Re amino-acid analysis [AAA]:
An NIH study citing an AAA for GH quantification AND HPLC as a secondary assay for the detection of PEP byproducts.
[The Somatropin Reference Standard (Control 951) of the National Institute of Health Sciences].
Yomota C, et al. Eisei Shikenjo Hokoku. 1996.
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Abstract
Somatropin material was examined for preparation of the "Somatropin Reference Standard". The candidate material was evaluated by a domestic collaborative study in which eight laboratories participated. The protein content was determined to be 4.5 mg/Vial based on amino acid analysis. Because of the possibility of application as a chemical reference standard for assay by the HPLC method, a physico-chemical evaluation of the candidate material was also performed. By SE-HPLC, the content of polymer, dimer were determined to be 0.54%, 0.98%, respectively. By RP-HPLC, the early peak area ascribed to desamido and sulfoxide form was 1.07% of the total peak area. And for informational data, the potency of the candidate material, being estimated by three different biological methods, weight gain assay, tibia test and adiposeconversion assay is 14.8 IU/vial. Based on the above results, the candidate was authorized as the Somatropin Reference Standard of the National Institute of Health Sciences.
Note the standard used to develop the WHO GH criteria was an AAA.
The First International Standard for Somatropin: report of an international collaborative study.
Bristow AF, et al. Growth Regul. 1995.
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Abstract
Following an earlier decision to move away from the in vivo bioassay for determination of the potency of therapeutic somatropin (recombinant DNA human growth hormone), 18 laboratories in 12 countries participated in an international collaborative study designed to establish an international standard for somatropin, calibrated both by bioassay and by physicochemical assays of somatropin content. The mean in vivo biological potency of preparation studied, coded 88/624, was 6.75 IU/ampoule (fiducial limits 6.30-7.23). Determination of the protein content by quantitative amino-acid analysis yielded a mean estimate of 1.98 mg protein per ampoule. (Relative standard deviation = 2.88%). Data from the study also yielded mean values of 97.2% +/- 0.8% for the monomer content of the preparation, and 8.18 (RSD = 4.00%) for A1% at 276 nm. At its 45th meeting, in October 1994, the ECBS of WHO formally established the preparation 88/624 as the First International Standard for Somatropin, with a defined content of 2.0 mg protein per ampoule, and a defined specific activity of 3.0 International Units per milligram.
This study assigned the currently used mg to IU ratio for rHGH.
Although SE/HPLC was the assay
used, quantification once again relied on the FIRST IRS which was based
on a AAA.
The Second International Standard for somatropin (recombinant DNA-derived human growth hormone): preparation and calibration in an international collaborative study.
Bristow AF, et al. Biologicals. 2001.
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Abstract
A preparation of somatropin (recombinant DNA-derived human growth hormone) was prepared as lyophilised ampoules according to WHO procedures for international biological standards. The candidate preparation (98/574) was evaluated in an international collaborative study (16 laboratories, nine countries), with the following aims: (i) to determine the suitability of the preparation to serve as the International Standard for somatropin by studying its performance in the current range of physico-chemical and biological assay methods employed for somatropin; (ii) to assign a content in terms of the existing (first) International Standard for somatropin, using the currently recognised assay procedure (Size Exclusion High Performance Liquid Chromatography, SE HPLC); (iii) to confirm the specific biological activity of the candidate preparation; (iv) to confirm the stability of the candidate preparation. On the basis of the collaborative study WHO agreed that: the preparation in ampoules coded 98/574 is suitable to serve as the next WHO International Standard for somatropin; the preparation in ampoules coded 98/574 should be established as the second International Standard for somatropin, with a defined ampoule content of 1.95 mg total somatropin plus somatropin-related proteins per ampoule; the specific activity of the preparation should be defined as 3.0 IU/mg somatropin.
