Strategies for the assessment of protein aggregates in pharmaceutical biotech product development.

Within the European Immunogenicity Platform (EIP) ( http://www.e-i-p.eu ), the Protein Characterization Subcommittee (EIP-PCS) has been established to discuss and exchange experience of protein characterization in relation to unwanted immunogenicity. In this commentary, we, as representatives of EIP-PCS, review the current state of methods for analysis of protein aggregates. Moreover, we elaborate on why these methods should be used during product development and make recommendations to the biotech community with regard to strategies for their application during the development of protein therapeutics.

Correlation of protein-protein interactions as assessed by affinity chromatography with colloidal protein stability: a case study with lysozyme.

Lysozyme-lysozyme interactions were assessed in the native state at 25 degrees C as well in the denatured state at 80 degrees C by affinity chromatography in order to measure the osmotic second virial coefficient (B). This parameter allows us to better understand protein aggregation pathways and colloidal protein stability. Repulsive interactions (B > 0) were weakened for both protein states by increasing salt concentration and by increasing the pH value toward lysozyme pI. This decrease was more pronounced in the denatured state, most likely caused by changes in electrostatic interactions and the formation of hydrophobic clusters. The lysozyme formulations presenting the more repulsive conditions (B > 0), as derived from the osmotic second virial coefficient, showed better colloidal stability under mechanical and thermal stresses. As expected, B values are much more negative for the interactions in the denatured state compared to the data obtained for the native state, reflecting a strong tendency of denatured lysozyme to aggregate. Thus, measurement of protein interactions by affinity chromatography allows us to gain information on protein interactions in both native and denatured states as well as to predict solution conditions prone for improving protein colloidal stability.

Systematic investigation of the effect of lyophilizate collapse on pharmaceutically relevant proteins, part 2: stability during storage at elevated temperatures.

The objective of this work was to investigate the effect of lyophilizate collapse on the stability of freeze-dried protein pharmaceuticals. In the first part of this study, it was shown that collapse has no negative impact either on the properties of the freeze-dried cake or on protein stability [Schersch K, Betz O, Garidel P, Muehlau S, Bassarab S, Winter G. 2010. J Pharm Sci 99(5):2256-2278]. In order to further investigate the effect of collapse, its impact on lyophilizate's long-term stability during storage at various temperatures was evaluated at 2°C-8°C, 25°C, 40°C, and 50°C for up to 6 months. Collapsed and noncollapsed lyophilizates of identical formulation and comparable residual moisture levels containing the following proteins were investigated: (1) a monoclonal immunoglobulin G antibody, (2) tissue-type plasminogen activator, and (3) the sensitive model protein l-lactic dehydrogenase. Protein stability was monitored using a comprehensive set of analytical techniques assessing the formation of soluble and insoluble aggregates, the biological activity, and the protein conformation. The properties of the freeze-dried cake--namely, the glass transition temperature, excipient crystallinity, reconstitution behavior, and the residual moisture content, were analyzed as well. Full protein stability in collapsed cakes was observed, and even enhanced protein stability was detected in collapsed cakes with regard to key stability-indicating parameters.

Development of a pilot-scale manufacturing process for protein-coated microcrystals (PCMC): mixing and precipitation - part I.

A novel protein-coated microcrystal (PCMC) technology offers the possibility to produce dry protein formulations suitable for inhalation or, after reconstitution, for injection. Micron-sized particles are hereby produced by co-precipitation via a rapid dehydration method. Thus, therapeutic proteins can be stabilised and immobilised on crystalline carrier surfaces. In this study, the development of a continuous manufacturing process is described, which can produce grams to kilograms of PCMC. The process chain comprises three steps: mixing/precipitation, solvent reduction (concentration) and final drying. The process is published in two parts. This part describes the mixing and precipitation performed using continuous impingement jet mixers. Mixing efficiency was improved by dividing the anti-solvent flow into two or four jets, which were combined again inside the mixer to achieve an embracing of the aqueous solution (sandwich effect). The jets provided high energy dissipation rates. The anti-solvent jets (95% of the total volume) efficiently mixed the protein-carrier containing aqueous solution (5% of the total volume), which was demonstrated with computational fluid dynamics and the Villermaux-Dushman reaction. The improved mixing performance of the double jet impingement (DJI) or the quadruple jet impingement (QJI) mixers showed a positive effect on easily crystallising carriers (e.g. dl-valine) at laminar flow rates. The mixer and outlet tube bore size was 2.0-3.2 mm, because smaller sizes showed a high tendency to block the mixer. The mixing effect by impaction was sufficiently high in the flow rate range of 250-2000 mL/min, which corresponds to the transition from laminar to turbulent flow characteristics. At lower flow rates, mixing was enhanced by ultrasound. 50-80L PCMC suspension was readily produced with the QJI mixer.

A critical evaluation of self-interaction chromatography as a predictive tool for the assessment of protein-protein interactions in protein formulation development: a case study of a therapeutic monoclonal antibody.

The aim of this study was to establish and evaluate a screening method for the physical characterization of protein-protein interactions of therapeutic proteins based on the determination of the osmotic second virial coefficient (B(22)). B(22) of an IgG1 was measured by self-interaction chromatography (SIC) and was compared to data obtained from static light scattering (SLS). As assessed by Fourier transform infrared spectroscopy (FTIR), the protein coupling to chromatography particles had no relevant influence on the three-dimensional native structure of the IgG1. B(22) variations could be measured for physiological relevant excipient concentrations. Significant positive B(22) values were observed for the following solution conditions of the investigated antibody: (i) acidic pH conditions, (ii) low buffer concentrations, (iii) low salt concentrations and (iv) high amino acid concentrations. B(22) was compared to IgG1 stability data derived from a study conducted for 12weeks at 40 degrees C. A concentration of 5mM histidine, which was the most promising buffer candidate according to B(22), showed a slightly better physical stability (as assessed by turbidity and size exclusion chromatography) compared to the other tested formulations. This is confirmed in a stress study investigating the colloidal stability. Thus, measuring protein-protein interactions with SIC appeared as a promising screening tool for physical characterization of protein formulations for cases in which the protein stability is governed by interparticle interactions.

A rapid, sensitive and economical assessment of monoclonal antibody conformational stability by intrinsic tryptophan fluorescence spectroscopy.

Steady-state intrinsic tryptophan fluorescence spectroscopy is used as a rapid, robust and economic way for screening the thermal protein conformational stability in various formulations used during the early biotechnology development phase. The most important parameters affecting protein stability in a liquid formulation, e. g. during the initial purification steps or preformulation development, are the pH of the solution, ionic strength, presence of excipients and combinations thereof. A well-defined protocol is presented for the investigation of the thermal conformational stability of proteins. This allows the determination of the denaturation temperature as a function of solution conditions. Using intrinsic tryptophan fluorescence spectroscopy for monitoring the denaturation and folding of proteins, it is crucial to understand the influence of different formulation parameters on the intrinsic fluorescence probes of proteins. Therefore, we have re-evaluated and re-assessed the influence of temperature, pH, ionic strength, buffer composition on the emission spectra of tryptophan, phenylalanine and tyrosine to correctly analyse and evaluate the data obtained from thermal-induced protein denaturation as a function of the solution parameters mentioned above. The results of this study are a prerequisite for using this method as a screening assay for analysing the conformational stability of proteins in solution. The data obtained from intrinsic protein fluorescence spectroscopy are compared to data derived from calorimetry. The advantage, challenges and applicability using intrinsic tryptophan fluorescence spectroscopy as a routine development method in pharmaceutical biotechnology are discussed.