Contains individual highly specific anti-human immunoglobulin class antibodies (anti-human IgA, anti-human IgD, anti-human IgE, anti-human gamma-chain specific IgG, and anti-human IgM) labeled with the enzyme horseradish peroxidase ( HRP).
When properly diluted, this antibody: HRP conjugates can be used to identify the type of antibody response to a particular antigen.
Due to the specificity of these conjugates, they can also be used as markers in ELISA for the quantification of individual immunoglobulin classes.
Host cell proteins (HCPs) constitute a major group of process-related impurities in biological drugs produced using cell culture technology. HCPs are produced inadvertently during the expression of recombinant biopharmaceuticals as secreted from host cells from aberrant cellular trafficking due to cell stress and cell lysis over the course of bioprocess manufacturing.
Host cell proteins (HCPs) constitute an important group of process-related impurities in biological drugs produced by cell culture technology. HCPs are inadvertently produced during the expression of recombinant biopharmaceuticals secreted by aberrant cell trafficking host cells due to cell stress and cell lysis in the course of bioprocess manufacturing.
Many PS are benign, but some are immunogenic; some may interact with the drug, and others, such as proteases and lipases, may reduce the effective dosage of the product through direct action on the drug or its stability by interfering with the formulation buffer. Since HCPs can pose a risk to patients and affect drug efficacy and stability, they constitute an important component of the overall risk management strategy of a biopharmaceutical drug developer.
A robust and widely reactive HCP enzyme-linked immunosorbent assay (ELISA) is a critical tool for monitoring purification process consistency, as well as final drug purity, and is the gold standard method for monitoring processes and product release tests for HCP (Figure 1). It is essential to ensure that the selected HCP ELISA method is suitable for its intended use. This is important from a regulatory perspective, as well as for process development and manufacturing.
Regulatory agencies around the world have implemented measures to ensure that the HCP ELISA used by a sponsor is suitable for monitoring the consistency of the purification process and batch release of products (1-3). A well-developed and qualified HCP ELISA will ensure that HCPs ANALYSIS KITS to safe levels and that the purification process is consistent from batch to batch.
Orthogonal methods to demonstrate antibody coverage to individual process-specific HCPs are recommended to support the use of a particular HCP immunoassay. HCP coverage evaluations help assess the ability of antibodies to recognize a wide range of HCPs in the calibration standard and those present in samples of pharmaceuticals and in-process substances. It is equally important to rate the assay for linearity, accuracy, and dilution precision.
In the absence of better coverage analysis methods, 2D Western Blot (2D WB) has traditionally been performed from large format two-dimensional polyacrylamide gel electrophoresis gels (2D PAGE) with the transfer of proteins to membranes for 2D comparison. Silver-stained WB. Due to the recognized sensitivity limitations of 2D WB, conventional acceptance criteria are that> 50% of total HCP must be reactive and that the antibody must recognize HCP in all four quadrants of a 2D PAGE gel.
While using 2D WB, coverage can only be estimated in upstream harvest samples where the concentration of most HCPs is still within the sensitivity limitations of various staining methods. Furthermore, the loading capacity of 2D PAGE, destruction of native epitopes by severe sample treatment, and possible steric hindrance of HCP antibody binding epitopes lead to low sensitivity and specificity of 2D WB. Due to these reasons, 2D WB significantly underestimates the true antibody coverage to ascending HCPs. More importantly, 2D WB does not predict how that antibody will quantitatively react to the most important HCPs, which are the ones that are co-purified with the drug substance.
Antibody affinity extraction (AAE ™), a type of immunoaffinity chromatography, is an advanced orthogonal method designed to evaluate the coverage of a polyclonal antibody against a series of HCPs present in a given process, as well as the reactivity of the antibody HCP versus downstream processes. Specific HCPs that can be co-purified with a drug. This method was developed by Cygnus Technologies in 2013 to overcome the analytical deficiencies of the traditional 2D WB and the two-dimensional differential in orthogonal transfer electrophoresis (2D-DIBE) methods used to evaluate the coverage of polyclonal antibodies to total cell protein. Guest.
Antibody Coverage Analysis by AAE
To perform AAE, the polyclonal antibody is covalently immobilized on chromatographic support. The column is then conditioned to avoid significant leaching of the antibody and to minimize any non-specific binding. The HCP sample in its undenatured native state is passed down the column for binding and then acid eluted. The HCP sample is cycled back onto the column by binding and eluting until no additional HCP has bound. All HCP elution fractions are pooled, buffers exchanged and concentrated back to the original sample volume.
The final sample is then separated using PAGE 2D and analyzed by comparison with a silver stain of the initial undrawn sample or by differential gel electrophoresis (DIGE) using Cy3 and Cy5 to mark the extracted and unextracted samples from departure (Figure 2). Coverage is assessed by the number of HCPs in the AAE elution fraction (silver stained or Cy3 stained) compared to those seen on PAGE 2D of the initial antigen sample (silver stained or Cy5 stained). ).