The phase-appropriate application of analytical methods to characterize, monitor, and control particles is an important aspect of the development of safe and efficacious biotherapeutics. The AAPS Product Attribute and Biological Consequences (PABC) focus group (which has since transformed into an AAPS community) conducted a survey where participating labs rated their method of choice to analyze protein aggregation/particle formation during the different stages of the product life cycle. The survey confirmed that pharmacopeial methods and SEC are the primary methods currently applied in earlier phases of the development to ensure that a product entering clinical trials is safe and efficacious. In later phases, additional techniques are added including those for non-GMP extended characterization for product and process characterization. Finally, only robust, globally-accepted, and stability-indicating methods are used for GMP quality control purposes. This was also consistent with the feedback during a webinar hosted by the group to discuss the survey results. In this white paper, the team shares the results of the survey and provides guidance on selecting phase-appropriate analytical methods and developing a robust particle control strategy.

Both, formulation parameters and the presence of liquid-air interfaces are known to affect the aggregation of protein drugs. In this study, the impact of pH on the liquid-air interfacial behavior of three proteins, a polyclonal and two monoclonal antibodies (IgG, mAB1 and mAB2) was investigated using different surface sensitive methods. Equilibrium surface pressure values revealed only a minor impact of pH. Infrared Reflectance Absorbance Spectroscopy (IRRAS) proved not only the presence of the proteins at the interface but also showed that the secondary structure was not considerably affected by the adsorption to the interface independent of pH between pH 3 and 9. Additionally, the physical resistance of the film as determined by the interfacial compressibility in a Langmuir trough was not affected by pH. Compression of the interfacial film caused the formation of telescoped areas which were no longer present after decompression at all pH values as investigated by underwater Atomic Force Microscopy (AFM). Brewster Angle Microscopy (BAM) showed some slight changes in the film reflectivity depending on pH, indicating changes in the interfacial film thickness. IRRAS experiments at different angles of incidence as well as section analysis of AFM images proved not only that the film thickness increased upon compression, but also that the interfacial film is thinner at pH 4 than at pH 9. Continuous compression and decompression of the protein film resulted in particle formation with increasing numbers of particles at higher pH value as detected by Light Obscuration (LO) and Micro-Flow Imaging (MFI). The use of different surface sensitive methods provides expedient information on how liquid-air interfacial events are affected by formulation pH. These findings enable a better understanding of not only the events and processes happening at the interface but can also be directly linked to the interface-related formation of particles.

The tendency of protein pharmaceuticals to form aggregates is a major challenge during formulation development, as aggregation affects quality and safety of the product. In particular, the formation of large native-like particles in the context of liquid-air interfacial stress is a well-known but not fully understood problem. Focusing on the two most fundamental criteria of protein formulation affecting protein-protein interaction, the impact of pH and ionic strength on the interaction parameter A(2)* and its link to aggregation upon mechanical stress was investigated. A(2)* of two monoclonal antibodies (mABs) and a polyclonal IgG was determined using dynamic light scattering and was correlated to the number of particles formed upon shaking in vials analyzed by visual inspection, turbidity analysis, light obscuration and micro-flow imaging. A good correlation between aggregation induced by interfacial stress and formulation pH was given. It could be shown that A(2)* was highest for mAB(1) and lowest for IgG, what was in good accordance with the number of particles formed. Shaking of IgG resulted in overall higher numbers of particles compared to the two mABs. A(2)* decreased and particle numbers increased with increasing pH. Different to pH, ionic strength only slightly affected A(2)* Nevertheless, at high ionic (100 mM) strength the samples exhibited more pronounced particle formation, particularly of large particles > 25 mu m, which was most pronounced at high pH. Protein solutions were identified to form continuous films with an inhomogeneous protein distribution at the liquid-air interface. These areas of agglomerated, native-like protein material can be transferred into the bulk solution by compression-decompression of the interface. Whether or not those clusters lead to the appearance of large protein aggregates or fall apart depends on the attractive or repulsive forces between protein molecules. Thus, protein aggregation due to interfacial stress is correlated with the protein-protein interactions as determined by A(2)*. This enables to differentiate different antibodies according to their propensity to form particles upon mechanical stress and to identify optimum formulation conditions.

The presence of liquid-air interfaces in protein pharmaceuticals is known to negatively impact product stability. Nevertheless, the mechanisms behind interface-related protein aggregation are not yet fully understood. Little is known about the physical-chemical behavior of proteins adsorbed to the interface. Therefore, the combinatorial use of appropriate surface-sensitive analytical methods such as Langmuir trough experiments, Infrared Reflection-Absorption Spectroscopy (IRRAS), Brewster Angle Microscopy (BAM), and Atomic Force Microscopy (AFM) is highly expedient to uncover structures and events at the liquid-air interface directly. Concentration-dependent adsorption of a human immunoglobulin G (IgG) and characteristic surface-pressure/area isotherms substantiated the amphiphilic nature of the protein molecules as well as the formation of a compressible protein film at the liquid-air interface. Upon compression, the IgG molecules do not readily desorb but form a highly compressible interfacial film. IRRA spectra proved not only the presence of the protein at the interface, but also showed that the secondary structure does not change considerably during adsorption or compression. IRRAS experiments at different angles of incidence indicated that the film thickness and/or packing density increases upon compression. Furthermore, BAM images exposed the presence of a coherent but heterogeneous distribution of the protein at the interface. Topographical differences within the protein film after adsorption, compression and decompression were revealed using underwater AFM. The combinatorial use of physical-chemical, spectroscopic and microscopic methods provided useful insights into the liquid-air interfacial protein behavior and revealed the formation of a continuous but inhomogeneous film of native-like protein molecules whose topographical appearance is affected by compressive forces.

Protein aggregation is a major challenge in the development of biopharmaceuticals. As the pathways of aggregation are manifold, good understanding of the mechanisms behind is essential. Particularly, the presence of liquid-air interfaces has been identified to trigger the formation of large protein particles. Investigations of two monoclonal antibodies (IgGs) at the liquid-air interface exhibited the formation of a highly compressible film. An inhomogeneous protein distribution across the interface with areas of increased packing density was discovered by Brewster-Angle microscopy. Repeated compression and decompression of the film resulted in a considerable hysteresis and in significantly elevated numbers of particles. Furthermore, the extent and speed of compression directly affected the mechanical properties of the film as well as the number of particles formed. Infrared reflection-absorption spectroscopy did not indicate considerable changes in secondary structure compared to FT-IR spectra in solution. Hence, the IgG remains in a native-like conformation at the interface. Consequently, the physical-chemical methods applied in combination with the newly-designed Mini-trough provided substantial new knowledge of the mechanisms of interface-related protein aggregation and enable testing of different formulations under controlled stress conditions. Pure compression and decompression with a Mini-Trough allows a much more controlled stressing than shaking.