Preparative purification of human monoclonal antibody isoforms in a multi-compartment electrolyser with immobiline membranes.
The performance of a multi-compartment electrolyser with isoelectric Immobiline membranes for large-scale protein purification is evaluated. Owing to the presence of isoelectric membranes possessing a high buffering capacity and ionic strength, isoelectric protein precipitation inside the membranes, one of the major drawbacks of present membrane uses, is fully avoided. In addition, owing to this novel membrane technology, pH gradient decay, typical of isoelectric focusing in carrier ampholytes, is fully eliminated and pH and conductivity constancy is guaranteed in all flow chambers for running periods of more than 11 days (160,000 V h). The membranes described possess a unique selectivity, in that they act by modulating the surface charge (i.e., the mobility) of macroions crossing or tangential to them. The concept of isoelectric Immobiline membranes acting like a pH-stat unit is introduced. Protein homogeneity in each chamber of the electrolyser can be achieved even when purifying human monoclonal antibodies against HIV-1, which possess high pI values (9.0-9.6), are large molecules (Mr 150,000) and are fractionated in the presence of large micelles of neutral detergents.
Righetti
P G
PG
Chair of Biochemistry, University of Milan, Italy.
Wenisch
E
E
Jungbauer
A
A
Katinger
H
H
Faupel
M
M
D000911:Antibodies, Monoclonal / Q000302:isolation & purification*; D006801:Humans; D007525:Isoelectric Focusing / Q000295:instrumentation / Q000379:methods*; D008567:Membranes, Artificial
D000911:Antibodies, Monoclonal; D008567:Membranes, Artificial