ORIGIN OF THE ISOELECTRIC HETEROGENEITY OF MONOCLONAL IMMUNOGLOBULIN H1B4

The origin of the microheterogeneity of a highly purified antiinflamma tory humanized monoclonal antibody prepared in mammalian cell culture has been investigated. This antibody is an IgG directed toward human C D18 (a subunit of leukocyte integrins). When the IgG preparation is su bjected to isoelectric focusing, it is found to contain four major spe cies with pI values ranging from 6 to 7. Although the relative amounts of each form differ and some species are present only in small quanti ties, each has been isolated by a combination of high-resolution anion -exchange chromatography and isoelectric focusing. Comparative studies reveal no detectable differences in overall secondary (far UV circula r dichroism) or tertiary (intrinsic fluorescence) structure, molecular weight (laser-desorption mass spectroscopy), or antigen binding activ ity. When each of the isolated species is incubated under conditions w hich favor deamidation, it is converted to forms of lower pI which app ear to correspond to naturally observed species. While the isolated li ght chain is relatively homogeneous, the heavy chain exhibits a patter n of isoelectric focusing bands similar to that of the intact immunogl obulin. These results suggest that in this case, charge microheterogen eity is due to the sequential deamidation of the immunoglobulin heavy chain.