*Written by expert: Gert, Associate Director Assays*

The matrix effect is the effect on an analytical assay caused by all other components of the sample except the specific compound (analyte) to be analyzed. Matrix effects are observed either as a loss in response, resulting in an underestimation of the amount of analyte, or as an increase in response, producing an overestimated result. These effects have long been associated with bioanalytical techniques. Their evaluation for each assay and each sample matrix can be very time consuming. Also, these extra evaluation steps bring additional costs. So, how important is evaluation of the matrix effects when analyzing bioprocess samples? What are the best approaches to evaluate the effect? And in what situations is the matrix effect acceptable?

## Importance of matrix effect evaluation

To release final product for use in patients, the product undergoes different quantitative or quantitative analytical procedures. Based on the obtained data, a decision for approval or rejection of the product batch is made. For that reason, it is necessary to show that generated analytical data is true and no matrix effect is involved. During the assay development and validation, the impact of sample matrix is investigated. Each assay is developed to allow monitoring of the matrix effect and, if possible, eliminate it.

## Methods to evaluate and eliminate

There are a few quick, qualitative options to determine whether a matrix effect is present, such as the dilution-based method. However, for this article we will focus on a few quantitative methods often used for bioanalytical techniques.

### Signal-based method

This method allows for quantification of the matrix effect for one specific concentration. At this concentration, the analyte is measured in the matrix and subsequently measured in a solvent known not to induce any effect. The analyte signal in the matrix is then divided by the analyte signal in the solvent and multiplied by hundred, resulting in the percentage of matrix effect. If the percentage is below hundred, the matrix effect results in a suppression of the result. When it is above hundred, it causes enhancement of the result. This method is useful when only this concentration is relevant. It doesn’t necessarily provide any indication for other analyte concentrations.

### Concentration-based method

In this method, the matrix effect is measured with the signal-based method, but for a range of analyte concentrations. The concentration-based method is used to show that the matrix effect is not analyte concentration dependent.

### Calibration-based method

This method is particularly relevant when blank matrix is not available. Different analyte concentrations are measured in solvent as well as the matrix and obtained data are plotted in a graph and linear regression model is used to generate a slope value. The slope of calibration analyzed in the matrix is then divided by the slope of calibration curve prepared in the solvent. The ratio is then multiplied by hundred to generate %ME. For %ME >100% matrix results in overestimation and for %ME < 100% tested matrix leads to signal suppression.

When a matrix effect turns out to be present, the effect causing component needs to be removed from the sample before analysis. Unfortunately, this is not always an option. In those cases, *matrix minimization *(*dilution) *will provide a way forward. Matrix minimization is especially useful when the analytical technique has sensitivity to spare. Elimination of matrix effect for particular matrix is proved during the assay development or validation.

## When the matrix effect can be ignored

Theoretically, samples consisting a pure compound could be ignored for matrix testing. In some cases, however, even a supposedly pure compound may contain other elements. Elements such as reaction impurities or by-products that may lead to matrix effects. Especially during process development, it is challenging to remove the matrix effect. This is due to the large number of matrices generated at each step of upstream or downstream process development. Analysis of PD samples usually serve to monitor processes and provide process developers with an indication whether changes in certain process parameters have beneficial or undesirable consequences. As the absolute value of the analysis is less important during the process development compared to batch release analysis, matrix effects are often not completely removed. Instead, they are only monitored using a spike recovery approach. This approach enables sample analysis with sufficient information on a potential matrix effect while saving time and resources.

*Batavia Biosciences offers a broad range of process development and manufacturing services. We are dedicated to help bring biopharmaceuticals to the market at higher speed, with reduced costs, and with a higher success rate. Batavia Biosciences has vast experience in developing product-specific assays for viral vectors, viral vaccines and protein-based biopharmaceuticals. With our team of experienced scientists and technicians, we are well equipped to take on any challenge associated with biopharmaceutical assay development.*

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