Purification of monoclonal antibodies derived from transgenic goat milk by ultrafiltration.
With the goal of recovering heterologous immunoglobulin (IgG), which comprises 10-15% of the total proteins, from transgenic goat milk at 80% yield and 80% purity, we have developed and tested a two-step membrane isolation and purification process. In the first step, reported earlier by Baruah and Belfort, microfiltration was used to fractionate the milk proteins and recover > 90% of the original IgG at a purity of about 15-20% in the permeate stream. Here, we focus on ultrafiltration (UF) to increase the purity of the target protein to 80%, while maintaining a relatively high IgG yield (80%). Tangential flow UF experiments in diafiltration mode were conducted with 100 kDa cellulosic membranes to evaluate the optimal pH, ionic strength, and uniform transmembrane pressure (TMP). The TMP was kept uniform by permeate circulation in co-flow mode. The traditional approach of conducting the UF process close to the pI of the predominant whey proteins (15-40 kDa, pI 5.2), to transmit these proteins while retaining heterologous IgG (155 kDa), could not be applied here because of precipitation of residual casein at pH values lower than 8.5. Instead, the packing characteristics of the cake layer on the membrane wall, as elucidated in the Aggregate Transport Model presented by Baruah et al. was utilized to achieve a selectivity of > 15, which was sufficient to meet the stated goals of purity and yield for this difficult separation. This combined process is expected to reduce the load on subsequent purification and polishing steps for eventual therapeutic use.
Baruah
Gautam Lal
GL
Howard P. Isermann Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy, New York 12180, USA.
Nayak
Arpan
A
Winkelman
Eric
E
Belfort
Georges
G
D000818:Animals; D030801:Animals, Genetically Modified; D000911:Antibodies, Monoclonal / Q000302:isolation & purification*; D006041:Goats / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007074:Immunoglobulin G / Q000302:isolation & purification*; D008892:Milk; D013678:Technology, Pharmaceutical; D014462:Ultrafiltration
D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G