Analyzing Aggregate-Detection Methods

The drug industry’s future could lie in large molecules. The introduction of new biopharmaceuticals and a predicted rise in patient spending on biosimilars indicate an industry with a strong potential for growth. Companies that manufacture biopharmaceuticals must ensure that their products do not contain protein aggregates, which can trigger harmful immune responses or potentially dangerous side effects. Two popular methods for detecting protein aggregates are analytical ultracentrifugation (AUC) and size-exclusion chromatography–multiangle light scattering (SEC–MALS). These techniques’ results correlate relatively well, but each one has its own strengths.

To perform AUC, scientists load a sample into a cell, spin the cell, and analyze the product with ultraviolet-light (UV) absorption or refractometric detection. “The big advantage of analytical ultracentrifugation over SEC–MALS is that it offers matrix-free separation and minimal sample handling,” says Mark Staples, principal and consultant for Cusp Pharmatech Consulting. The technique does not rely on calibration standards or interactions between the drug surface and chromatography media.

Because AUC can detect aggregates within the matrix of the final-product formulation, some scientists believe that it presents a truer analysis of aggregation in the product than SEC–MALS does. “With AUC, you’re more likely to get a much more accurate picture as far as the type of aggregates that are present and the quantity of these aggregates,” says Mario DiPaola, chief scientific officer and chief operating officer of BlueStream Labs.

Scientists who need quick results are more likely to choose SEC–MALS, however. The time required per run for adequate SEC resolution of each species limits SEC throughput, but may produce results in minutes—usually less than an hour—per run, says Staples. In contrast, it may take between six and 48 h to complete AUC analysis, depending on the experiment.

In SEC–MALS, a scientist separates a sample by passing it through an SEC column. Next, the scientist places the sample into a cell and shoots a laser light through it. Detectors catch the beams as they exit the sample, and a computer indicates the presence of aggregates based on the angles at which light is refracted.

SEC–MALS is much easier to adapt and use than AUC, says Michelle Chen, director of analytical services at Wyatt Technology. The method also covers a wider molecular weight range (i.e., 500–10 million Da) than AUC does. For smaller molecules, the sample concentration must be higher. In addition, MALS is often sensitive in detecting the aggregates and is a good technique for measuring the aggregate size.

But the method’s biggest drawback is that it may filter certain types of aggregates out of the sample. During SEC, the column frits remove insoluble aggregates, and the sample is diluted by the mobile phase. By the end of the process, the sample’s matrix composition has changed, and scientists may have an inaccurate picture of the number and types of aggregates in the product.

Manufacturers are not limited to these two techniques. Microflow imaging (MFI), which Brightwell Technologies originated, could be a valuable complement to SEC–MALS, says Wendy Saffell-Clemmer, director of research at Baxter Healthcare. MFI captures digital images of particles, displays them in real time, and stores them for further analysis. The technique detects particles between 0.75 and 400 µm and stores information particle count, size, level of transparency, and shape. MFI requires only 1 mL of sample, and it allows users to distinguish between bubbles, silicone droplets, and protein aggregates. MFI may require samples to be diluted, however, and it may have difficulty handling highly viscous samples.

SEC–MALS and AUC would be difficult to adapt for on-line analysis. But MFI “seems ideal for a [process analytical technology] application because of the speed of analysis, the lack of a requirement for sample preparation, and the small volume of sample required,” says Saffell-Clemmer.

AUC, SEC–MALS, and MFI each have strengths and weaknesses, but a combination of these methods could give scientists needed information about a final drug product. Equipment improvements are enhancing the quality of the information that these techniques can provide. In addition, other analytical techniques are available, and companies can compare the results of various analyses for a broader perspective. As the biopharmaceutical market is poised to expand, manufacturers can be fairly confident about their ability to detect potentially harmful protein aggregates.