Light Scattering in Protein Aggregation Studies

The past 15 years has seen a renewed dedication to quality by biopharmaceutical manufacturers and regulators. The U.S. FDA’s Quality by Design initiative, now well into its second decade, focuses on building quality into a process versus “testing in” quality through post-production analytics.

Quality in consumer markets is based on attributes desirable to customers. For drugs, quality reduces to a molecule’s suitability for the intended purpose (e.g. reducing inflammation, relieving pain, fighting pathogens), and most importantly, safety. Protein aggregation—a strong physical association or “stickiness” between and among protein molecules resulting in dimers, trimers, etc. —is a key quality attribute affecting a drug’s effectiveness and safety.

The most obvious effect of aggregation is it replaces active drug in circulation with inactive molecular junk, but the loss is usually less than one percent. Immunogenicity is a much more serious concern. Due to their size and dissimilarity to natural proteins, aggregates can cause undesirable and sometimes serious immune responses. The antibodies generated against aggregates can also neutralize the native drug, further diminishing its efficacy.

Due to the complexity of interactions between and among proteins, and between proteins and their physical media, aggregation may occur at numerous stages during a protein’s commercial life. Moreover the nature and composition of aggregates, for example size, shape, and morphology, may differ substantially.

“It is therefore important to understand the interactions, causes, and analyses of such aggregates in order to control protein aggregation to enable successful products,” writes Hans-Christian Mahler in Journal of Pharmaceutical Sciences. “A major challenge for the analysis of protein aggregates is that no single analytical method exists to cover the entire size range or type of aggregates which may appear,” he adds. Leading methods have their advantages, but also limitations, specifically in their limits of detection and the potential for introduction of artifacts during sample preparation. “Therefore, it may also be advisable to carefully compare analytical results of orthogonal methods for similar size ranges to evaluate method performance.”

That no definitive, single method exists is a testament to the complexity of protein structure, particularly in secondary and tertiary domains. While analytical scientists enjoy a comprehensive toolbox of methods for determining a protein’s primary structure, “these tools cannot tell us whether the protein is in the correct, folded structure in solution,” according to a published study by John Philo, director of biophysical chemistry at Alliance Protein Laboratories. “Proteins also participate in noncovalent self-association ... [which] may be either desirable (for example, the native functional state may be a dimer) or undesirable (producing aggregates, a common and often vexing degradation pathway).”

For this reason, Philo advises those undertaking protein aggregation studies to establish the protein’s native, biologically active, state of association, and compare experimental batches against this reference. Philo’s methods of choice are sedimentation velocity and sedimentation equilibrium, both of which are available through Beckman Coulter’s ProteomeLab Optima XL-A/XL-I.

Different stages, different methods

Malvern Panalytical provides a suite of systems utilized within biotherapeutic development to minimize immunogenic risk associated with protein aggregation. Choice of instrument, and what information it is capable of providing, depends on the point in the biomolecule’s development lifecycle.

For early development screening for aggregation propensity, the Zetasizer instrument provides the diffusion interaction parameter (k_D) using dynamic light scattering, the 2^nd virial coefficient (B₂₂) using static light scattering, and the effective charge (Z_Eff) and valence (Z_DHH) using electrophoretic light scattering.

“Large positive k_D and B₂₂ values, and large Z_Eff numbers, indicate candidates and formulation conditions with high colloidal stability and low protein aggregation propensity,” says Kevin Mattison, Ph.D., principal scientist at Malvern. “All three parameters are used to screen for bioformulation developability.”

The Zetasizer also provides T_Agg, representative of protein structural stability, and our PEAQ DSC system utilizes differential scanning calorimetry to measure the melting point and unfolding enthalpy, both of which are used to screen for protein structural stability and manufacturability.”

Malvern’s OMNISEC/MALS system provides the mass distribution across the oligomeric size region of 1 to 200 nm, along with the molecular weights of each component. OMNISEC measures percent monomeric purity, and provides a baseline chromatogram for comparison at later development stages.

The company’s PEAQ ITC is another product utilized in early bioformulation development, providing the binding affinity, stoichiometry, and energetics of biological APIs using isothermal titration calorimetry. “It is used to screen for efficacy and/or to validate binding affinity screens collected from other technologies such as SPR,” Mattison says.

These tools play different roles during late-stage development, where aggregate quantification during stress testing and process development is critical. For example the Zetasizer provides the particle size distribution from 1 nm to 10 μm. At this development stage Zetasizer rapidly assesses aggregate generation, and helps identify subsequent technologies for aggregate quantification. Through size-exclusion chromatography, OMNISEC/MALS provides mass distributions across the size region of 1 to 200 nm, along with detection of high molecular weight species below the concentration detector limit.

Other Malvern Panalytical systems utilized in late-stage development include the NanoSight, which utilizes nanoparticle tracking analysis and provides the particle concentration across the 100 to 500 nm size range; the Archimedes, which utilizes resonance mass measurement to provide the particle concentration across the 500 nm to 2 um size range and can distinguish protein aggregates from silicon oil droplets; and the G4, which utilizes morphology directed Raman spectroscopy to provide chemical identification of particles in the subvisible size region greater than 10 um. According to Mattison, all three of these technologies are routinely utilized by formulation scientists for the development of protein aggregation control strategies outlined in recent FDA guidelines to the biotherapeutic industry.

“No single technology is absolute for the prediction of aggregation propensity in early development or the full quantification of protein aggregates in late development,” Mattison explains. In recognition of this, the FDA recommends orthogonal verification be utilized for all release specifications.

Table 1. Orthogonal methods that complement light-scattering technologies for protein aggregate characterization. Table courtesy of Kevin Mattison/Malvern.