize the systems: two pituitary extracts (an old freeze-dried purified extract A, and a freshly extracted frozen pituitary B); two purified forms (hGH20K and dimer); and two chemically modified monomeric forms (reduced and deamidated). Gel filtration chromatography (pH 8) separated the two pituitary extracts A and B into four components (monomeric, dimeric, aggregate and void material), the relative compositions of which were very similar in both extracts. Reversed-phase HPLC under acid dissociating conditions (pH 2) separated the extracts into four peaks (M1, M2, D and A). The first two components are both monomers: M1 contains all those forms where no major conformational change has occurred; M2 comprises forms with substantial conformational alteration (e.g. disulphide bridge cleavage). Component D includes the interchain disulphide dimer, whilst A is an uncharacterized oligomeric form. Anion-exchange HPLC (pH 8) separated extract A into four regions of immunoreactivity. Region 1 contains hGH20K separated from hGH22K; region 2 includes other monomeric charge variants; region 3 is a broad peak which includes true dimers of hGH20K and hGH22K, as well as loosely aggregated monomer. Region 4 is another broad peak, presumably containing a higher-molecular-weight hGH form or forms. All the hGH forms (identified and unidentified) can be separated by sequential use of two out of three chromatographic methods. No two forms found so far co-eluted in all three systems.