PrismA ELISA™ : Get the whole picture

The challenge of protein A purification of monoclonal antibodies

Protein A-based affinity chromatography is a widely used separation and purification technology because of its high purity and recovery (1). It’s primarily used to purify monoclonal antibodies (mAbs) for use as therapeutics, though it’s also possible to purify antibody fragments with protein A based technologies. The therapeutic must be suitable for use in humans, meaning that it can’t elicit an unwanted immune response or contain potentially toxic substances. Impurities introduced during the purification process must be removed to meet regulatory guidelines.

Process development managers or scientists involved in the production of mAbs are pressured to meet regulatory requirements, while also getting their product to market quickly. Removal and quantification of impurities such as host cell proteins (HCP) is necessary throughout process development and manufacturing. The purification of process can cause leaching of additional impurities, such as ligands from chromatography resins, and therefore increase time and expenses.

In this article we discuss how to use the MabSelect PrismA™ resin and the importance of accurate quantification of leached PrismA ligand during development and production. We give an overview of existing strategies and discuss improvements to existing workflows to quantify leached PrismA ligand.

Why do we need to quantify PrismA ligand?

During purification, protein A and its derivatives such as PrismA ligand commonly leach from the affinity resin where it co-elutes as a ProteinA-Immunoglobulin (IgG) complex. Protein A is potentially immunogenic, mitogenic, and can result in serious safety concerns for patients (2). Leached protein A in mAbs that are intended for in vivo administration must be monitored precisely to parts-per-million (ppm) or even to parts-per-billion (ppb) where it is possible so they comply with regional and global regulations.

Most regulatory agencies, such as the Food and Drug Administration (FDA) and the European Medicines Agency (EMA), require documented evidence of leached protein A clearance, which may be below the detection limit of a given assay.

Monitoring strategies for PrismA ligand

The ligand leakage from MabSelect PrismA™ resin is generally low, but must still be carefully monitored throughout each purification step and in the final product. ELISAs remain the most effective and commonly used method for the quantification of protein A and the PrismA ligand.

Many ELISAs are commercially available to detect and quantify protein A, but there were previously no ELISA kits specifically targeted for the PrismA ligand. Commercial kits use antibodies that are not raised against the PrismA ligand. Despite extensive modification and validation, there is still a risk that the measured amount of PrismA ligand is not accurate.

Commercial and “in-house” ELISAs require the PrismA ligand as a protein standard to get accurate and consistent data. Regulatory guidelines such as UPS <1103> recommend the use of a homologous molecule that accurately represents the sample analyte as a protein standard. As commercially available kits do not include the PrismA ligand as a protein standard, it needs to be procured independently.

Benefits of the PrismA ELISA™ kit

To facilitate process development workflows and enhance quantification of the PrismA ligand, we developed PrismA ELISA™ kit. Our kit includes the PrismA ligand and a customized antibody created specifically for use with the PrismA ligand.

Future-proof your assay with specificity and sensitivity

Leached protein A and PrismA ligand concentration in downstream materials is usually low, especially at the final stages of purification. This makes detection challenging. Our PrismA ELISA™ kit uses polyclonal antibodies raised specifically against the PrismA ligand used in MabSelect PrismA™ resins and Fibro™ PrismA Units. This increases confidence in an accurate quantification, even in the final purified product.

Regulatory guidelines are becoming more stringent. While pre-existing methods for measuring PrismA leakage are currently sufficient for regulators, this may change in the future. Access to a highly sensitive ligand ELISA that can detect extremely low amounts of ligand in challenging samples can help you feel secure that your production release workflow is ready to meet future regulatory changes.

Improving accuracy with high IgG tolerance

Protein A and its modified versions like PrismA ligand have a high affinity for IgG. They often co-elute as a complex, which can cause the protein A ELISA data to be misleading. Dissociating the interactions between leached PrismA ligand and IgG can address this, allowing for more efficient binding of the ELISA antibody to the leachate. Though this process offers better accuracy, it’s still challenging due to excess of IgG compared to PrismA ligand.

The PrismA ELISA™ can detect PrismA ligand in samples containing IgG at concentrations up to 20 mg/mL, with a sensitivity below 10 parts per billion (Fig 1).

Fig 1. IgG tolerance testing. Three concentrations (high, medium, and low) of PrismA ligand spiked into different levels of IgG, demonstrating that the assay is compatible with up to 20 mg/mL IgG in the initial sample. The dotted lines show the upper and lower acceptance limits of the assay (100% +/- 20%).

Consistency is key

Intra- and inter-plate variability can give inconsistent ELISA data. It is essential that ELISAs provide accurate and consistent data because of stringent regulatory guidelines. The PrismA ELISA™ kit minimizes intra- and inter-plate variability to ensure your assay results are reproducible.

The PrismA ELISA™ kit provides reliable and reproducible data, demonstrated by a mean intra-assay CV of 2.88% with 96% of data point CVs < 10%, and mean inter-assay CV of 5.95% with 78% of data points CVs < 10% (Fig 2).

Fig 2.Fig 2. Demonstrating precision of the PrismA ELISA™ as coefficient of variation (CV). Intra-assay variation from 127 in-process sample measurements within a single assay (green line) and inter-assay variation from 51 in-process sample measurements across triplicate assays (blue line).

Accelerate your workflow with an all-in-one kit

While customized or in-house developed ELISA kits for PrismA ligand can provide you with usable data, designing a custom assay and procuring a separate PrismA ligand is time-consuming. You can also run into issues with supply chains, item storage operations, and lot control in QMS.

When you use our PrismA ELISA™ kit, all required reagents are included. Everything arrives in one box with a single lot number and the assurance of a standardized and validated protocol. Supply chain security makes scaling up your mAbs development process straightforward.

Get the whole picture with PrismA ELISA™ kit

Meeting regulatory requirements and quickly bringing products to market requires efficient process development and manufacturing workflows. When using MabSelect PrismA™ resin to purify your product, our PrismA ELISA™ kit streamlines your workflow and provides you with confidence in the detection and quantification of leached ligand.

The PrismA ELISA™ kit is specifically designed for detecting the PrismA ligand at all purification steps and in the final product. This gives you a comprehensive view of the presence of PrismA ligand in your workflow. The all-in-one solution delivers accurate, timely and consistent ligand results required to ensure the quality of your final mAb product and meet current and future regulatory guidelines.

Discover how the PrismA ELISA™ kit can enhance your mAb process development workflow.

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