Comparison of assays for determination of peptide content for lyophilized thymalfasin*

Abstract

Precise determination of the peptide content in drug substance samples depends highly upon the particular peptide compound and methodology used. Four independent methods were evaluated and compared to determine which would produce the best experimental precision for analysis of thymalfasin (thymosin alpha-1). Four different methods were evaluated including elemental analysis (CHN), quantitative amino acid analysis (AAA), high-performance liquid chromatography (HPLC), and Kjeldahl. This study demonstrates that the AAA method is highly variable in one laboratory while quite precise in another laboratory. Similarly, HPLC results depended on the laboratory conducting the study with more precise values obtained under cGMP. On the contrary, the CHN method yielded highly precise [i.e. <2% coefficient of variation (CV)] values. As precise knowledge of protein content is fundamental for the compounding of final pharmaceutical product of a specific potency, the CHN analysis is recommended for peptide content determination of the drug substance thymalfasin.

Comparative analysis of internalisation, haemolytic, cytotoxic and antibacterial effect of membrane-active cationic peptides: aspects of experimental setup

“…After standard solid phase synthesis and purification, cationic peptides are often isolated as trifluoroacetate salts (Roux et al 2008;Vemuri 2005). In the case of lysine and arginine rich peptide, the high amount of TFA can modify the biological and physicochemical properties (i.e.…”

Solid‐phase Synthesis of a Peptide Derivative of Thymosin alpha1 and Initial Studies on its 99mTc‐Radiolabelling

“…A derivative (1) of the immunopotentiating 28-peptide thymosin alpha1 has been especially designed, so that it can be 99m Tc-radiolabelled, and synthesized following the Fmoc solid-phase peptide synthesis approach. Derivative 1 contains the N-terminal fragment Ta1 [1][2][3][4][5][6][7][8][9][10][11][12][13][14] as a bioactive segment, at the C-terminus of which a 99m Tc-chelating moiety consisting of N a ,N a -dimethylglycine, serine and cysteine is linked through the N e -amino group of a 'bifunctional' lysine residue; the latter is indirectly anchored on the solid-phase peptide synthesis resin through 6-aminocaproic acid (dmGSCK{N e -Ta1 [1][2][3][4][5][6][7][8][9][10][11][12][13][14]}Aca). Synthetic derivative 1 was obtained at high overall yield (approximately 35%) and purity (>95%) and shown to be efficiently radiolabelled with 99m Tc, thus resulting in the first, to our knowledge, so far reported 99m Tc-radiolabelled derivative of thymosin alpha1, which may be eventually used as a specific molecular tool for the in vitro/in vivo study of the mode of action of the parent bioactive peptide.…”

“…Key words: 99m Tc-chelating moiety, 99m Tc-radiolabelling, Fmoc solidphase peptide synthesis, thymosin alpha1 Abbreviations: dm, N a ,N a -dimethyl; dmGSCK{Ta1 [1][2][3][4][5][6][7][8][9][10][11][12][13][14]}Aca, peptide derivative of thymosin alpha1 (¼derivative 1) the primary structure of which is shown in Figure 1; m, N a -methyl; Aca, 6-aminocaproic acid; Boc, t-butyloxycarbonyl; Bu t , t-butyl; DCM, dichloromethane; Dde, 1(4,4dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl; DIC, N,N¢-diisopropylcarbodii-mide; DIEA, diisopropylethylamine; DMF, N,N¢-dimethylformamide; Fmoc, 9-fluorenylmethoxycarbonyl; HOBt, 1-hydroxybenzotriazole; ID, internal diameter; LC-ESI-MS, liquid chromatography-electrospray ionization-mass spectrometry; PDA, photodiode array; ProTa, prothymosin alpha; PyBOP, benzotriazol-1-yloxytripyrrolidino-phosphonium hexafluorophosphate; RP-HPLC, reversed-phase high-performance liquid chromatography; RT, room temperature; SPPS, solid-phase peptide synthesis; Ta1, thymosin alpha1; TFA, trifluoroacetic acid; THF, tetrahydrofuran; Trt, triphenylmethyl (trityl). Thymosin alpha1 (Ta1) is a peptide of 28 amino acid residues ( Figure 1) which has been isolated (1) from an extract of bovine thymus gland known as thymosin fraction 5 (2) and shown in vitro and in vivo immunoenhancing activity (3) as well as a positive effect on the angiogenesis process (4).…”

“…Thymosin alpha1 (Ta1) is a peptide of 28 amino acid residues ( Figure 1) which has been isolated (1) from an extract of bovine thymus gland known as thymosin fraction 5 (2) and shown in vitro and in vivo immunoenhancing activity (3) as well as a positive effect on the angiogenesis process (4). Pharmaceutical formulations of Ta1 are currently being evaluated in the clinic as immunopotentiating therapeutics for hepatitis and cancer (5), either in monotherapy or in combination with interferon (6). Ta1 is presumably the proteolytic product of prothymosin alpha (ProTa), a polypeptide of 109 amino acid residues (human sequence, Figure 1) isolated from rat thymus, the N-terminal fragment of which is identical with Ta1 (7-9).…”

“…Derivative 1 contains the N-terminal fragment of Ta1, Ta1 [1][2][3][4][5][6][7][8][9][10][11][12][13][14], as a 'bioactive segment'. At the C-terminus of this bioactive sequence, the 99m Tc chelating moiety, N a ,N a -dimethylglycine-serine-cysteine (17), is linked through the N e -amino group of a 'bifunctional' lysine residue which has been indirectly anchored on the SPPS resin through an 6-aminocaproic acid residue (derivative 1, dmGSCK{Ta1 [1][2][3][4][5][6][7][8][9][10][11][12][13][14]}Aca, Figure 1). As preliminary studies have indicated, derivative 1 can be directly 99m Tc-radiolabelled with high efficiency.…”