Anti-Human TNF-α Adalimumab [Clone D2E7] — APC

Research-grade Adalimumab biosimilars are commonly used in pharmacokinetic (PK) bridging ELISA assays as calibration standards or reference controls to quantify drug concentration in serum samples. They provide a means to establish standard curves for quantification and to harmonize assay results across different batches and laboratories.

When used in a PK bridging ELISA:

• Biosimilar adalimumab is serially diluted in an appropriate matrix (such as drug-free human serum) to create a standard curve, against which the concentration of adalimumab in patient serum samples is measured.
• These standards serve as the quantitative reference, allowing accurate determination of the drug concentration based on the absorbance or signal response measured by the ELISA.
• The biosimilar standard must be carefully matched and characterized against the reference product (e.g., Humira®) to ensure accurate and parallel dose-response behavior in the ELISA system.

Supporting details:

• Validation data have shown that research-grade adalimumab biosimilars (such as ABP501) produce similar results as reference adalimumab (Humira®) in well-validated assays, enabling interchangeability as calibration standards in PK ELISAs.
• The World Health Organization (WHO) International Standard (IS) for adalimumab and similar research-grade biosimilars have been shown in multi-center studies to align laboratory results and reduce inter-laboratory variability in quantifying adalimumab levels, supporting their use as harmonizing and robust reference materials.
• Specifically, in ELISAs, dose-response curves using biosimilar, reference, or in-house standards are highly comparable, except for minor variation at extreme concentrations.

Summary process:

1. Prepare the standard curve by serial dilution of the adalimumab biosimilar in assay buffer or drug-free serum.
2. Include the biosimilar standard in each assay run alongside patient samples to calibrate quantification.
3. Compare patient sample responses to the curve to calculate serum adalimumab concentration.
4. Employ biosimilars as quality controls to monitor assay performance and reproducibility across different runs, kits, or laboratories.

Research-grade adalimumab biosimilars are thus reliable and suitable reference materials for PK bridging ELISA, essential for both therapeutic drug monitoring and biosimilar development.

Standard flow cytometry protocols using a conjugated Adalimumab biosimilar (e.g., PE or APC-labeled) to validate the expression levels or binding capacity of the TNF-α target involve several key steps:

Steps for Flow Cytometry Analysis

1. Sample Preparation:

   • Stimulation of Cells: Stimulate human peripheral blood mononuclear cells (PBMCs) with agents like Phorbol 12-Myristate 13-Acetate (PMA) and Ionomycin to induce TNF-α expression. This step may also involve the addition of GolgiStop or its equivalent to inhibit protein transport.
   • Harvesting and Washing: Collect the cells, wash them with appropriate buffers (e.g., BD Pharmingen Stain Buffer), and proceed to fixation and permeabilization if necessary.

2. Fixation and Permeabilization:

   • For intracellular staining of TNF-α, fix the cells using BD Cytofix Fixation Buffer and permeabilize them with BD Perm/Wash Buffer.

3. Staining:

   • Conjugated Antibody Use: Use the conjugated Adalimumab biosimilar (e.g., PE or APC-labeled) to stain the cells. This biosimilar is designed to bind to TNF-α, allowing for the detection of TNF-α expression or bound Adalimumab on the cell surface.
   • Control Staining: Include appropriate isotype controls or staining for other cell markers (e.g., CD3) to ensure specificity and help in cell identification.

4. Data Acquisition and Analysis:

   • Acquire data using flow cytometry analyzers like the BD FACSymphony system.
   • Analyze data using software such as FlowJo to examine the expression levels of TNF-α based on the fluorescence signals from the conjugated Adalimumab biosimilar.

