Andrea Hawe

Extrinsic Fluorescent Dyes as Tools for Protein Characterization

Noncovalent, extrinsic fluorescent dyes are applied in various fields of protein analysis, e.g. to characterize folding intermediates, measure surface hydrophobicity, and detect aggregation or fibrillation. The main underlying mechanisms, which explain the fluorescence properties of many extrinsic dyes, are solvent relaxation processes and (twisted) intramolecular charge transfer reactions, which are affected by the environment and by interactions of the dyes with proteins. In recent time, the use of extrinsic fluorescent dyes such as ANS, Bis-ANS, Nile Red, Thioflavin T and others has increased, because of their versatility, sensitivity and suitability for high-throughput screening. The intention of this review is to give an overview of available extrinsic dyes, explain their spectral properties, and show illustrative examples of their various applications in protein characterization.

Structural properties of monoclonal antibody aggregates induced by freeze-thawing and thermal stress.

Aggregation of monoclonal antibodies can be induced by freeze-thawing and elevated temperature, typical stress factors during development, production and storage. Our aim was to characterize structural properties of aggregates formed after freeze-thawing and thermal stressing of humanized monoclonal IgG(1) antibody (IgG). Formulations with 1.0mg/ml IgG in 100mM phosphate pH 7.2 were subjected to freeze-thawing and heating and characterized by spectroscopic techniques (UV-absorption, CD, ATR-FTIR and fluorescence), light obscuration, dynamic light scattering, SDS-PAGE, AF4 with UV and MALLS detection, and HP-SEC with UV and online fluorescent dye detection. Thermal stress led to an increased formation of dimers and soluble oligomers (HP-SEC, AF4). Aggregates smaller than 30nm were measured (DLS), next to slightly elevated particle levels in the mum range (light obscuration). Aggregates created by heating were in part covalently linked (SDS-PAGE) and made up of conformationally perturbed monomers (CD, ATR-FTIR, extrinsic dye fluorescence). Aggregation after freeze-thawing was manifested primarily in particle formation in the mum range. These aggregates were noncovalently linked (SDS-PAGE) and composed of native-like monomers, as obvious from CD, ATR-FTIR and extrinsic dye fluorescence spectroscopy. In conclusion, the complementary methods used in this study revealed that heating and freeze-thawing induced aggregates differ significantly in their physico-chemical characteristics.

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.

Particles in Therapeutic Protein Formulations, Part 1: Overview of Analytical Methods

The presence of particles is a major issue during therapeutic protein formulation development. Both proteinaceous and nonproteinaceous particles need to be analyzed not only due to the requirements of the Pharmacopeias but also to monitor the stability of the protein formulation. Increasing concerns about the immunogenic potential together with new developments in particle analysis make a comparative description of established and novel analytical methods useful. Our review aims to provide a comprehensive overview on analytical methods for the detection and characterization of visible and subvisible particles in therapeutic protein formulations. We describe the underlying theory, benefits, shortcomings, and illustrative examples for quantification techniques, as well as characterization techniques for particle shape, morphology, structure, and identity.

Quality of original and biosimilar epoetin products.

ABSTRACTPurposeTo compare the quality of therapeutic erythropoietin (EPO) products, including two biosimilars, with respect to content, aggregation, isoform profile and potency.MethodsTwo original products, Eprex (epoetin alfa) and Dynepo (epoetin delta), and two biosimilar products, Binocrit (epoetin alfa) and Retacrit (epoetin zeta), were compared using (1) high performance size exclusion chromatography, (2) ELISA, (3) SDS-PAGE, (4) capillary zone electrophoresis and (5) in-vivo potency.ResultsTested EPO products differed in content, isoform composition, and potency.ConclusionOf the tested products, the biosimilars have the same or even better quality as the originals. Especially, the potency of originals may significantly differ from the value on the label.

Micro-flow imaging and resonant mass measurement (Archimedes)--complementary methods to quantitatively differentiate protein particles and silicone oil droplets.

Our study aimed to comparatively evaluate Micro-Flow Imaging (MFI) and the recently introduced technique of resonant mass measurement (Archimedes, RMM) as orthogonal methods for the quantitative differentiation of silicone oil droplets and protein particles. This distinction in the submicron and micron size range is highly relevant for the development of biopharmaceuticals, in particular for products in prefilled syringes. Samples of artificially generated silicone oil droplets and protein particles were quantified individually and in defined mixtures to assess the performance of the two techniques. The built-in MFI software solution proved to be suitable to discriminate between droplets and particles for sizes above 2 μm at moderate droplet/particle ratios (70:30-30:70). A customized filter developed specifically for this study greatly improved the results and enabled reliable discrimination also for more extreme mixing ratios (95:5-15:85). RMM showed highly accurate discrimination in the size range of about 0.5-2 μm independent of the ratio, provided that a sufficient number of particles (>50 counted particles) were counted. We recommend applying both techniques for a comprehensive analysis of biotherapeutics potentially containing silicone oil droplets and protein particles in the submicron and micron size range.

