Anti-Human IL 12/23 (Briakinumab) Dylight® 488

Research-grade Briakinumab biosimilars are commonly used as calibration standards or reference controls in pharmacokinetic (PK) bridging ELISAs to enable accurate measurement of drug concentration in serum samples. These biosimilars are produced to closely match the originator drug’s biochemical and biophysical properties, enabling precise and comparable quantification of both biosimilar and reference product concentrations.

Usage in PK Bridging ELISA:

• Calibration Standard Construction:

  • The biosimilar is serially diluted in a relevant matrix (often human serum) to create a standard curve across a wide concentration range (e.g., 50–12,800 ng/mL).
  • Each run includes multiple points of the standard curve, which serves as the reference for quantitating unknown samples.
  • Using a well-characterized research-grade biosimilar as the calibrator ensures that assay quantification directly relates to the structure and epitope of the therapeutic antibody being measured.

• Reference Controls and Assay Validation:

  • The same biosimilar batch may serve as a reference control to validate the assay’s accuracy and precision and to enable cross-platform or cross-lot comparisons.
  • Both the biosimilar and the originator/reference product are spiked into serum matrix and assayed using the calibration curve created with the biosimilar standard to assess bioanalytical equivalence.
  • Regulatory and best practice guidelines recommend using a single analytical standard (commonly the biosimilar candidate) for both the biosimilar and originator comparability assessment. This minimizes variability and eliminates the need for complex cross-calibration or correction factors.

• Bridging ELISA Principle:

  • In the typical sandwich ELISA, anti-Briakinumab capture antibodies (which may be the biosimilar itself or a different specific anti-idiotype antibody) are coated onto the plate.
  • Serum samples and biosimilar standards are added; Briakinumab in the samples or standards binds to the capture antibody.
  • A secondary, often enzyme-labeled, detection antibody is added to complete the sandwich and allow quantitation by colorimetric readout, which is directly proportional to Briakinumab concentration.

Summary Table: Biosimilar Use in PK ELISA

Additional Notes:

• The biosimilar should be highly purified, stable, and characterized (with low endotoxin, high purity, and defined concentration).
• Performing method qualification ensures that the single-standard approach provides equivalent measurement for both test and reference products, as required for regulatory submissions.
• The use of a single calibration standard (the biosimilar) for both biosimilar and reference product minimizes assay variability and simplifies validation during biosimilar PK studies.

In short, research-grade Briakinumab biosimilars serve as both calibration standards and reference controls in PK bridging ELISAs—providing consistency, reducing variability, and enabling direct measurement of drug levels in serum samples for both biosimilar and reference comparability studies.

Standard Flow Cytometry Protocol Using Conjugated Briakinumab Biosimilar

Objective:

To validate the expression levels or binding capacity of IL-12/IL-23 p40 using a conjugated Briakinumab biosimilar (e.g., PE or APC-labeled) in flow cytometry.

Protocol Overview:

1. Preparation of Cells:

   • Sample Collection: Collect cells from the appropriate source (e.g., peripheral blood, tissue cultures).
   • Cell Preparation: Ensure cells are in a single-cell suspension and in a suitable buffer for flow cytometry, such as PBS with 0.1% BSA.

2. Staining:

   • Blocking: Pre-treat cells with Fc block (e.g., human FcR Blocking Reagent) to prevent non-specific binding.
   • Primary Antibody Staining: Add the PE or APC-conjugated Briakinumab biosimilar antibody to the cells. The recommended concentration for staining is typically ≤ 1.0 μg per 10^6 cells in a volume of 100 μl.
   • Incubation: Incubate the cells with the antibody at 4°C for 30 minutes to 1 hour.
   • Washing: Wash the cells with PBS + 0.1% BSA to remove excess antibody.

3. Flow Cytometry Analysis:

   • Instrument Setup: Use a flow cytometer compatible with the fluorophore used (e.g., PE requires an excitation wavelength around 488 nm and emission detection around 575 nm).
   • Gating Strategy: Gate on the population of interest based on forward/side scatter. Use appropriate gating controls (e.g., unstained cells, isotype controls) to set the threshold for positivity.
   • Data Analysis: Analyze the fluorescence intensity of the gated population using software like FlowJo or BD FACSDiva.

4. Controls:

   • Isotype Control: Use an appropriate human IgG1 isotype control to account for non-specific binding.
   • Unstained Control: Include a sample without the conjugated antibody to set the fluorescence threshold.

5. Optimization:

   • Perform titration experiments to determine the optimal concentration of the conjugated antibody for each specific application.

Applications and Considerations:

• ELISA and Neutralization Assays: Beyond flow cytometry, the Briakinumab biosimilar can be used in ELISA and neutralization assays to further characterize IL-12/IL-23 activity.
• Cellular Targets: The antibody binds to the p40 subunit of IL-12/IL-23, which is produced by cells like dendritic cells and macrophages.

Storage and Handling:

• Store the conjugated Briakinumab biosimilar according to the manufacturer's instructions, typically at 2-8°C for short-term storage and at -20°C for long-term storage. Avoid repeated freeze-thaw cycles.

Biopharma companies typically perform a variety of analytical assays to confirm the structural and functional similarity of a proposed biosimilar to the originator drug. These assays include:

Structural Assays

• Primary, Secondary, and Tertiary Structure Analysis: Techniques such as mass spectrometry (MS), nuclear magnetic resonance (NMR), and circular dichroism (CD) are used to assess the primary, secondary, and tertiary structures of proteins.
• Peptide Mapping: This involves analyzing the peptide fragments of a protein to assess its primary structure and post-translational modifications.
• Glycosylation Analysis: Techniques like high-performance liquid chromatography (HPLC) and mass spectrometry are used to evaluate glycosylation patterns, which are crucial for protein function.

Functional Assays

• Binding Assays: These measure the ability of the biosimilar to bind to its target, such as cell surface receptors, which is crucial for its efficacy.
• Biological Potency Assays: These assess the biological activity of the biosimilar, ensuring it functions similarly to the originator.
• Enzyme Kinetics: Studies of enzyme activity and kinetics help to confirm that the biosimilar molecule behaves like the reference product in terms of its enzymatic activity.

Impurity and Purity Assays

• Impurity Profiling: This involves comparing the profiles of product-related variants, aggregates, and impurities in both the biosimilar and the reference product to ensure manufacturing process consistency.
• Chromatographic Techniques: Methods such as HPLC are used to assess purity and impurity levels.

The Leinco biosimilar is not specifically mentioned in the available search results. However, in general, biosimilars like those potentially developed by Leinco (if they are involved in such projects) would undergo these comprehensive analytical assessments to prove their structural and functional similarity to the originator drugs. Companies like Leinco might contribute to these studies by providing biosimilar candidates or technological expertise in performing these assays.

To confirm the involvement of Leinco in these studies, further specific information or clarification would be required.