Improved recovery of insulin-like growth factors (IGFs) from bovine colostrum using alkaline diafiltration.
Studies to develop a rapid, bioprocess-compatible method to recover low-molecular-mass growth factors from bovine colostrum are reported. Defatted bovine colostrum was subjected to tangential-flow filtration (TFF) using two different filters [polyether sulphone (PES) and regenerated cellulose (RC)] at pH 5.8, pH 8.0 and pH 8.0+0. 01 M NaCl. Single-pass TFF at pH 5.8 using a 100 kDa RC filter resulted in the loss of approx. 90% of insulin-like growth factor I (IGF-I) to non-specific filter adsorption. Comparison of 30 kDa RC and PES filters under single-pass conditions showed that yields of IGF-1 and IGF-II were highest with RC filters. Yields of IGF-I and protein from both filter types were increased at pH 8.0 and were greatest for the 30 kDa RC filter. Effects of adding large diluent volumes continuously during TFF (diafiltration) were tested at pH 5. 0 and 8.0. The use of 10 diafiltrate vols. at pH 8.0 resulted in the recovery of 15-28% of colostral IGF-1 from the RC 30 kDa permeates, 2-4-fold greater than under acidic conditions. Yields of IGF-II (39.6%) were unaffected by pH and at least 97% of total protein was retained by the 30 kDa filter at pH 8.0. Denaturing SDS/PAGE analysis of the alkaline RC 30 kDa permeates demonstrated two major regions of stained proteins at 10-13 kDa and 17-19 kDa. Acidic TFF permeates contained additional stained proteins at approximately 90, 48 and 37 kDa. Isoelectric focusing of these samples demonstrated the presence of proteins with isoelectric points of 8.2 and 8.6. The current study demonstrates a one-step bioprocess-compatible technique for the recovery of low-molecular-mass polypeptides from bovine colostrum. By using alkaline diafiltration with RC filters TFF provided optimal recovery of IGF-1 from colostrum.
Hossner
K L
KL
Department of Animal Sciences, Colorado State University, Fort Collins, CO 80523-1171, USA. khossner@agsci.colostate.edu
Yemm
R S
RS
D000468:Alkalies; D000818:Animals; D002417:Cattle; D003126:Colostrum / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007334:Insulin-Like Growth Factor I / Q000737:chemistry / Q000302:isolation & purification*; D007335:Insulin-Like Growth Factor II / Q000737:chemistry / Q000302:isolation & purification*; D008851:Micropore Filters; D008970:Molecular Weight; D010539:Permeability; D012492:Salts / Q000494:pharmacology; D014462:Ultrafiltration / Q000379:methods*
D000468:Alkalies; D012492:Salts; D007334:Insulin-Like Growth Factor I; D007335:Insulin-Like Growth Factor II