Flow Imaging Microscopy for Protein Particle Analysis—A Comparative Evaluation of Four Different Analytical Instruments

Flow imaging microscopy was introduced as a technique for protein particle analysis a few years ago and has strongly gained in importance ever since. The aim of the present study was a comparative evaluation of four of the most relevant flow imaging microscopy systems for biopharmaceuticals on the market: Micro-Flow Imaging (MFI)4100, MFI5200, Flow Cytometer And Microscope (FlowCAM) VS1, and FlowCAM PV. Polystyrene standards, particles generated from therapeutic monoclonal antibodies, and silicone oil droplets were analyzed by all systems. The performance was critically assessed regarding quantification, characterization, image quality, differentiation of protein particles and silicone oil droplets, and handling of the systems. The FlowCAM systems, especially the FlowCAM VS1, showed high-resolution images. The FlowCAM PV system provided the most precise quantification of particles of therapeutic monoclonal antibodies, also under impaired optical conditions by an increased refractive index of the formulation. Furthermore, the most accurate differentiation of protein particles and silicone oil droplets could be achieved with this instrument. The MFI systems provided excellent size and count accuracy (evaluated with polystyrene standards) especially the MFI5200 system. This instrument also showed very good performance for protein particles, also in case of an increased refractive index of the formulation. Both MFI systems were easier to use and appeared more standardized regarding measurement and data analysis as compared to the FlowCAM systems. Our study shows that the selection of the appropriate flow imaging microscopy system depends strongly on the main output parameters of interest and it is recommended to decide based on the intended application.

Asymmetrical Flow Field-Flow Fractionation Method for the Analysis of Submicron Protein Aggregates

For the analysis of protein aggregates in the submicron size range, there is still a need for reliable, quantitative methods that can assist the development of therapeutic protein formulations. The aim of our study was to develop an asymmetrical flow field-flow fractionation (AF4) method for the analysis of protein aggregates in the size range of up to approximately 1000 nm. Method development was performed with polystyrene standard beads (60, 200, and 1000 nm) and a heat-stressed IgG formulation containing a substantial amount of submicron aggregates. By AF4, the analysis of these heterodisperse submicron IgG aggregates, as well as the monomer, could be achieved by a stepwise reduction of the cross-flow. The suitability of the developed AF4 method for aggregate analysis in general was demonstrated by analyzing several other stressed therapeutic protein samples (another IgG and etanercept). In each case, a clearly better separation and a more reproducible (although in some cases incomplete) recovery was achieved with AF4 as compared with high-performance size-exclusion chromatography. In conclusion, AF4 proved to be a valuable method for the characterization and quantification of submicron protein aggregates.

Transient Molten Globules and Metastable Aggregates Induced by Brief Exposure of a Monoclonal IgG to Low pH

The presence of aggregates in therapeutic protein formulations is of great concern due to quality, safety, and efficacy issues. Nonetheless, the mechanisms and kinetics of protein aggregation are only partly understood. In this study, metastable immunoglobulin G (IgG) aggregates induced by a brief exposure to pH 1 were kept at 4°C and analyzed over time by size-exclusion chromatography (SEC), nanoparticle tracking analysis, light obscuration, dynamic light scattering, fluorescence spectroscopy, and circular dichroism. The results show the formation of polydisperse aggregates (from dimers to 10-μm particles) shortly after the pH-shift stress. These aggregates increased in size and number over time until a pseudo-equilibrium was reached after 5-7 days. The presence of transient, partially unfolded monomers (molten globules) was detected by SEC with online fluorescent dye detection. The molten globules seemed to either refold into the native state or become involved in aggregation pathways. Seeding pH-shift-induced aggregates into unstressed IgG did not accelerate aggregation during incubation for 3 weeks at 55°C. These results reinforce the role of partially unfolded species in the aggregation of therapeutic proteins. We conclude that the formation of pH-shift-induced IgG aggregates is likely driven by downhill polymerization, as a consequence of successive additions of molten globular monomers.

Optimization of a pharmaceutical freeze-dried product and its process using an experimental design approach and innovative process analyzers.

The aim of the present study was to examine the possibilities/advantages of using recently introduced in-line spectroscopic process analyzers (Raman, NIR and plasma emission spectroscopy), within well-designed experiments, for the optimization of a pharmaceutical formulation and its freeze-drying process. The formulation under investigation was a mannitol (crystalline bulking agent)-sucrose (lyo- and cryoprotector) excipient system. The effects of two formulation variables (mannitol/sucrose ratio and amount of NaCl) and three process variables (freezing rate, annealing temperature and secondary drying temperature) upon several critical process and product responses (onset and duration of ice crystallization, onset and duration of mannitol crystallization, duration of primary drying, residual moisture content and amount of mannitol hemi-hydrate in end product) were examined using a design of experiments (DOE) methodology. A 2-level fractional factorial design (2(5-1)=16 experiments+3 center points=19 experiments) was employed. All experiments were monitored in-line using Raman, NIR and plasma emission spectroscopy, which supply continuous process and product information during freeze-drying. Off-line X-ray powder diffraction analysis and Karl-Fisher titration were performed to determine the morphology and residual moisture content of the end product, respectively. In first instance, the results showed that - besides the previous described findings in De Beer et al., Anal. Chem. 81 (2009) 7639-7649 - Raman and NIR spectroscopy are able to monitor the product behavior throughout the complete annealing step during freeze-drying. The DOE approach allowed predicting the optimum combination of process and formulation parameters leading to the desired responses. Applying a mannitol/sucrose ratio of 4, without adding NaCl and processing the formulation without an annealing step, using a freezing rate of 0.9°C/min and a secondary drying temperature of 40°C resulted in efficient freeze-drying supplying end products with a residual moisture content below 2% and a mannitol hemi-hydrate content below 20%. Finally, using Monte Carlo simulations it became possible to determine how varying the factor settings around their optimum still leads to fulfilled response criteria, herewith having an idea about the probability to exceed the acceptable response limits. This multi-dimensional combination and interaction of input variables (factor ranges) leading to acceptable response criteria with an acceptable probability reflects the process design space.