Protocol Example

Reagents:

• Adalimumab Biosimilar (e.g., PE or APC-conjugated)
• PMA and Ionomycin
• GolgiStop
• BD Cytofix Fixation Buffer
• BD Perm/Wash Buffer
• Isotype Controls (e.g., PE or APC-conjugated IgG1 κ)
• Flow Cytometry Fixation Buffer (if needed)

Procedure:

1. Stimulate PBMCs with PMA (50 ng/mL) and Ionomycin (1 μg/mL) for 5 hours, adding GolgiStop during the last 4 hours.
2. Harvest and wash cells with appropriate buffers.
3. Fix and permeabilize cells using BD Cytofix and Perm/Wash buffers if necessary for intracellular staining.
4. Stain cells with the conjugated Adalimumab biosimilar (using appropriate dilutions) and any additional markers (e.g., CD3).
5. Acquire and analyze data using flow cytometry software.

This protocol assesses TNF-α expression or the binding capacity of the Adalimumab biosimilar, which is crucial for understanding its mechanism of action and therapeutic efficacy.

Biopharma companies use a comprehensive suite of analytical assays to confirm the structural and functional similarity of a proposed biosimilar to its originator (reference) drug; these include detailed physiochemical and functional studies focusing on critical quality attributes (CQAs) relevant to clinical outcomes.

Key analytical assays and methods performed include:

• Structural (Physicochemical) Characterization:

  • Primary structure: Peptide mapping, amino acid analysis, and sequence confirmation by mass spectrometry.
  • Higher order structure: Circular dichroism, nuclear magnetic resonance (NMR), X-ray crystallography, and hydrogen-deuterium exchange to confirm secondary, tertiary, and quaternary structures.
  • Post-translational modifications: Detailed evaluation of glycosylation patterns (N- and O-glycans), phosphorylation, acetylation, and other modifications using mass spectrometry and chromatography.
  • Purity and impurities: Comparison of aggregates, fragments, process-related and product-related impurities using size-exclusion chromatography, capillary electrophoresis, and related methods.

• Functional Characterization:

  • In vitro biological activity/potency assays: Cell-based assays to demonstrate comparable biological functionality to the originator.
  • Binding assays: Surface plasmon resonance (SPR), ELISA, or other ligand binding tests to assess binding to target antigens or receptors, including Fc receptor binding for monoclonal antibodies.
  • Enzyme kinetics or activity studies: Assess whether slight structural differences affect activity.
  • Orthogonal methods: Multiple different but complementary techniques are applied to robustly assess each CQA and confirm similarity from multiple analytical perspectives.

Additional key points:

• These studies are designed to be highly sensitive and can reveal even minor differences between biosimilar and reference product, focusing on those differences that could affect safety, efficacy, or immunogenicity.
• All relevant critical quality attributes (CQAs) are assessed rigorously according to ICH Q6B guidelines and regulatory expectations.

Purity and Consistency:

• Extended analyses are done for process impurities and aggregates, since manufacturing differences can introduce product variants. Demonstrating similar impurity profiles indirectly confirms process replication success.

Leinco Biosimilar—Role in Analytical Similarity Studies

No direct evidence in the provided search results specifically details how Leinco biosimilars are used in these studies. Leinco Technologies is known in the industry as a supplier of reference standards, recombinant antibodies, and biosimilar proteins, which can be used by developers as critical reagents or assay controls in analytical and functional comparability assessments. However, the specific application of a "Leinco biosimilar" would depend on the reagent type (for example, as a comparator standard, negative/positive control in assays, or benchmarking tool in potency/binding studies).

If biosimilar products from Leinco are used, typical usage scenarios would include:

• Reference standard for calibration curves in binding or bioactivity assays.
• In parallel comparability runs with the originator and the developer’s own biosimilar to benchmark performance in specific assays.

If more detail is needed on Leinco's biosimilars’ exact applications, this information may need confirmation directly from product datasheets or Leinco's technical documentation, as it was not available in the retrieved search results.

In summary:
Biosimilar developers confirm similarity through an extensive array of complementary structural and functional assays targeting CQAs. Products from suppliers like Leinco may serve as standards or controls in these assays, facilitating rigorous, well-controlled comparative analysis, though specific details on Leinco biosimilar application were not identified in the results provided.