Anti-Human IL 12/23 (Briakinumab) – Biotin

Research-grade Briakinumab biosimilars are used as calibration standards or reference controls in PK bridging ELISAs by serving as the analytical standard against which unknown serum sample concentrations are quantified, ensuring assay precision and comparability between biosimilar and reference products.

In a PK bridging ELISA, the biosimilar (e.g., Briakinumab) is first thoroughly characterized for purity and concentration, often exceeding 95% purity and low endotoxin levels, as confirmed by multi-step affinity chromatography and rigorous quality control assessments. For the assay:

• Standard Curve Creation: Serial dilutions of the research-grade Briakinumab biosimilar are prepared in serum or relevant buffer to generate a standard curve covering the expected concentration range in clinical samples. These calibrators are typically used in multiple concentrations, spanning low to high ng/mL values.
• Reference Control/Assay Validation: The biosimilar is rigorously compared to the originator (reference product) for bioanalytical equivalence within the assay. This involves qualification studies where both are spiked into serum and quantified to ensure analytical equivalence based on performance parameters like accuracy and precision. If equivalence is confirmed, the biosimilar can be selected as the sole calibrator for subsequent PK sample analysis, reducing variability and simplifying the assay workflow.
• ELISA Workflow: The biosimilar standard and serum samples are loaded into ELISA plates pre-coated with anti-Briakinumab antibodies. Detection is achieved via labeled secondary antibody (e.g., HRP-conjugated), and the signal readout (absorbance) is proportional to the Briakinumab concentration in each sample. The standard curve generated from biosimilar calibrators is used to interpolate the drug concentration in unknown samples.

Benefits of Using Biosimilars as Calibration Standards:

• Ensures comparability in quantifying both biosimilar and reference drug concentrations, which is critical for regulatory PK bridging studies.
• Establishes analytical consistency and minimizes assay-related bias by validating a single standard curve for both products.
• Reduces resource use and complexity when only one standard is required for diverse sample sets.

Regulatory and Best Practice Notes:

• The method and performance of the biosimilar-based calibration must meet stringent validation criteria consistent with FDA/EMA bioanalytical guidance, including assessments of accuracy, precision, range, and specificity.
• The biosimilar standard should only be used for research, not as a clinical diagnostic, unless such use is validated and approved.

In summary, research-grade Briakinumab biosimilars are critical reagents as calibration standards/reference controls in PK bridging ELISA, enabling the accurate, reproducible measurement of drug concentration in serum while supporting PK comparability between biosimilar and originator molecules.

A standard Flow Cytometry protocol using a PE or APC-conjugated Briakinumab biosimilar to assess the expression or binding capacity of the IL-12/IL-23 p40 target involves the following key steps and considerations:

1. Sample Preparation

• Collect and prepare single-cell suspensions of the relevant human cells, which express IL-12/IL-23 p40 (commonly dendritic cells, macrophages, neutrophils, monocytes, or B-lymphoblastoid cells).
• Wash cells with flow buffer (e.g., PBS containing 1% BSA and 0.09% sodium azide) to minimize nonspecific antibody binding and preserve cell integrity.

2. Antibody Staining

• Use the PE- or APC-conjugated Briakinumab biosimilar at a recommended concentration of ≤ 1.0 μg per 10⁶ cells in 100 μl volume. However, titration of the antibody is highly recommended to optimize signal-to-noise ratio for your specific assay conditions.
• Incubate the cells with antibody for 30 minutes at 2–8°C protected from light to enable maximum binding.
• Include appropriate isotype controls to assess background/unspecific binding (e.g., Human IgG1 conjugated to PE/APC).

3. Washing

• Wash cells at least twice with flow buffer to remove unbound antibody, then resuspend in buffer suitable for flow cytometry analysis (e.g., PBS/BSA/azide).

