Bioanalytical Assays to support Denosumab
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
1. SPEAD method
- 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
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
Somru Value Proposition
PK Assay
- Meaement of Bioactive Denosumab- Somru has developed a method that uses RANKL as the capture for high sensitivity and high specificity for the measurement of bioactive denosumab which is critical for establishment of biosimilarity
- Handling of RANKL- Requires adept and skilled handling of RANKL in the laboratory – Somru years of experience in handing recombinant RANKL and able to transfer this knowledge for seamless execution of the method
- Minimize reagent variability -It is important to limit the number of lots used for RANKL for the entire study. Somru can secure a large lot of RANKL to reduce assay variation within a study for a cost.
- Detection antibody consistency – this method requires highly purified (multi stage purification) detection antibody conjugated to biotin. Somru has developed methods for the generation of detection antibody and conjugation process that generated detection antibody with high lot to lot consistency
Somru Value Proposition
ADA Assay
- This assay requires high drug tolerance. To achieve this high tolerance, Somru has developed SPEAD method that delivers required sensitivity in presence of high drug concentrations
- The assay requires a highly purified positive control. Somru has developed a multi stage purification process that ensures a highly purified positive control that will achieve required sensitivity. Please note due to polycloal nature of the antibody it is expected to have lot to lot variability. We recommend securing sufficient amount of positive control to ensure minimal lot to lot variability. It should be noted that new positive control generation can take 90-120 days
NAb
- The use of a reporter cell-based assay reduces assay variability. For EMA or FDA submission we recommend using reporter cell based NAb assay
- Somru has also developed LBA assay that is sufficient for Indian regulatory submission. The assay is sufficiently drug tolerant. We have also developed a protocol to minimize interference from endogenous RANKL which is a significant issue in most widely published methods
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