Hanns-Christian Mahler

Protein aggregation: Pathways, induction factors and analysis

Control and analysis of protein aggregation is an increasing challenge to pharmaceutical research and development. Due to the nature of protein interactions, protein aggregation may occur at various points throughout the lifetime of a protein and may be of different quantity and quality such as size, shape, morphology. It is therefore important to understand the interactions, causes and analyses of such aggregates in order to control protein aggregation to enable successful products. This review gives a short outline of currently discussed pathways and induction methods for protein aggregation and describes currently employed set of analytical techniques and emerging technologies for aggregate detection, characterization and quantification. 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. Each analytical method not only shows its specific advantages but also has its limitations. The limits of detection and the possibility of creating artifacts through sample preparation by inducing or destroying aggregates need to be considered with each method used. Therefore, it may also be advisable to carefully compare analytical results of orthogonal methods for similar size ranges to evaluate method performance.

An Industry Perspective on the Monitoring of Subvisible Particles as a Quality Attribute for Protein Therapeutics

Concern around the lack of monitoring of proteinaceous subvisible particulates in the 0.1-10 microm range has been heightened (Carpenter et al., 2009, J Pharm Sci 98: 1202-1205), primarily due to uncertainty around the potential immunogenicity risk from these particles. This article, representing the opinions of a number of industry scientists, aims to further the discussion by developing a common understanding around the technical capabilities, limitations, as well as utility of monitoring this size range; reiterating that the link between aggregation and clinical immunogenicity has not been unequivocally established; and emphasizing that such particles are present in marketed products which remain safe and efficacious despite the lack of monitoring. Measurement of subvisible particulates in the <10 microm size range has value as an aid in product development and characterization. Limitations in measurement technologies, variability from container/closure, concentration, viscosity, history, and inherent batch heterogeneity, make these measurements unsuitable as specification for release and stability or for comparability, at the present time. Such particles constitute microgram levels of protein with currently monitored sizes >or=10 microm representing the largest fraction. These levels are well below what is detected or reported for other product quality attributes. Subvisible particles remain a product quality attribute that is also qualified in clinical trials.

Forced Degradation of Therapeutic Proteins

The scope of this paper is to review approaches used for forced degradation (synonym, stress testing) of therapeutic proteins. Forced degradation studies play a central role in the development of therapeutic proteins, for example, for candidate selection, molecule characterization, formulation development, assay development, and comparability studies. Typical stress methods are addressed within this review, such as exposure to elevated temperatures, freeze-thawing, mechanical stress, oxidation, light, as well as various materials and devices used in the clinics during final administration. Stability testing is briefly described as far as relevant to the discussion of forced degradation studies. Whereas stability-testing requirements are defined in regulatory guidelines, standard procedures for forced degradation of therapeutic proteins are largely unavailable, except for photostability. Possible selection criteria to identify appropriate stress conditions and recommendations for setting up forced degradation studies for the different phases of development of therapeutic proteins are presented.

The Degradation of Polysorbates 20 and 80 and its Potential Impact on the Stability of Biotherapeutics

ABSTRACTPurposeTo study the potential impact of the degradation of Polysorbates (PS) 20 and 80 on the stability of therapeutic proteins in parenteral formulations.MethodFirst, degradation products of PS20 and 80 were identified. Subsequently, the effect of degraded polysorbate on physical characteristics and long-term stability of protein formulations was assessed. Further, the impact of polysorbate degradation on protein stability was evaluated via shaking stress studies on formulations spiked with artificially degraded polysorbate or degradants like fatty acids. Additionally, aged formulations with reduced polysorbate content were shaken.ResultsThe degradation of polysorbate leads to a buildup of various molecules, some of which are poorly soluble, including fatty acids and polyoxyethylene (POE) esters of fatty acids. Spiking studies showed that the insoluble degradants could potentially impact protein stability and that the presence of sufficient intact polysorbate was crucial to prevent this. End-of-shelf-life shaking of protein formulations showed that the stability of various monoclonal antibodies was, however, not affected.ConclusionsAlthough some degradants can potentially influence the stability of the protein (as discerned from spiking studies), degradation of polysorbates did not impact the stability of the different proteins tested in pharmaceutically relevant temperature and storage conditions.

