Prevalence and isotypic complexity of the anti-Chinese hamster ovary host cell protein antibodies in normal human serum

Abstract

Host cell-derived protein impurities may be present at low levels in biopharmaceutical products. Antibodies to host cell proteins are present in individuals with no known exposure to these products. In this study, antibodies to drug process-specific Chinese hamster ovary host cell-derived proteins (CHO-HCP) were measured in unexposed individuals using a validated enzyme-linked immunosorbent assay. Samples that tested positive for anti-CHO-HCP reactivity were further characterized to determine the isotypes and IgG subclasses expressed in each positive individual. The specificity of the detected anti-CHO-HCP antibody isotypes was confirmed by the competitive inhibition assay and the uncoated plate specificity testing. These antibody characterization experiments revealed that the prevalent anti-CHO-HCP antibody subclasses were predominantly IgG1 (present in 66.6% of individuals) and IgG2 (60%), with 33.3% positive for IgG3 while IgG4 was detected in low abundance. Forty percent (40%) of the individuals with IgG type reactivity were also identified as IgM-positive. Anti-CHO-HCP-specific IgE was not detected in the assays used in this study. Some individuals exhibited single isotype anti-CHO-HCP reactivity; others contained a combination of multiple antibody isotypes. Data presented in this study demonstrate the isotypic complexity and the high prevalence of anti-CHO-HCP antibodies in human serum with no known exposure to CHO cell-derived therapeutic biologics.

Fig. 1

Prevalence of anti-CHO-HCP reactivity in normal human serum (HS) samples. A validated ELISA was used to detect the reactivity to the drug process-specific CHO-HCP in 83 serum samples collected from individuals with no known prior exposure to therapeutic biologics. a Frequency of normal individual serum samples with anti-CHO-HCP reactivity. b Anti-CHO-HCP reactivity distribution. Mean negative control OD = 0.0762. Plate cut point ODs equal to two times the plate mean negative control ODs

Fig. 2

Confirmation of the positive reactivity to CHO-HCP in normal human serum (HS) samples—competitive inhibition specificity testing. Normal human serum samples were pre-incubated with purified CHO-HCP at concentrations of 0, 10, 100, and 500 μg/ml, respectively, prior to being tested in the anti-CHO-HCP ELISA. The anti-CHO-HCP-negative sample HS-23 was used as negative control for the testing. Observation of the dose-dependent readout signal inhibition in the tested samples indicates specific reactivity to CHO-HCP

Fig. 3

Identification of the IgG4 type reactivity in anti-CHO-HCP-positive serum samples using the biotin mouse anti-human IgG4 detector. IgG4 isotypic cut point OD is defined as two times the mean of the negative sample OD. Samples with OD values greater than the IgG4 cut point were considered positive; samples with OD values lower than the IgG4 cut point were considered negative. Samples were tested at dilutions of 1:10 and 1:25. *Identified positive for IgG4 reactivity

Fig. 4

Confirmation of the specificity of the anti-CHO-HCP antibody isotypes in normal human serum (HS) samples—competitive inhibition specificity testing. Serum samples were pre-incubated with purified CHO-HCP at concentrations of 0, 10/ml, 100, and 500 μg/ml, respectively, prior to being analyzed in the antibody isotyping assays using the mouse anti-human Ig isotype antibody–HRP conjugates detectors

Fig. 5

Confirmation of the specificity of the IgG4 type anti-CHO-HCP antibody in normal human serum (HS) samples—competitive inhibition specificity testing. Serum samples were pre-incubated with purified CHO-HCP at concentrations of 0, 10, 100, and 500 μg/ml, respectively, prior to being analyzed in the antibody isotyping assays using the biotin mouse anti-human IgG4 antibody detector in conjunction with avidin-D-HRP