Purification of glucagon-like peptide 1 analogs
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example 1
tion of Purification Conditions
[0162]Small scale experiments were carried out to identify suitable purification conditions. The seven mobile phase buffers given in column 2 of Table 2 were tested each on four different stationary phases as indicated in line 1, columns 3-6 of Table 2. Each mobile phase buffer was used to prepare a Buffer A consisting of 3% (v / v) acetonitrile in an aqueous solution of said buffer, and a Buffer B consisting of 67 or 80% (v / v) acetonitrile in an aqueous solution of said buffer. The buffer concentrations in the aqueous solutions were between 20 and 400 mM, depending on the nature of the mobile phase buffer.
[0163]Each line of Table 2 represents four different one dimensional RP-HPLC runs, namely one run for each of the four stationary phases tested. For each of said runs, the Buffers A and B prepared with the mobile phase buffer indicated in column 2 the respective line were used. The following general protocol applied: Crude Liraglutide peptide produced ...
example 2
sional RP-HPLC Purification
[0167]The purification involved a chromatographic purification at pH 7.5 in the first dimension, followed by a chromatographic purification under acidic conditions in the second dimension.
[0168]A 5 cm MODcol column (Grace) packed with C8-bonded silica (approx. bed-depth 32 cm) was used on a preparative HPLC system (Knauer HPLC pump 1800) with detection at 220 nm (Knauer smartline UV detector 2500) and an automated fraction collector (Büchi C-660). The same stationary phase was used in both dimensions of the purification protocol. Crude Liraglutide produced by Fmoc SPPS (purity>60%) was used as a starting material. The sample was loaded on the column at a flow rate of 90 ml / min. The detailed elution protocols for each step are given in Tables 3 and 4 below. The buffer concentration in the aqueous part of the mobile phase used in the first dimension was 20 mM.
TABLE 3Parameters first purification dimensionSample loading bufferaqueous ammonium phosphate pH 7.5...
example 3
sional RP-HPLC Purification
[0171]The purification involved a chromatographic purification at pH 7.7 in the first dimension, followed by a chromatographic purification under acidic conditions in the second dimension.
[0172]A 5 cm MODcol column (Grace) packed with C8-bonded silica (approx. bed-depth 32 cm) was used on a preparative HPLC system (Knauer HPLC pump 1800) with detection at 220 nm (Knauer smartline UV detector 2500) and an automated fraction collector (Büchi C-660). The same stationary phase was used in both dimensions of the purification protocol. Crude Liraglutide produced by Fmoc SPPS was used as a starting material. The sample was loaded on the column at a flow rate of 43 ml / min (1st dimension) or 64 ml / min (2nd dimension). The detailed elution protocols for each step are given in Tables 5 and 6 below.
TABLE 5Parameters first purification dimensionSample loading bufferaqueous ammonium phosphate pH 7.7Buffer A3% (m / m) acetonitrile, aqueousammonium phosphate pH 7.7Buffer B6...
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