Novel purification process of gonadotropin
A gonadotropin, hydrophobic technology, applied in the field of gonadotropin improvement
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Embodiment 1
[0095] Embodiment 1: Purification of recombinant FSH
[0096] Step 1: Cell isolation and reconditioning
[0097] After batch harvest, cells were first separated from the culture broth by centrifugation followed by depth filtration in order to obtain a clarified supernatant containing the protein under study along with other soluble contaminants. Centrifugation was performed at about 10,000 g x 30 minutes. pass A 0.45 0.22 μm membrane was depth filtered for further clarification. The clarified supernatant was reconditioned to adjust to the next affinity column equilibration buffer conditions such as pH and conductivity. This step is not required when gonadotropins obtained from urine are to be purified.
[0098] Step 2: Affinity Column Chromatography
[0099] After reconditioning, the clarified supernatant was passed through the affinity column matrix to selectively capture the desired protein. The affinity matrix is subjected to midi column washes prior to elution of ...
Embodiment 2
[0120] Example 2: Purification of Urinary HCG
[0121] Step 1: Affinity Column Chromatography
[0122] After reconditioning, the u-HCG crude mixture was passed through the affinity column matrix to selectively capture the desired protein and thereafter eluted. The affinity matrix is subjected to midi column washes prior to elution of the desired protein. The desired protein was eluted from the column at acidic pH. The column chromatogram is shown in Figure 8 middle.
[0123] Step 2: Ultrafiltration - Diafiltration and Reconditioning
[0124] Proteins eluted from the affinity column were reconditioned by UF / DF to match the next column (Q column) step equilibration buffer conditions using a 10kDa MWCO membrane filter for a pH 7.0 buffer of low ionic strength (e.g. , pH and conductivity). The diafiltered protein solution was passed through a 0.22 μm filter before loading onto the Q column.
[0125] Step 3: Virus Inactivation
[0126] The diafiltered protein solution wa...
Embodiment 3
[0135] Example 3: Purification of urinary FSH
[0136] The purification method of u-FSH was carried out in the manner as described in Example 2. Purified u-FSH exhibited a single band purity in the gel as assessed by SDS-PAGE ( Figure 11 ) and greater than 98% purity as assessed by HP-SEC ( Figure 12 ).
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