Degradation of polysorbates 20 and 80: Studies on thermal autoxidation and hydrolysis

The purpose of this work was to study the mechanistic pathways of degradation of polysorbates (PS) 20 and PS80 in parenteral formulations. The fate of PS in typical protein formulations was monitored and analyzed by a variety of methods, including (1)H NMR, high-performance liquid chromatography/evaporative light scattering detection, and ultraviolet-visible spectroscopy. Oxidative degradation of PS in neat raw material was studied using thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), and headspace gas chromatography-mass spectrometry. TGA-DSC studies revealed that autoxidation via a radical mechanism is dominated by statistical random scission in PS20 and PS80. Thermal initiation of radical formation occurs at the polyoxyethylene (POE) as well as the olefin sites. In PS80, radical initiation at the olefinic site precedes initiation at the POE site, leading to modified degradation profile. Corresponding to these results, in aqueous formulations, a surge peroxide content was detected in PS20-containing samples and in higher concentrations in those containing PS80. Hydrolysis in aqueous formulations, as followed by (1)H NMR, was found to have a half-life of 5 months at 40°C. On the basis of the obtained results, PSs degrade mainly via autoxidation and also via hydrolysis at higher temperatures. Further studies are required to investigate on potential effects of degradation on surface activity and protein stability in PS-containing formulations.

Freeze drying of human serum albumin (HSA) nanoparticles with different excipients

Freeze drying is a suitable technique to improve the long-term storage stability of colloidal drug carrier systems such as nanoparticles. Aim of this study was to systematically evaluate excipients for the freeze drying and long-term stability of albumin-based nanoparticles. In our study, nanoparticles made of human serum albumin (HSA) were freeze dried in the presence of different cryoprotective agents and after reconstitution were evaluated with regard to their physico-chemical characteristics. Empty, doxorubicin-loaded, and PEGylated nanoparticles were prepared and were freeze dried in the presence of different concentrations of sucrose, trehalose, and mannitol, respectively. The samples were physico-chemically characterised with regard to lyophilisate appearance, particle size, and polydispersity using photon correlation spectroscopy. For evaluation of long-term stability, the samples were stored at 2-8, 25, and 40 degrees C over predetermined time intervals. In the absence of cryoprotectants, particle growth was observed in all freeze-dried formulations. In the presence of sucrose, mannitol, and trehalose aggregation of HSA nanoparticles during the freeze-drying procedure was prevented. Although all of the excipients were identified to be suitable stabilisers for freeze drying of HSA nanoparticles, sucrose and trehalose were superior to mannitol, especially with regard to the long-term storage stability results.

Glycation during storage and administration of monoclonal antibody formulations

Covalent modifications of therapeutic proteins are of interest for the biotech industry as they potentially impact the quality of the material. This study focuses on covalent protein modifications by the reducing monosaccharide glucose via the glycation reaction. In particular, we examined (i) to which extent different therapeutic monoclonal antibodies are glycated, (ii) the glycation during storage in sucrose-containing formulation buffers where non-reducing sucrose potentially could hydrolyze into its reducing constituent monosaccharides and (iii) the risk of glycation in the course of short-term incubation in Dextrose infusion bags in simulated administration testing. A boronate affinity chromatography method was employed to detect and quantify glycation products in different antibody formulations. For confirmation and to determine the degree of glycation per molecule, selected samples were analyzed via LC-ESI-MS. We could demonstrate that different antibodies differed drastically regarding the degree of glycation, probably a result of their respective fermentation conditions and protein glycation susceptibility. We also demonstrated that sucrose is a non-critical excipient with respect to glycation when stored long-term at intended storage conditions (2-8 degrees C). Finally, we could show that short-term incubation of antibodies in Dextrose infusion bags might lead to huMAb glycation, suggesting to test on glycated products when considering diluting protein drug products in infusion media containing reducing sugars.

