Size Exclusion Chromatography
“Over the years our company has worked on over 250 different protein products including most of the major biotech products, and that gives us a unique perspective,” said John Philo, Ph.D., vp and director of biophysical chemistry at Alliance Protein Laboratories.
“Aggregates cover a huge range of sizes and types, from small oligomers up to large particles and both covalent and noncovalent species. No single analytical method is good for the entire range.
“The first line of defense for measuring aggregation is size exclusion chromatography. That is the routine method everyone uses that is really suitable for QC in a GMP environment. One thing our clients forget is that SEC separates according to size, but this size is not molecular weight, it is a hydrodynamic size,” he explained.
“Normally when you see a peak eluting earlier than your main peak, then that earlier peak must be an aggregate. But I show several examples where particular proteins, when stressed, form partially unfolded monomers that are hydrodynamically bigger than the native state so they will look bigger and are very easy to mistake as an aggregate,” Dr. Philo noted.
“If you have a classical light scattering detector in line after your SEC column, that detector will tell you the true molecular weight of the peak that comes off.
“Another technique that can be useful in distinguishing these conformational differences from aggregation differences is sedimentation velocity. A conformation change affects the sedimentation in the opposite way from the shift in SEC elution, and that orthogonality can be very useful.”
Automation Is Key
Finally, there is the issue of reversible versus irreversible associations. Time, temperature, salt conditions, and sample dilution can all contribute to changing chromatographic profiles that can cause significant problems.
“In early formulation and analytical development work, we generally have limited sample, many candidates, multiple projects, and not enough time,” said Darryl Davis, principal scientist, pharmaceutical development & manufacturing sciences, Janssen Research & Development.
“Automation of the sample preparation and analysis techniques can be useful and provide unexpected benefits when used in an integrated development environment where cell-line selection, purification, and formulation have intersecting data points from the same sample.
“We have an automated high-throughput screening platform in place that can screen the formulation space to look for aggregation, turbidity, solubility, and product quality. The platform can use dynamic light scattering, differential scanning calorimetry, UV absorbance, and liquid chromatography/liquid chromatography-mass spectrometry (LC/LC-MS) assays to test critical quality attributes,” he explained.
“The cell biology group has used the LC-MS technique to screen for glycosylation and clipping of 96 well-plate transfectants. They have also used it to look at issues of scale and media changes. The amount of samples being run and analyzed would not be possible without using these automated platforms. The push toward standardization or platforming of techniques allows for streamlining the overall process and ensures a lower failure rate,” Davis remarked.
“We are looking at the fundamental mechanisms of folding and unfolding. When does a protein begin to unfold to form the first seeds, the first nuclei that will begin to aggregate?”
“We are not looking at the assembly of the aggregates; there are other technologies that are being used for that. We are looking at the weak points in a protein where unfolding begins and where aggregation begins,” said Peter Wright, Ph.D., professor and chairman, department of molecular biology, The Scripps Research Institute, in describing one of the research areas in his laboratory.
“Some of our current work has been enabled by advances in NMR technology. Using relaxation dispersion techniques allows us to study the small populations of short-lived higher energy folding intermediates.
“Using this and other methods, my lab has been able to put together an extremely detailed picture of the folding and spontaneous unfolding of the model protein apomyoglobin,” he said.
“One area that I think has great promise is to study the spontaneous unfolding processes in proteins associated with disease. Relaxation dispersion methods could prove particularly powerful for the characterization of the earliest events that occur as a protein spontaneously unfolds and forms protein aggregates, which in turn can lead to amyloid formation and disease.”