A High Throughput Protein Formulation Platform: Case Study of Salmon Calcitonin

Purpose: The feasibility of using high throughput spectroscopy for characterization and selection of physically stable protein formulations was studied. Materials and Methods: A hundred aqueous formulations of salmon calcitonin (sCT) were prepared using 20 buffer compositions. The solutions had pH values between 2.5 and 10.5. The stability of the sCT formulations was analyzed over 1week by the following assays: (1) protein concentration, (2) volume control by measuring pathlength, (3) turbidity (absorbance at 350nm), (4) intrinsic tyrosine fluorescence, (5) 1-anilino-naphthalene-8-sulfonate (ANS) fluorescence, (6) Nile Red fluorescence. Addition of the dyes (Nile Red and ANS) was used to study protein conformational changes. Results: After 1day, 27 out of the 100 formulations of salmon calcitonin were stable. After 7days, 12 stable sCT formulations remained. The best salmon calcitonin formulation was in 10mM sodium acetate buffer with pH values between 3.5 and 5.5. Conclusions: The findings are in accordance with the sCT formulations that were patented and used commercially. This can be considered as a proof of concept for the high throughput protein formulation platfor

Inhibition of Succinimide Formation in Aqueous Zn-rHirudin Suspensions1

Purpose. The formation of succinimide intermediates at Asp-Gly sites and their hydrolysis products, e.g., isoAsp isomers, represents a common source of microheterogeneity in therapeutic proteins. Here we report on the stabilization effect of a zinc chloride induced precipitation of recombinant hirudin HV1 (rHir), an anticoagulant protein. Methods. rHir was precipitated by zinc chloride at neutral pH to form a Zn-rHir suspension. An Arrhenius-type study (at 50, 40, 30, and 25°C) and a 4°C stability study were performed. Monitoring of rHir, rHir succinimides at Asp33-Gly34 (Q5) and Asp53-Gly54 (Q4), and further side products was by capillary electrophoresis (CE). Results. The activation energies of rHir degradation in both aqueous rHir solution and Zn-rHir suspension were similar, i.e. 104.5 and 110.3 kJ/mol, respectively. Zn-rHir suspension demonstrated improved shelf-life stability (t90%, 95% confidence limit) versus rHir solution, i.e., 23 versus 3 days at 25°C and 292 versus 147 days at 4°C, respectively. In rHir solution, Q4 (Asp53-Gly54 succinimide) levels were slightly above Q5 (Asp33-Gly34 succinimide) levels. In Zn-rHir suspension, however, Q4 succinimide levels dropped markedly whereas Q5 levels were not affected. Correspondingly, in Zn-rHir isoAsp53-rHir levels were reduced but not isoAsp33-rHir levels. Conclusions. In Zn-rHir suspensions, interactions of zinc and rHir show site-specific inhibition of succinimide formation only at Asp53-Gly54 (Q4), located in the highly flexible C-terminal tail of rHir. In contrast, succinimide formation at Asp33-Gly34 (Q5), located in a less flexible loop domain is not affected, reflecting steric hindranc

Where disease pathogenesis meets protein formulation: renal deposition of immunoglobulin aggregates

Aggregation is one of the important issues encountered during the development of immunoglobulin-based drugs. The aim of the current review is to discuss the causes and consequences of immunoglobulin aggregation as well as the relevance of immunoglobulin aggregation to disease pathogenesis. Extracellular deposition of immunoglobulins, either monoclonal light chains or intact polyclonal antibodies, induces renal failure in various nephropathies. The aggregates can present fibrillar or amorphous structures. In this review, factors known to influence protein aggregation, such as the primary structure of the protein, local environment and glycosylation are assessed, as well as the subsequent altered clearance, fibril formation and toxicity. The role of the protein local environment is emphasized. Even if the local environment causes only minor perturbations in the protein structure, these perturbations might be sufficient to trigger aggregate formation. This fact underlines the importance of choosing appropriate formulations for protein drugs. If the formulation provides a slightly destabilizing environment to the protein, the long-term stability of the drug may be compromised by aggregate formation

