Somatotropin
The reagent is a human somatotropin, a recombinant protein with a molecular weight of 22 kDa, which is genetically engineered using an E. Coli BL31 culture. It is a frozen solution with a target protein concentration of 3.5 mg / ml.
As a producer of somatotropin, an E. coli strain containing a multi-copy plasmid of a wide range of rT-type hosts with a human somatotropin gene cloned in it is used. The morphological, cultural, and physico-biochemical properties of the producer strain should be stable during the year of storage in a lyophilized form at a temperature of minus (19 ± 1) ° C.
Control methods:
General requirements for testing reagent - in accordance with the requirements of GOST 27025, TU 9358-001-34341095-2014 and in accordance with test methods approved in the prescribed manner.
Reagent quality control is carried out according to the following indicators: appearance, transparency, authenticity, mechanical impurities, hydrogen ion concentration, protein, sterility, pyrogenicity, toxicity, specific activity, specific activity, producer strain impurities, nucleic acid impurities, reagent purity and its homogeneity, molecular weight.
Подлинность — методом ВЭЖХ — Высокоэффективная жидкостная хроматография. Время удерживания основного пика в растворах и стандартного образца должны совпадать.
The concentration of hydrogen ions (pH) is determined potentiometrically.
Determination of protein - according to the method of Lowry. Before determining the reagent is diluted with water twice.
Sterility (bacterial contamination) — an indicator indicating the complete absence of pathogenic and non-pathogenic microflora, checked by the method of sowing or membrane filtration.
The reagent under test conditions does not have an antimicrobial effect. Microbiological purity test of the reagent is carried out by the two-layer agar method.
Toxicity - an indicator characterizing the adverse effects of the reagent used on animals, is determined in a human L-68 human lung embryo cell line or white mice. When determining the toxicity in white mice, the test dose per animal is 0.2 mg.
The protein content of the E.coli producer strain is determined by enzyme immunoassay
The content of impurities in the DNA of the producer strain and impurities of nucleic acids is determined by real-time PCR. The sensitivity of the method is at least 3 pg. Impurities of non-protein origin are detected by staining the gel with silver.
Посторонние примеси, его гомогенность и молекулярную массу определяют по результатам электрофореза в полиакриламидном геле (ПААГ), проведенного в редуцирующих условиях. В редуцирующих условиях, выявляемое красителем Coomassie Brilliant Blue, содержание соматотропина (ММ 22+0,6 КД) должно составлять не менее 98 %; суммарное количество примесей — димера, тримера и других фрагментов соматотропина не более 2 %.
The volume of the reagent is checked by measuring the volume in a graduated container.
The use of other certified control methods with metrological characteristics not lower than the characteristics of the methods specified in this section is allowed.
Physical and biochemical parameters
- Appearance: odorless transparent colorless solution. Light opalescence and sediment are allowed.
- Authenticity: The retention time of the main peak on the chromatogram of the test solution should be the same as the retention time of the main peak on the chromatogram of a standard sample.
- PH 7.0+/-1
- Protein content not less than 3 mg / ml
- Impurities not more than 2%
- Microbiological indicators of safety and toxicity: the reagent must be sterile, must not be toxic
- Protein content of E.coli producer strain is not more than 200 ng / mg
- Content of DNA impurities of the producer strain, not more than 300 pg per 1 mg of recombinant protein
Safety and environment
The reagent is a sterile substance, fire-and-explosion-proof, and does not have a harmful effect on humans and the environment.
Reagent belongs to hazard class IV according to GOST 12.1.007-76
Microbiological work and work on the preparation of the reagent is carried out in specially adapted isolated rooms (sterile boxes) with the provision of aseptic rules in accordance with technological documents and sanitary and epidemiological requirements.
Transportation and storage
Somatotropin is transported by all types of covered transport in a transport container that ensures the safety of products, in accordance with the rules in force for this type of transport. Storage of the reagent should be carried out at a temperature of 2-8 ° C.
The manufacturer guarantees the compliance of somatotropin with the requirements of this technical specification under the conditions of transportation and storage established by these technical conditions. Expiration date - 2 years from the date of production.
