Purpose
To evaluate the nanoparticle tracking analysis (NTA) technique, compare it with dynamic light scattering (DLS) and test its performance in characterizing drug delivery nanoparticles and protein aggregates.

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Critical Evaluation of Nanoparticle Tracking Analysis (NTA) by NanoSight for the Measurement of Nanoparticles and Protein Aggregates

In 1989, Manning, Patel, and Borchardt wrote a review of protein stability (Manning et al, Pharm Res 6:903-918, 1989), which has been widely referenced ever since At the time, recombinant protein therapy was still in its infancy This review summarizes the advances that have been made since then regarding protein stabilization and formulation In addition to a discussion of the current understanding of chemical and physical instability, sections are included on stabilization in aqueous solution and the dried state, the use of chemical modification and mutagenesis to improve stability, and the interrelationship between chemical and physical instability

Stability of Protein Pharmaceuticals: An Update

http://wolfson.huji.ac.il/purification/Course92632_2014/Aggregation/MAHLER2009.pdf

Protein aggregation: Pathways, induction factors and analysis

Protein–excipient interactions: Mechanisms and biophysical characterization applied to protein formulation development

Biologics such as monoclonal antibodies are much more complex than small-molecule drugs, which raises challenging questions for the development and regulatory evaluation of follow-on versions of such biopharmaceutical products (also known as biosimilars) and their clinical use once patent protection for the pioneering biologic has expired. With the recent introduction of regulatory pathways for follow-on versions of complex biologics, the role of analytical technologies in comparing biosimilars with the corresponding reference product is attracting substantial interest in establishing the development requirements for biosimilars. Here, we discuss the current state of the art in analytical technologies to assess three characteristics of protein biopharmaceuticals that regulatory authorities have identified as being important in development strategies for biosimilars: post-translational modifications, three-dimensional structures and protein aggregation.

Analytical tools for characterizing biopharmaceuticals and the implications for biosimilars

Shaken, not stirred: mechanical stress testing of an IgG1 antibody.

To study the potential impact of the degradation of Polysorbates (PS) 20 and 80 on the stability of therapeutic proteins in parenteral formulations. First, 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. The 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. Although 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.

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

Equilibrium studies of protein aggregates and homogeneous nucleation in protein formulation

Shaking or heat stress may induce protein aggregates. Aggregation beha- vior of an IgG1 stressed by shaking or heat following static storage at 5 and 258C 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. Sig- nificant 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, inde- pendent of the spiking concentration over time. Thus, neither mechanically stressed native nor temperature-induced denatured aggregates were involved in nucleating or propagating aggregation. 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 99:632-644, 2010

Sylvia Kiese

Papers

Protein aggregation: Pathways, induction factors and analysis

Shaken, not stirred: mechanical stress testing of an IgG1 antibody.

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

Equilibrium studies of protein aggregates and homogeneous nucleation in protein formulation

BIOTECHNOLOGY Equilibrium Studies of Protein Aggregates and Homogeneous Nucleation in Protein Formulation