Purpose: SKY59 is a humanized recombinant monoclonal antibody directed against complement component C5, which exists at high concentration in circulation. A direct ELISA is typically used to quantify a therapeutic antibody. However, when relevant amount of soluble target exists in circulation, this assay may underestimate the values. The purpose of our study was to develop and validate an assay that measures the total drug capable of binding to the target in the presence of a high concentration target, C5, in cynomolgus monkey plasma. Moreover, we aimed to develop and validate the format as a total target assay for C5, which was required for pre-clinical study.
Methods: The total drug assay format is shown in Figure 1. Goat anti-human IgG pAb was used as the capture antibody. SKY59 in standard, QC, and plasma samples (diluted 1:100) was pre-incubated with C5 from plasma to form an antigen-antibody immune complex. The immune complex was captured on the plates coated with anti-human IgG, and then cynomolgus monkey C5 was added to saturate the binding sites on SKY59. A rabbit anti-C5 mAb that did not compete with SKY59 and a POD-labeled anti-rabbit IgG pAb were added for detection. ABTS was used for visualization. All incubation steps were performed at room temperature under shaking at 600 rpm. In the total C5 assay, C5 in standard, QC, and plasma samples (diluted 1:25000) were incubated with an excess amount of SKY59 to form an immune complex. The immune complex was captured on the plates coated with rabbit anti-C5 mAb which is the same antibody as that used in the total drug assay. POD-labeled anti-human pAb and ABTS were used for detection.
Results: The total drug assay method was validated by assessing calibration range, accuracy and precision, selectivity, prozone effect, dilutional linearity, target tolerance, stability, and by incurred sample re-analysis according to FDA guidance. The calibration range was from 0.05 to 3.2 ug/mL in plasma. Between-run and within-run accuracy and precision met the acceptance criteria. This method showed no prozone effect and good dilutional linearity up to 1/150000. High target tolerance was also shown. The accuracy and precision of the determined value in samples containing 400 ug/mL of C5 were within 100%±10% and within 10% (CV), respectively. Thus, this method was able to measure a wide concentration range of SKY59 in the presence of C5. The total C5 assay method was validated by assessing calibration range, accuracy and precision, selectivity, prozone effect, parallelism, drug tolerance, and stability. The calibration range was from 6.25 to 400 ug/mL in plasma. Each validation item satisfied the criterion set for evaluating C5 levels in pre-clinical study. The total drug assay and the total target assay were used to measure samples in pre-clinical studies in cynomolgus monkey and were used to assess exposure and analyze changes in target levels.
Conclusion: A total drug assay was developed and validated that could quantify SKY59 in the presence of a high concentration of its target, C5, in cynomolgus monkey plasma. A total target assay for quantifying C5 that was required for pre-clinical study was also developed and validated. Both assays were utilized in pre-clinical studies to assess exposure and to evaluate change in target concentration.
Hiroo Watanabe– Chugai Pharmaceutical Co., Ltd.
Norihito Shibahara– Chugai Pharmaceutical Co., Ltd., Gotemba
Masahiko Nanami– Chugai Pharmaceutical Co., Ltd
Naoaki Murao– Chugai Pharmaceutical Co., Ltd.
Masaki Ishigai– Chugai Pharmaceutical Co., Ltd.