title: Origin of the isoelectric heterogeneity of monoclonal immunoglobulin h1B4.
abstract: The origin of the microheterogeneity of a highly purified antiinflammatory humanized monoclonal antibody prepared in mammalian cell culture has been investigated. This antibody is an IgG directed toward human CD18 (a subunit of leukocyte integrins). When the IgG preparation is subjected to isoelectric focusing, it is found to contain four major species with pI values ranging from 6 to 7. Although the relative amounts of each form differ and some species are present only in small quantities, 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 circular dichroism) or tertiary (intrinsic fluorescence) structure, molecular weight (laser-desorption mass spectroscopy), or antigen binding activity. When each of the isolated species is incubated under conditions which favor deamidation, it is converted to forms of lower pI which appear to correspond to naturally observed species. While the isolated light chain is relatively homogeneous, the heavy chain exhibits a pattern of isoelectric focusing bands similar to that of the intact immunoglobulin. These results suggest that in this case, charge microheterogeneity is due to the sequential deamidation of the immunoglobulin heavy chain.
authors.affiliation: Department of Pharmaceutical Research, Merck Research Laboratories, West Point, Pennsylvania 19486.
authors.forename: P K
authors.initials: PK
authors.lastname: Tsai
authors.forename: M W
authors.initials: MW
authors.lastname: Bruner
authors.forename: J I
authors.initials: JI
authors.lastname: Irwin
authors.forename: C C
authors.initials: CC
authors.lastname: Ip
authors.forename: C N
authors.initials: CN
authors.lastname: Oliver
authors.forename: R W
authors.initials: RW
authors.lastname: Nelson
authors.forename: D B
authors.initials: DB
authors.lastname: Volkin
authors.forename: C R
authors.initials: CR
authors.lastname: Middaugh
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry*; D015703:Antigens, CD / Q000276:immunology*; D018821:CD18 Antigens; D007525:Isoelectric Focusing
chemical_list: D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D018821:CD18 Antigens