February 1, 2012 (Vol. 32, No. 3)

Starting with the first protein drug, insulin, in 1982, recombinant proteins have played a vital and growing role in the treatment of human disease.Yet inherent to the production of protein drugs is the possibility of aggregation, which may increase the risk of an immunogenic response to the dosed drug.

The expression, purification, formulation, and storage of protein therapeutics can result in the formation of dimers, trimers, and multimer aggregates that can range in size from the nanometer range all the way up to visible particles of 100 microns or larger. There is no single method capable of measuring and quantifying aggregates throughout this entire size range.

Although all biotherapeutics contain some level of aggregates, there has been no direct demonstration that aggregates are a major risk factor contributing to immunogenicity. This is the result of the complexities of the potential causes of an immunogenetic response and the inability to measure particulates.

Because protein product aggregates represent a potential risk factor for immunogenicity, industry and the FDA seek a method to accurately count and characterize these particles. The challenges faced in measuring and characterizing these protein aggregates were discussed at the recent “PepTalk: The Protein Science Week” conference.

“Levels of protein aggregates have long been mentioned as a critical quality attribute and have potential clinical consequences. Today we have size exclusion chromatography (SEC) to look at the increase in the dimers, trimers, and oligomers of proteins. On the other extreme we look at particles visible to the naked eye that are 80–100 microns or larger.

“Despite its drawbacks, light obscuration is the current accepted method for monitoring of subvisible particulates during lot release. So we are missing the gap between what we can detect with SEC and light obscuration and what the human eye can see,” said Danny Chou, Ph.D., chief scientific officer, NorthStar Bio.

“The quality of a protein pharmaceutical is defined by the analytical capability that is available. In the last few years the regulatory agencies have been making a push for the industry to assess subvisible particles (SVPs) and their relevance to product quality, as well as to evaluate the technologies that can be used to measure them.”

According to Dr. Chou, NorthStar Bio is the first company created to better understand SVPs. Functioning as a CRO, NorthStar Bio can characterize protein production samples using state-of-the-art microflow imaging, fluid imaging particle analysis, the Coulter principle, and other emerging technologies.

“These orthogonal technologies will be used to characterize SVPs in a sample, and to illustrate how to use the technology to develop protein formulations and decide which technology can better address regulatory concerns,” he continued.

“In five years we will have a more diverse set of analytical tools as well as a much better understanding of what type of particles we need to control to minimize immunogenicity in the wide range of protein product types.”

Subvisible particles can be quite heterogeneous and can come from a variety of sources, either externally from the production environment or from the protein itself.

“Ideal samples for detecting particles would consist of a clear, water-like fluid in which individual particles of high contrast and moderate buoyancy are effectively recognized,” said Tobias Frommknecht, formulation scientist, F. Hoffmann-La Roche.

“The color, refractive index, and opalescence of actual product samples can provide erroneous data.” To study the effect of matrix irregularities on light obscuration and microflow digital imaging (MDI), Fromm­knecht used a number of artificial matrices and particle standards. Polystyrene and glass particles and pseudo protein particles were added to opalescent, viscous, and colored matrices to access sizing and counting effects of the methods.

“FlowCam (Fluid Imaging Technologies) showed low counting and sizing ability overall, but it has the advantage of higher quality particle images and is really useful for differentiating silicone oil droplets from other particles,” he said. “MDI has not been validated for automated morphological analysis.” New particle standards mimicking protein particles will be needed to assess the limits of these technologies.

Sizing and counting by the light obscuration method was only slightly affected by the nonideal sample conditions. “Light obscuration remains the gold standard for quantification in a QC environment. Both light obscuration and MDI are used during formulation development to get the most information on our product.”

NorthStar Bio characterizes protein production samples using state-of-the-art microflow imaging, fluid imaging particle analysis, the Coulter principle, and other emerging technologies.

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?”

Exposing Weaknesses

“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.”

The molten globule folding intermediate of apomyoglobin. Color code: folded helical regions, blue; flexible regions, red; unstructured regions, white. [The Scripps Research Institute]

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