Anti-Human CD279 (PD-1) (Nivolumab) [Clone 5C4.B8] — APC

Research-grade Nivolumab biosimilars are used as calibration standards or reference controls in pharmacokinetic (PK) ELISA assays by serving as the known concentration samples that establish the standard curve, which is then used to quantify Nivolumab levels in serum samples of patients.

In a PK bridging ELISA for Nivolumab:

• The biosimilar or reference standard is diluted to several known concentrations to generate a standard calibration curve (often ranging from low ng/mL to several hundred ng/mL, with quintuplicate analysis for precision).
• These standards are prepared in a matrix (often buffer, sometimes serum) matrix-matched to the sample environment to ensure accuracy and comparability.
• The ELISA readout (typically absorbance) for each standard concentration is plotted against the known concentrations to create a curve, often fitted to a polynomial or 4-parameter logistic (4PL) model (e.g., quadratic polynomial model, as with Y = −0.0002x² + 0.0322x + 0.0387 in a published ELISA for Nivolumab).
• Unknown serum samples from study subjects are then analyzed under the same conditions, and their absorbances are used to interpolate drug concentration from the established standard curve.
• The single calibrator approach is industry standard for biosimilar bridging PK assays: the same research-grade biosimilar or reference Nivolumab is used as the calibrator for quantifying both the biosimilar and reference arms, minimizing assay variability and eliminating the need for separate calibration for each arm.
• Calibration standards must be validated for precision, accuracy, and recovery in the presence of serum matrix, as matrix effects can influence assay performance.
• For official bioequivalence studies, the calibration strategy and biosimilar standard must be rigorously validated, including intra- and inter-assay precision, functional binding equivalence, and lot-to-lot consistency.

In summary:
Research-grade Nivolumab biosimilars, assuming they are analytically comparable to the innovator product (Opdivo), are serially diluted to make calibration standards. These are essential to generate a reliable calibration curve in ELISA-based PK assays, serving as the reference against which serum drug concentrations are quantitatively measured in patient samples. This is considered a regulatory best practice for immunoassays supporting biosimilar PK bridging studies.

Standard flow cytometry protocols using a PE or APC-conjugated Nivolumab biosimilar are designed to quantify PD-1 expression levels and confirm binding capacity directly on the cell surface, particularly for human immune cells such as T lymphocytes. These protocols are typically used in clinical and preclinical research to assess immune checkpoint blockade effectiveness or to validate reagents for immunomonitoring.

Protocol Overview:

• Sample Type: Human peripheral blood mononuclear cells (PBMC), whole blood, or other mononuclear cell preparations.
• Antibody Reagent: Nivolumab biosimilar directly conjugated to a fluorophore (e.g., PE, APC), which binds PD-1 on the cell surface.
• Controls: Include isotype-matched fluorochrome controls for gating and background, and potentially unconjugated Nivolumab to block specific binding as a competitive control.

Core Procedure Steps:

1. Cell Preparation: Isolate PBMCs by Ficoll-Hypaque density centrifugation or use freshly drawn, minimally processed whole blood (less than 100 μL can be sufficient).
2. Blocking (optional): Incubate cells with Fc receptor block or excess unconjugated human IgG to minimize nonspecific binding.
3. Staining:
   • Incubate cells with PE- or APC-labeled Nivolumab biosimilar at an optimized dilution (typically titrated in pilot experiments; each investigator must determine this for their system).
   • Include relevant surface marker antibodies in a multiparameter panel (e.g., CD3, CD4, CD8, CD19) if needed for immune subset identification.
4. Washing: Wash cells to remove unbound antibody, often using PBS with 1% BSA and 0.09% sodium azide as preservative.
5. Data Acquisition: Acquire samples on an appropriate fluorescence-activated cell sorter (FACS), detecting the relevant emission spectrum for PE (excitation at 488/561 nm) or APC (excitation at 650 nm).
6. Analysis:
   • Gate on lymphocyte populations and quantify PD-1 expression as mean fluorescence intensity (MFI) or percent PD-1+ cells.
   • To assess binding capacity or receptor occupancy, compare fluorescence levels before and after blockade with unconjugated Nivolumab, or use competitive ELISA data in parallel.

