Chip-based gel electrophoresis method for the quantification of half-antibody species in IgG4 and their by- and degradation products.
The inter-heavy-chain disulfide bonds of the IgG4 subclass can be described as being at equilibrium with the intra-chain disulfide bonds. This means that a fraction of IgG4 has noncovalently linked heavy chains (half-antibody). The percentage of half-antibodies produced depends upon the expression system used. Nondenaturing assays fail to separate the half-antibodies from the native form because two half-molecules are held together by noncovalent forces. The pharmaceutical industry needs a reliable denaturing assay for checking batch-to-batch consistency. Until now sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) has been the standard method used to detect the presence of half-antibodies. However, this technique is laborious and cannot be automated. Furthermore, cumbersome densitometric measurements are necessary for quantification. To overcome these disadvantages a chip-based gel electrophoresis method was investigated. In the nonreduced format the separation profile is compared with that from SDS-PAGE. The limit of quantification as a percentage of the amount applied, repeatability, reproducibility, and linearity are compared with those of SDS-PAGE. The amounts of half-antibody and of by- and degradation products were determined for several batches by using area percentage and by external calibration with IgG4 as a reference standard. Both methods allow avoidance of error introduction for the quantification as is the case by application of myosin as reference concentration. Both sets of results are compared with each other and with the results from SDS-PAGE. In the reduced format it is noted that the reduction of the inter-heavy-chain disulfide bridges proceeds faster than the reduction of the heavy-light-chain bonds. Therefore optimized conditions are necessary to obtain a complete reduction.
Forrer
Kurt
K
Novartis Pharma AG, Biotechnology Development, 4002 Basel, Switzerland. kurt.forrer@pharma.novartis.com
Hammer
Sandrine
S
Helk
Bernhard
B
D000906:Antibodies / Q000032:analysis* / Q000378:metabolism; D004220:Disulfides; D046209:Electrophoresis, Microchip / Q000295:instrumentation / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D004867:Equipment Design; D007128:Immunoglobulin Fragments / Q000032:analysis*; D007074:Immunoglobulin G / Q000032:analysis / Q000378:metabolism
D000906:Antibodies; D004220:Disulfides; D007128:Immunoglobulin Fragments; D007074:Immunoglobulin G