4. Data Acquisition

• Analyze stained cells using a flow cytometer equipped with the appropriate lasers and detectors:
  • PE excitation: 488 nm (blue), 532 nm (green), or 561 nm (yellow-green) laser
  • APC excitation: 650 nm (red) laser
• Collect data for fluorescence intensity of the labeled antibody, which corresponds to the amount of cell-associated IL-12/IL-23 p40.

5. Controls and Validation

• Include unstained cells and cells stained with isotype control antibodies (PE/APC conjugated IgG1) to set gates and verify specificity.
• Validate that observed staining correlates with known patterns of IL-12/IL-23 p40 expression using positive and negative cell populations (e.g., stimulated dendritic cells vs. unstimulated controls).

6. Data Analysis

• Quantify expression levels (mean fluorescence intensity, MFI) and/or percentage of positive cells using standard flow cytometry software.
• Compare binding in test groups versus controls to assess relative expression or target engagement.

Additional Notes

• Each investigator should experimentally determine optimal dilutions and incubation times for their specific cell type and cytometer, as noted in manufacturer instructions.
• These Briakinumab biosimilar conjugates are for research use only and are not for therapeutic application.
• Storage: Both PE and APC conjugates should be stored at 2–8°C and must not be frozen.

This protocol aligns with general best practices for antibody-based flow cytometry, specifically validated for PE and APC-conjugated Briakinumab biosimilar detection of the IL-12/IL-23 p40 target.

Biopharma companies use a comprehensive range of analytical assays to confirm the structural and functional similarity of a proposed biosimilar to the originator drug. These include detailed structural analyses and functional (biological activity) assays.

Key analytical assays typically performed:

• Structural (Physicochemical) Characterization:

  • Primary, secondary, tertiary, and quaternary structure analysis: Assessed by mass spectrometry, chromatographic techniques, peptide mapping, and higher-order structural methods such as circular dichroism and NMR spectroscopy.
  • Post-translational modifications: Including detailed glycosylation profiling, charge variant analysis, and determination of deamidation, oxidation, etc.
  • Impurity and aggregate analysis: Identifies process- and product-related impurities using HPLC, SEC, CE, and other orthogonal methods.
  • Product-related variants: Such as aggregates, fragments, and other modified forms are rigorously compared.

• Functional Assays:

  • Potency assays: Measure the biological activity related to clinical effect, often via cell-based bioassays that reflect the mechanism of action.
  • Binding assays: Assess the binding affinity to the target antigen and secondary receptors (e.g., FcγR binding for monoclonal antibodies).
  • Enzyme kinetics and biological responses: Evaluate downstream functional effects closely tied to therapeutic efficacy.

• Critical Quality Attribute (CQA) Assessment:

  • Attributes most relevant to clinical outcomes (e.g., binding, effector functions, glycosylation) are identified and compared using orthogonal (complementary) assays across multiple lots.
  • The goal is to demonstrate that any minor structural differences do not translate into clinically meaningful differences in function or safety.

How Leinco biosimilars are used in these studies:Leinco provides highly characterized biosimilar monoclonal antibodies and proteins that are used as reference standards, controls, or comparators in the analytical workflows. These biosimilars serve multiple key roles:

• As a reference material against which the structure and function of the test biosimilar are directly compared.
• In method development and validation—ensuring that the analytical tools used for comparison are robust and selective.
• For assay calibration, ensuring consistency and allowing assessment under harmonized conditions.

The use of Leinco biosimilars allows developers to conduct head-to-head comparisons (analytical similarity assessments) to verify that their candidate biosimilar matches the reference in both structure and function, as required by regulatory authorities. This is critical in both early- and late-stage characterization studies to ensure reliability, reproducibility, and regulatory compliance.

Summary Table: Analytical Assays for Biosimilar Assessment

In summary, confirming biosimilarity requires a strategic, multifactorial analytical approach focused on both structural and functional attributes, with validated materials such as Leinco’s biosimilars providing a critical foundation for comparability and regulatory acceptance.