Detection and characterization of protein aggregates by fluorescence microscopy

Aggregation may compromise the stability as well as the biological activity of protein drugs. Detection of protein aggregates is needed in the process of protein characterization and during optimization of pharmaceutical formulations. This paper describes a technique, which consists of analysing protein aggregates by fluorescence microscopy after staining with the hydrophobic probe Nile Red. Dilution, filtration or other modifications of the sample are not needed. Assessment of aggregation was possible in highly concentrated protein samples (193 mg/ml). Fluorescence microscopy observations allowed the detection and characterization of protein aggregates not easily detected by spectroscopic techniques. Nile Red was shown to be very sensitive for the detection and analysis of immunoglobulin aggregates. Nile Red, Congo red and Thioflavine T stainings were compared. Nile Red and Thioflavine T fluorescence were colocalized. The diameter of immunoglobulin aggregates was determined, and the number of aggregates was correlated with 90 degrees light scattering measurements. Studies of human calcitonin aggregates brought to light new aspects of the human calcitonin aggregation mechanisms. Thus, Nile Red staining not only allows detection of very low levels of protein aggregates, but also contributes to a better understanding of the complex mechanisms governing protein aggregation

A high throughput protein formulation platform: case study of salmon calcitonin

PURPOSE: The feasibility of using high throughput spectroscopy for characterization and selection of physically stable protein formulations was studied. MATERIALS AND METHODS: A hundred aqueous formulations of salmon calcitonin (sCT) were prepared using 20 buffer compositions. The solutions had pH values between 2.5 and 10.5. The stability of the sCT formulations was analyzed over 1 week by the following assays: (1) protein concentration, (2) volume control by measuring pathlength, (3) turbidity (absorbance at 350 nm), (4) intrinsic tyrosine fluorescence, (5) 1-anilino-naphthalene 8-sulfonate (ANS) fluorescence, (6) Nile Red fluorescence. Addition of the dyes (Nile Red and ANS) was used to study protein conformational changes. RESULTS: After 1 day, 27 out of the 100 formulations of salmon calcitonin were stable. After 7 days, 12 stable sCT formulations remained. The best salmon calcitonin formulation was in 10 mM sodium acetate buffer with pH values between 3.5 and 5.5. CONCLUSIONS: The findings are in accordance with the sCT formulations that were patented and used commercially. This can be considered as a proof of concept for the high throughput protein formulation platform

High throughput screening of protein formulation stability: practical considerations

The formulation of protein drugs is a difficult and time-consuming process, mainly due to the complexity of protein structure and the very specific physical and chemical properties involved. Understanding protein degradation pathways is essential for the success of a biopharmaceutical drug. The present review concerns the application of high throughput screening techniques in protein formulation development. A protein high throughput formulation (HTF) platform is based on the use of microplates. Basically, the HTF platform consists of two parts: (i) sample preparation and (ii) sample analysis. Sample preparation involves automated systems for dispensing the drug and the formulation ingredients in both liquid and powder form. The sample analysis involves specific methods developed for each protein to investigate physical and chemical properties of the formulations in microplates. Examples are presented of the use of protein intrinsic fluorescence for the analysis of protein aqueous properties (e.g., conformation and aggregation). Different techniques suitable for HTF analysis are discussed and some of the issues concerning implementation are presented with reference to the use of microplates

High throughput methods to characterize protein permeation and release

oai:unige.ch:unige:1Spectroscopic methods have been developed to study protein permeation and release kinetics in multi-well plates. The permeation of bovine serum albumin (BSA) through a membrane, which separated a 96-well plate in two compartments, was characterized. A change in fluorescence intensity was measured corresponding to the permeation of BSA from one compartment to another. The permeation of BSA was influenced by the pore size and pore density size of the membrane. The multi-well plates were also used to study the release of a protein drug, hirudin, from an agar hydrogel. A hirudin formulation was mixed at 60 degrees C with liquid agar and the mixture turned to a gel by cooling at room temperature. The gel entrapping hirudin was formed inside the wells of a 96-well plate. On top of the 100microl agar-hirudin gel a volume of 200microl of 10mM phosphate buffer pH 7.4, 140mM NaCl was added. The release kinetics of hirudin from the gel were measured following the changes in the hirudin intrinsic tyrosine fluorescence. The release of hirudin over 12h was measured at three positions: at the bottom of the agar gel, at the interface of the gel with the solution, and in the middle of the receiver solution. The data presented in this paper indicate that high throughput methods can be applied in the characterization of protein drug release from drug delivery systems using small sample volumes

Inhibition of Succinimide Formation in Aqueous Zn-rHirudin Suspensions1

ISSN:0724-8741ISSN:1573-904