A rapid method to monitor structural perturbations of high-concentrated therapeutic antibody solutions using Intrinsic Tryptophan Fluorescence Emission spectroscopy.

Drug product development of therapeutic antibody formulations is still dictated by the risk of protein particle formation during processing or storage, which can lead to loss of potency and potential immunogenic reactions. Since structural perturbations are the main driver for irreversible protein aggregation, the conformational integrity of antibodies should be closely monitored. The present study evaluated the applicability of a plate reader-based high throughput method for Intrinsic Tryptophan Fluorescence Emission (ITFE) spectroscopy to detect protein aggregation due to protein unfolding in high-concentrated therapeutic antibody samples. The impact of fluorophore concentration on the ITFE signal in microplate readers was investigated by analysis of dilution series of two therapeutic antibodies and pure tryptophan. At low antibody concentrations (< 5 mg/mL, equivalent to 0.8 mM tryptophan), the low inner filter effect suggests a quasi-linear relationship between antibody concentration and ITFE intensity. In contrast, the constant ITFE intensity at high protein concentrations (> 40 mg/mL, equivalent to 6.1 mM tryptophan) indicate that ITFE spectroscopy measurements of IgG1 antibodies are feasible in therapeutically relevant concentrations (up to 223 mg/mL). Furthermore, the capability of the method to detect low levels of unfolding (around 1 %) was confirmed by limit of detection (LOD) determination with temperature-stressed antibody samples as degradation standards. Change of fluorescence intensity at the maximum (ΔIaM) was identified as sensitive descriptor for protein degradation, providing the lowest LOD values. The results demonstrate that ITFE spectroscopy performed in a microplate reader is a valuable tool for high-throughput monitoring of protein degradation in therapeutic antibody formulations.

Process optimization and transfer of freeze-drying in nested vial systems.

Scale-up and transfer of freeze-drying processes is a crucial challenge in biopharma industry. With the success of small batch processing lines utilizing rack vial holding systems, further detailed knowledge about freeze-drying cycles and their scale-up for vials in a rack is required. Therefore, product temperature (TP) profiles as well as Kv values of vials nested in a Polyetheretherketon (PEEK) rack were compared to those of vials placed in a commonly used stainless steel tray. Additionally, both setups were challenged with varying fill volume and partially versus fully loaded rack. Additionally, a process developed for rack was compared to a tray freeze-drying cycle. Freeze-drying in vials placed in the rack is markedly faster for center vials and more homogeneous compared to vials in bulk tray setting, as indicated by TP and Kv values. Due to the more homogeneous drying the rack is more flexible regarding variation of the fill volume. The key point for the transfer of a freeze-drying cycle from rack to tray is to consider the higher sublimation rates in the rack by adapting chamber pressure or shelf temperature for the tray. Furthermore, transfer from one rack per shelf in a laboratory freeze-dryer to pilot scale with four racks per shelf was successful. Thus, understanding of the process in rack and tray setup was enhanced to ensure efficient scale-up and transfer of freeze-drying processes.

Impact of chamber wall temperature on energy transfer during freeze-drying.

Minimization of radiation coming from the chamber wall during lyophilization has the potential to reduce the edge-vial-effect. The edge-vial-effect is a phenomenon in which vials positioned at the shelf edges and corners tend to run warmer compared to center vials. A higher product temperature may result in product collapse in these vials. Consequently, more conservative and time-consuming freeze-drying cycles with lower shelf temperatures and pressures are chosen to ensure a product temperature below the collapse temperature in all vials. The edge-vial-effect is of even higher impact in small batches, where the ratio of corner and edge to center vials is higher compared to large scale manufacturing. The chamber wall is often discussed as the primary source of radiation impacting corner and edge vials. A radiation cage was set at different low temperatures to determine the impact of chamber wall temperatures below 0 °C on product temperature. At the end of primary drying, product temperature of corner vials could be reduced by 6 °C through the radiation cage but primary drying was elongated. Compared to vials in a tray, the chamber wall temperature had less impact on vials nested in a rack system due to a shielding effect of the rack itself. Corner and center vials ran more homogeneous with radiation cage since the edge and corner vials were slowed down. The difference in primary drying time between corner and center vials in the tray could be significantly reduced by 18% by means of 7 h when the radiation cage was controlled at product temperature and combined with a higher shelf temperature. In summary, the radiation cage is a useful tool for a more homogeneous batch with the potential to reduce primary drying time. Nevertheless, the drying difference between corner and center vials could only be reduced and was not completely eliminated.

Trouble With the Neighbor During Freeze-Drying: Rivalry About Energy.

Batch homogeneity during lyophilization is crucial to ensure products with high quality. Known as edge-vial-effect, vials at the corners and edges tend to run warmer than center vials during primary drying. This is associated with risk of collapse or increased costs due to use of more conservative, longer drying conditions resulting in lower product temperature. The edge-vial-effect has been attributed to radiation coming from the chamber wall. We could show that the neighbor vial has a dominant impact on product temperature during lyophilization. Depending on the number of neighbors as well as the distance to a neighbor vial, the neighbor vial exerts a remarkable cooling effect. Energy transfer by gas conduction enables the cooling effect of a neighboring vial over a distance up to 10 mm. This not only leads to prolonged primary drying but also impacts cake appearance. Thus, to avoid trouble during lyophilization you have to watch out for the neighborhood.

Energy Transfer in Vials Nested in a Rack System During Lyophilization.

Small batch sizes are a consequence of more personalized medicine and reflect a trend in the biopharmaceutical industry. Freeze drying of vials nested in a rack system is a tool used in new flexible pilot scale processing lines. Understanding of heat transfer mechanisms in the rack loaded with vials not in direct contact with each other is necessary to ensure high quality. Lyophilization in the rack vial system enables a homogeneous drying with a reduced edge-vial-effect and shielding against radiation from surrounding components, e.g., the chamber wall. Due to the separation effect of the rack, direct shelf contact contributes approx. 40% to the overall energy transfer to the product during primary drying. Hence overall the rack is a flexible, robust tool for small batch production, which ensures a controlled heat transfer resulting in a uniform product.

The missing piece in the puzzle: Prediction of aggregation via the protein-protein interaction parameter A∗2.

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 mAB1 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 µm, 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.

Impact of formulation pH on physicochemical protein characteristics at the liquid-air interface.

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.

Notorious but not understood: How liquid-air interfacial stress triggers protein aggregation.

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.

The film tells the story: Physical-chemical characteristics of IgG at the liquid-air interface.

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.