Fluorescence Spectroscopy

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Fluorescence Spectroscopy

Method Introduction

  • The intrinsic fluorescence of proteins in solution is commonly measured to detect changes in protein conformation, for instance, due to different solution conditions (pH, excipients, etc.), elevated temperature and storage. Upon excitation at 280 nm, a protein’s fluorescence signal arises primarily from tryptophan and tyrosine residues. For example, selective excitation of tryptophan residues at 295 nm is also possible and commonly applied to proteins because tryptophan fluorescence is particularly sensitive to subtle conformational changes. Tyrosine and tryptophan residues are typically buried in the core of a folded protein (not solvent exposed). Partial unfolding and aggregation lead to changes in the local environment of these residues (e.g., solvent exposure), leading to changes in fluorescence intensity and emission maximum, amongst others.

  • Extrinsic fluorescence can also be used to detect changes in protein structure and the formation of aggregates. Various fluorescent dyes, such as Nile Red, ANS and Bis-ANS, can be used as hydrophobic probes. If a protein unfolds or aggregates, e.g., due to destabilizing solution conditions or increased temperature, the dye binds to hydrophobic patches on the protein (aggregates). It fluoresces with much greater intensity than free dye in solution.

Applications

Fluorescence spectroscopy is a frequently used and versatile technique for the analysis of higher-order structures of proteins.

Quality and Biosafety Level

We provide all our analytical services with the highest quality standards. Experienced scientists carry out each project, and a scientific reviewer comprehensively checks every report or data presentation.

We offer this technology with the following quality and biosafety levels:

R&D level

We offer this method under R&D. Our GRP system assures the highest-quality research standards.

Up to biosafety level 2

This method can be applied to nucleic acids, viruses, cells, viral vectors, including lentiviruses and more.

Analytical Method Development, Qualification and Validation

For common sample types, we can often apply standardized methods with little setup effort. However, when needed, our experienced analytical experts create or optimize custom methods tailored to your active pharmaceutical ingredient, product type and development phase.

Method Development

Our method development approach tailors sample preparation, method settings and data analysis to the needs of your project and sample.

We include representative samples and, where available, suitable reference standards and stressed/degraded materials, allowing our analytical scientists to design a highly suitable, stability-indicating, robust and repeatable method. Upon request, we will compile a detailed description of the method for your records.

Method Qualification

Method qualification is the initial assessment of an analytical procedure’s performance to show its suitability for its intended purpose.

During method qualification, our analytical scientists perform documented testing demonstrating that the analytical procedure meets criteria in several categories. Criteria may include factors such as repeatability, specificity and robustness. We compile a qualification plan and report, including all relevant data.

Method Validation

Under GMP conditions, method validation confirms that an analytical procedure’s performance suits its intended purpose. Depending on the method’s scope, a broad range of method characteristics, such as specificity, accuracy, precision, limit of detection/limit of quantification (LOD/LOQ), linearity and range, is considered.

During method validation, our analytical scientists perform documented testing demonstrating that the analytical procedure consistently produces a result that meets the predetermined acceptance criteria. We compile a validation plan and report that includes all relevant data.

Depending on the development phase, a fit-for-purpose validation approach can be offered, adjusting the validation required efforts in a phase-appropriate way to meet the method’s needs.

Method Verification

Compendial method verification confirms that a compendial method (e.g., from Ph. Eur. or USP) is suitable and reliable for its intended purpose under the specific conditions of the laboratory.

Unlike full method validation, compendial method verification is often considered a partial validation since the method has already undergone extensive testing and validation during its inclusion in the compendium. The extent of method verification depends on the type of method.

During method verification, our analytical scientists perform documented testing demonstrating that the developed analytical method performs adequately for the specific product or matrix being tested and within the specific laboratory where the method will be employed.

Talk to Our Experts or Request a Quote

Our expert team is ready to answer your questions and guide you to the services best suited to your program’s modality, stage and challenge. If your needs are well-defined, we’ll begin the quotation process.

Description

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