OR03-2 Anovel GH1 mutation resulting in deletion of amino acids 188–190 is associated with retarded growth

Abstract

There is therefore need for a platform technology which can generate variable clearance of biological drugs. Hypothesis: Does the introduction of increasing numbers of N-linked glycosylation motifs between two growth hormone molecules lead to variable increased half-life whilst retaining biological activity? Methods: GH tandem molecules with variable glycosylated linkers were cloned, sequenced and expressed in a CHO cell line. SDS-PAGE was used to verify increases in molecular weight and an in house dual luciferase reporter assay used to test bioactivity. Protein was purified using immobilised metal affinity chromatography (IMAC). In vivo PK analysis of GHT-0 (no glycosylation control), GHT-5 (contains 4 glycosylation motifs) and rhGH (recombinant human GH) was conducted by giving a single s.c. injection of 4nMoles specific protein/Kg/animal to male Wistar rats. Serum samples were taken routinely over a 120hrs period and analysed for both GH and IGF-I levels by immunoassays. Total body weight, fat-pad weights and liver weights were also analysed (full ethical approval was granted). Results: SDS-PAGE analysis showed that with increasing number of glycosylation motifs there was a concomitant increase in molecular weight (MW). An in increase in MW was observed from 42kDa for GHT-0 up to 60–70kDa for GHT-5. GH tandem GHT-0 and GHT-5 were purified using IMAC from suspension adapted serum free cultures (1.9mg from 1L GHT0, 1.5mg from 700ml GHT5). Both proteins showed comparable bioactivity to rhGh when used at 5nM [6.53 (SE 0.63), 5.65 (SE 0.27), 6.36 (SE 0.30)]. In vivo PK analysis showed no differences in IGF-I levels. No differences were also observed in total body weight, fat-pad weight and liver weight between groups. GH analysis of serum samples showed that increasing glycosylation using GHT-5 had a positive effect on delaying clearance compared to GHT-0 and rhGH. GHT-5 was still detected 24hrs post injection giving a peak detection of 0.48nM (SE 0.11) at 8 hrs post injection. GHT-0 and rhGH both showed a peak detection at 1hr post injection of 0.88nM (SE 0.17) for rhGH and 0.92nM (SE 0.25) for GHT-0 respectively and could not be detected at 8hrs post injection. Conclusion: It is possible to increase MW of hormone tandems using glycosylated-linkers whilst maintaining bioactivity and that this also improves the PK profile of the molecule in rat model system providing a potential platform technology for generating long acting biologicals.