Surface Activity of a Monoclonal Antibody

The development of high concentration antibody formulations presents a major challenge for the formulation scientist, as physical characteristics and stability behavior change compared to low concentration protein formulations. The aim of this study was to investigate the potential correlation between surface activity and shaking stress stability of a model antibody-polysorbate 20 formulation. The surface activities of pure antibody and polysorbate 20 were compared, followed by a study on the influence of a model antibody on the apparent critical micelle concentration (CMC) of polysorbate 20 over a protein concentration range from 10 to 150 mg/mL. In a shaking stress experiment, the stability of 10, 75, and 150 mg/mL antibody formulations was investigated containing different concentrations of polysorbate 20, both below and above the CMC. The antibody increased significantly the apparent CMC of antibody-polysorbate 20 mixtures in comparison to the protein-free buffer. However, the concentration of polysorbate required for stabilization of the model antibody in a shaking stress experiment did not show dependence on the CMC. A polysorbate 20 level of 0.005% was found sufficient to stabilize both at low and high antibody concentration against antibody aggregation and precipitation.

Equilibrium studies of protein aggregates and homogeneous nucleation in protein formulation

Shaking or heat stress may induce protein aggregates. Aggregation behavior of an IgG1 stressed by shaking or heat following static storage at 5 and 25 degrees C was investigated to determine whether protein aggregates exist in equilibrium. Aggregates were detected using different analytical methods including visual inspection, turbidity, light obscuration, size exclusion chromatography, and dynamic light scattering. Significant differences were evident between shaken and heated samples upon storage. Visible and subvisible particles (insoluble aggregates), turbidity and z-average diameter decreased whilst soluble aggregate content increased in shaken samples over time. Insoluble aggregates were considered to be reversible and dissociate into soluble aggregates and both aggregate types existed in equilibrium. Heat-induced aggregates had a denatured protein structure and upon static storage, no significant change in insoluble aggregates content was shown, whilst changes in soluble aggregates content occurred. This suggested that heat-induced insoluble aggregates were irreversible and not in equilibrium with soluble aggregates. Additionally, the aggregation behavior of unstressed IgG1 after spiking with heavily aggregated material (shaken or heat stressed) was studied. The aggregation behavior was not significantly altered, independent of the spiking concentration over time. Thus, neither mechanically stressed native nor temperature-induced denatured aggregates were involved in nucleating or propagating aggregation.

Excipient Effects on Humanized Monoclonal Antibody Interactions with Silicone oil Emulsions

The interfacial adsorption of three humanized monoclonal antibodies to emulsions of microdroplets of silicone oil was examined using indirect measurement via integrated peak areas in size-exclusion high-performance liquid chromatograms. The level of silicone oil far exceeded the typical levels used in prefillable syringes. The three antibodies rapidly adsorbed to silicone oil-water interfaces in various buffer formulations. Addition of 140 mM NaCl to solutions buffered with 10 mM l-histidine, pH 6.0, increased the amount of protein adsorbed. Conversely, the extent of adsorption was significantly decreased by the addition of 0.03% (w/v) Tween® 20. Stern-Volmer constants determined from intrinsic tryptophan fluorescence quenching by acrylamide suggested that the tertiary structure of the adsorbed antibodies was significantly perturbed. However, no aggregation or precipitation of the antibodies was detected. Flow cytometric analysis of emulsions of fluorescently stained silicone oil in solutions containing fluorescently labeled antibodies and light microscopy experiments suggested that agglomeration of silicone oil droplets in the emulsions occurred. Zeta potentials measured for silicone oil microdroplets with adsorbed antibodies suggested that droplet agglomeration was probably the result of reduced electrostatic energy barriers to droplet collisions.