Somru developed the following comprehensive list of bioanalytical assays to support Denosumab biosimilar development
- Pharmacokinetic Assay (PK) assay
- ADA assay
- Binding ADA assay
- Cell-based NAb assay(s)
- Ligand Binding NAb Assay
- Pharmacodynamic Assays
- sCTX
- TRAP-5b
- P1NP
PK Assays to support Denosumab
- Somru has developed an assay to support the pharmacokinetic measurement of Denosumab
- This assay utilizes the target (RANKL) as the capture and anti Denosumab for detection
- Sensitivity – 25 ng/mL ELISA
- Range: 2000 – 25 ng/mL
ADA Assay to support Denosumab
- SPEAD method involves extraction of ADA from solid phase bound to biotin-Drug
- The assay utilizes acid dissociation
- Depending on lot of positive control, ELISA sensitivity is less than 100 ng/ml using a polyclonal rabbit affinity purified antibody
- Drug tolerance – 20 µg/mL
1. SPEAD method
2. Somru does not recommend the use of SPR method due to the sensitivity requirements of regulatory authorities and matrix effect
Neutralizing Antibody Assay
Cell-based Nab Assay
- Utilizes RAW 267.4 cell line – This is macrophage-like, Abelson leukemia virus transformed cell line derived from BALB/c mice. The end point is measurement of TRAP using an ELISA assay. The assay is highly variable. To overcome this, we have developed a reporter-cell line based NAb assay.
- Reporter-cell line-based NAB assay – utilizes modified cell line with reporter gene utilizing RANK/RANKL interaction to verify neutralizing antibody
- Ligand binding NAb assay – Somru has also developed a ligand binding method for the detection of NAbs.
- Sensitivity – 500 ng/mL
Pharmacodynamic Assay
Somru has validated antibody pairs for the measurement of the following biomarkers in ELISA or ECL format in a functionally relevant manner to support Denosumab biosimilar development
- sCTX
- TRAP-5b
- P1NP