Special Notes and Best Practices:

• When monitoring patients treated with Nivolumab or other anti–PD-1 antibodies, only directly labeled Nivolumab (rather than diagnostic anti-PD-1 clones) will reliably detect PD-1 occupied by therapeutic antibody.
• The method can distinguish between fully, partially, or non–antibody-bound PD-1 on T cells by analyzing MFI shifts upon competitive staining.
• Incorporate isotype-matched controls and unstained cells to identify background signal and set analytical gates.
• For clinical monitoring, serial sampling can assess receptor occupancy dynamics and correlate with therapeutic response.

References to Protocol Details:

• Detailed, validated protocol steps and controls for 12-parameter multiparameter flow cytometry (including Nivolumab receptor occupancy and PD-1 quantification) are described and published.
• Commercial biosimilar research reagents (PE- and APC-Nivolumab) specify use in flow cytometry with stability and formulation details, emphasizing investigator titration for optimal detection.

This approach is a gold-standard for validating PD-1 expression and confirming Nivolumab biosimilar binding and functionality in immunophenotyping and drug development contexts.

Biopharma companies perform a comprehensive set of analytical assays to confirm that a proposed biosimilar is structurally and functionally similar to the originator (reference) drug. These include highly sensitive, orthogonal (complementary) methods targeting all relevant critical quality attributes (CQAs).

Typical Analytical Assays Performed:

• Structural Characterization:

  • Primary structure: Peptide mapping and mass spectrometry for amino acid sequence confirmation.
  • Higher-order structure: Circular dichroism, NMR, and differential scanning calorimetry to assess folding and tertiary/quaternary structure.
  • Post-translational modifications: Glycosylation patterns, oxidation, deamidation, and other modifications analyzed by LC-MS, capillary electrophoresis, and HPLC.
  • Impurity and variant analysis: Chromatography and electrophoresis to identify aggregates, fragments, and process- or product-related impurities.

• Functional Characterization:

  • Biological potency assays: Cell-based assays that evaluate the product’s in vitro bioactivity.
  • Binding assays: ELISA or SPR-based assays to confirm binding to the target antigen and Fc receptor interactions for antibody products.
  • Enzyme kinetics and effector function tests: As appropriate for mechanism of action (e.g., ADCC and CDC for antibodies).
  • Orthogonal functional assays: Multiple methods to functionally confirm that minor structural differences have no clinically meaningful impact.

Workflow:

• Analytical studies compare the biosimilar and originator head-to-head, across multiple lots, to assess that any minor differences fall within an acceptable range (i.e., "no clinically meaningful differences").
• Results focus especially on attributes affecting safety, efficacy, pharmacokinetics, and immunogenicity.
• Functional testing acts as the link between observed molecular similarity and expected clinical performance.

Leinco Biosimilars in Analytical Studies:

The search results do not specifically reference Leinco’s biosimilar products. Based on industry practice and Leinco’s reputation as a supplier of high-quality reagents (including biosimilar antibodies for research use), it is likely that Leinco biosimilars are used as control materials, reference standards, or test articles in analytical comparability assays. Such biosimilars may be employed for:

• Method development and qualification of analytical assays
• Comparative studies in academic or early-stage R&D settings when innovators are not available or for benchmarking assay performance

This role would be based on their quality and characterization, but unless explicitly cited in study protocols or regulatory filings, Leinco biosimilars themselves are generally not submitted as clinical biosimilar products for regulatory approval. Instead, they serve mainly as well-characterized, representative materials for robust, reproducible assay development and comparative analysis.

If a more specific application of Leinco biosimilars in regulatory submissions or pivotal biosimilarity studies is required, direct information from Leinco or cited regulatory filings would be necessary.