Heterogeneity of Monoclonal Antibodies(5)
发布时间:2021-06-07
发布时间:2021-06-07
C-terminal lysine residues can also be separated by HIC60,166and RP.43Treatment with carboxy-peptidase B readily removes C-terminal lysine and reduces charge heterogeneity caused by the partial removal of the C-terminal lysine residues. Deamidation
Nonenzymatic deamidation of asparagines is another common modification of proteins.167,168 Deamidation proceeding through the formation of succinimide intermediate followed by hydrolysis results in the formation of isoaspartate and aspartate in a molar ratio of approximately 3:1.169Succinimide is not stable,but its presence has been reported for several monoclonal anti-bodies.40,52,170Asparagine followed by glycine is by far the most susceptible site for deamida-tion.167,171,172Protein secondary and tertiary structures are also very important in modulating the deamidation rate.173,174In addition,buffer composition,ionic strength,and pH can all affect the rate of deamidation.175–177
For recombinant monoclonal antibodies,deami-dation can occur at any stage from inside the cells,after secretion,during purification,during storage,and under different conditions of stress.Deamidation of asparagines has been reported to be involved in charge heterogeneity of mono-clonal antibodies by the observation of more acidic species after incubating antibodies or their separated fractions under basic pH at elevated temperatures.17,24,26,27,51A more direct method of measurement is the detection of deamidation products such as ammonia,32isoaspartate,26,41 and the change in susceptibility to Asp-N clea-vage.24,122Deamidation sites have been identified by Edman sequencing and LC-MS/MS in several monoclonal antibodies including murine mono-clonal antibodies,26,51,123a CD4-IgG hybrid mole-cule,162and recombinant humanized monoclonal antibodies40,47,52,122,158including in vivo deami-dation of an antibody in the CDR region after administration in monkeys44(Table3). Deamidation of glutamine is kinetically slower than the deamidation of asparagine178and this degradation can only happen enzymatically or under very harsh conditions.Deamidation of a glutamine residue of a recombinant human monoclonal antibody has been reported,158but it was only detected after prolonged stress at408C for6months.
Deamidation of asparagine and glutamine residues increases the molecular weight by1Da. More importantly,deamidation introduces one
Table2.C-Terminal Lysine of Monoclonal Antibodies
Molecules States of C-Terminal Lysine References CD4-IgG1fusion protein Complete removal162 Murine monoclonal IgG2a Partial/complete removal(Hybridoma/ascites)22 Humanized IgG1Partial removal163 Humanized IgG1Partial removal113 Humanized IgG1Partial removal114 Recombinant human monoclonal IgG1Partial removal60 Mouse monoclonal IgG1Partial removal156 Mouse/human chimeric Partial removal29,30 Humanized IgG1Partial removal34,35 Recombinant human IgG1Partial removal36 Murine monoclonal IgG1Partial removal26 Humanized IgG1Partial removal119 Humanized IgG1Complete removal40 Recombinant monoclonal antibody Partial removal126 Recombinant monoclonal antibody Partial removal164 Recombinant monoclonal antibody Partial removal43 Recombinant monoclonal antibody Partial removal44 Humanized IgG1Complete removal122 Humanized IgG1Partial removal46 Recombinant monoclonal IgG1Partial removal63 Humanized IgG Partial removal47 Humanized IgG Partial removal48 JOURNAL OF PHARMACEUTICAL SCIENCES,VOL.97,NO.7,JULY2008DOI10.1002/jps 2430LIU ET AL.
上一篇:作文集序言