Delineating Protein Particles
Contending with various forms of junk that arise during protein purification requires a number of different strategies. Alla Polozova, a senior scientist in analytical biochemistry at MedImmune, described her approach to detecting and eliminating the variety of particulate contaminants in protein solutions that harry investigators.
“We need to identify and enumerate the contaminating particles,” insisted Polozova as she related her group’s experiences with two contentious monoclonal antibody projects. Common types of contaminating materials include polyethylene, cellulose, and various proteinaceous particles, and these are studied using various types of microscopy, including atomic force, electron, light scanning, and chemical.
Particles are counted using light obscuration, which enumerates particles one at a time, or optically with a conventional hemocytometer. Although each method has its advantages and disadvantages, combining them assures that an accurate census of the offenders, ranging in size from 2 to 300 µm, will be obtained. Sub-micron particles are detected using the NanoSight (www.nano?sight.com) system in which the particles in suspension are detected by light scattering of a laser beam using visual microscopy techniques.
The most noisome particles are protein aggregates, formed by irreversible recruitment of monomers and smaller units. Their genesis can be triggered by a variety of unfortunate events, including dilution, shaking, low pH, and the presence of silicon oil and other materials. Because of their propensity to block capillaries and act as immunogens, they pose substantial risk to patients.
Some visual techniques may be inaccurate because of the state of the solution, requiring multiple approaches. Light obscuration was found to fail at higher protein concentrations, because the particles become “invisible” due to loss in contrast. This can be ameliorated by calibration with protein-like particles, as was done in the characterization of a monoclonal antibody in which particle counts were found to be erratic and inconsistent.
An investigation, carried out to determine the root cause of particle formation, showed that the solutions are dynamics and over time new particles are forming and older ones are merging into larger particles. These particles may be composed of protein, or of proteins fused with contaminating oils or polypropylene.
“The characterization of particles in protein solutions offers unique challenges,” said Polozova. “Interference of background protein, the fragile nature and transparency of particles, and sample handling may result in formation of new particles. This requires a variety of orthogonal methods for reliable characterization.
“For reliable counting, avoid dilutions, but keep in mind that there are multiple particle-formation mechanisms and multiple triggering factors,” Polozova concluded.