Accurate topological comparison of two recombinant human growth hormones by optical surface plasmon resonance.
A strategy for the comparison of two recombinant derived human growth hormones (r-hGH) has been developed using surface plasmon resonance (SPR). Statistical analysis was systematically used on the results obtained with several batches derived from two different Escherichia coli strains. Monoclonal antibodies (MAb) directed against four different domains in the tertiary structure of natural human growth hormone were used to compare the epitopic maps of the three (two recombinant and one natural) hGH by SPR analysis. Topological studies show the homogeneity of the epitopic maps of the three hGH. The kinetic parameters, association rate, and dissociation rate constants were also analyzed for the binding of each hGH batch to all MAbs. They were found to be homogeneous between the three hormones. Furthermore, the two r-hGH were compared by more classical approaches examining recognition of lactogenic or somatogenic receptors using, respectively, a bioassay of Nb2 cell proliferation and binding to rat liver microsomes. Specific bioactivities and IC50 values calculated in radioreceptor assays did not significantly differ between different r-hGH. The method was sensitive enough to show slight differences on koff value for one MAb (3C11) between (natural) hormone and two r-hGH. These differences are discussed in relation to previous observation made in the literature and the presence of isoforms in the natural product. The strategy developed here was very useful as a new tool to establish the equivalence of the two r-hGH.
Mani
J C
JC
CNRS UMR9921, Faculté de Pharmacie, Immunoanalyse et Innovation en Biologie clinique, Montpellier, France.
Bras
J M
JM
Agut
C
C
Pau
B
B
Vita
N
N
Ferrara
P
P
Bayol
A
A
D000704:Analysis of Variance; D000818:Animals; D000911:Antibodies, Monoclonal; D001681:Biological Assay; D015374:Biosensing Techniques; D018604:Epitope Mapping / Q000379:methods*; D019382:Human Growth Hormone / Q000032:analysis*; D006801:Humans; D007700:Kinetics; D008862:Microsomes, Liver / Q000378:metabolism; D055095:Optics and Photonics; D011869:Radioligand Assay; D051381:Rats; D017208:Rats, Wistar; D015203:Reproducibility of Results; D013499:Surface Properties; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D019382:Human Growth Hormone