Fig 1.
The schematic structure of human insulin.
Fig 2.
The construction scheme for the expression plasmid pIBAINS.
The resulting expression plasmid pIBAINS contains a hybrid protein SOD::INS gene under the control of the deoP1P2 promoter. Abbreviations used: AmpR, ampicillin resistant; TetR, tetracycline resistant; trpA—transcription terminator trpA; SOD, superoxide dismutase gene; A chain, B chain—chains of human insulin gene; ORI, replication origin.
Table 1.
Nucleotide sequences of primers used for the point mutagenesis reaction.
Table 2.
Changes in amino acid sequence in new insulin analogs in reference to human insulin.
Fig 3.
a-d. Glycemia profiles after single dose administration of tested insulin analogs (GKR, GEKR, SR and AKR), positive (insulin glargine or insulin detemir) and negative (0.9% NaCl) control in streptozotocin-induced diabetic rats. Mean glycemia (± SEM). Star–statistical significance (p<0.05) confirmed in a Newman-Keuls test between one of the tested groups (GKR, GEKR, SR or AKR) and a negative control group (0.9% NaCl) at the individual sampling time point. Diamond–statistical significance (p<0.05) confirmed in a Newman-Keuls test between one of the tested groups (GKR, GEKR, SR or AKR) and a positive control group (insulin glargine or detemir) at the individual sampling time point. N–Number of tested animals per group.
Fig 4.
a-d. The influence of multiple dose administration of tested insulin analogs (GKR, GEKR, SR and AKR) positive (insulin glargine or insulin detemir) and negative (0.9% NaCl) controls on glucose concentration in streptozotocin-induced diabetic rats, together with the period after termination of analogs administration. Mean glycemia (± SEM). Star–statistical significance (p<0.05) confirmed in a Newman-Keuls between one of the tested groups (GKR, GEKR, SR or AKR) and a negative control group (0.9% NaCl) at the individual sampling time point. Diamond–statistical significance (p<0.05), confirmed in a Newman-Keuls test during the administration period or in a t-test after stopping insulin administration, between one of the tested groups (GKR, GEKR, SR or AKR) and a positive control (insulin glargine or detemir) at the individual sampling time point. N–Number of tested animals per group.
Fig 5.
The scheme for development and production of new analogs.
Table 3.
Physicochemical properties of insulin analogs.
Table 4.
Chemical stability of drug product of insulin analogs during storage at 37°C: Formation of related proteins (content of related proteins in total including desamido A21).
Table 5.
Chemical stability of drug product of insulin analogs during storage at 37°C: Formation of covalent dimers and polymers (content of covalent dimers and polymers in total).