Biologics Development:

Analytical

Overview of Analytical Methods Used for Characterization of Biologics

Chromatographic Methods

Spectroscopic Techniques

Peptide Mapping

Other Characterization Techniques

Overview of Analytical Methods Used for Characterization of Biologics

The success of the drug development process is directly dependent on the utilization of accurate analytical methods for identification, quantitation and purity analysis of the drug substance (DS) and the drug product (DP). Wolfe Laboratories performs these and other evaluations by characterizing key biophysical and chemical properties of the drug substance under various conditions, performed throughout the development process, as summarized in the following table.

Characterizations required during the drug development process for biologics

Analytical method development progresses in parallel with the progress of the program. In general, discovery-stage development can be supported with a limited number of analytical methods, for example SE-HPLC, ELISA, UV-Vis spectroscopy, SDS-PAGE and peptide mapping combined with LC-MS. As the program advances, however, additional techniques are implemented to better characterize the drug substance, and additional chromatographic methods are developed to support formulation development. Wolfe Laboratories develops the requisite methods de novo or installs existing methods. With complex molecules, whether they areproteins, antibodies or various conjugates, a battery of orthogonal methods typically determines all relevant properties of either the drug substance or the drug product. Wolfe utilizes typical biophysical and chemical methods during the development of biologics, which are summarized in the following table.

Typical battery of analytical methods used for development and release testing of biologic therapeutics

Chromatographic Methods

Chromatographic methods (HPLC, UPLC, LC-MS) characterize biophysical and chemical properties during preformulation characterization, formulation development and in release testing and are common tools for determination of physical and chemical degradation. These methods must be accurate and robust and indicate stability. Depending on the nature of the compound, an array of orthogonal analytical techniques is required to assess stability. Wolfe Laboratories analyzes biologics with such chromatographic methods as summarized in the following table.

Chromatographic methods to determine biophysical and chemical properties of biologics

LC-MS is a valuable tool for the characterization of peptide/protein fragments, chemical degradants and impurities. MS detection is used to assign mass to peaks, which allows for the establishment of correlation with theoretical fragments expected to be identified in a biologic drug substance. The data can then be used for structural assignment. MS detection is preceded by a digest after which the HPLC-MS typically analyzes samples using the chromatographic conditions capable of resolving the present peaks. Wolfe Laboratories obtains the MSn patterns to aid with structure elucidation by conducting additional fragmentation experiments.

Spectroscopic Techniques

Spectroscopic techniques, such as UV-Vis (UV) and fluorescence, are used for quantitative and qualitative analysis of conformational structure of proteins, their behavior in solution as a function of solution composition and their stability in development and release testing. Aside from the great simplicity of the actual testing, an added benefit to spectroscopic methods is their ability to analyze peptides/proteins in a non-destructive manner. UV spectroscopy determines protein concentration, aggregation and conformational stability of proteins by second-derivative analysis. UV also is used to analyze the drug substance during initial development and preformulation characterization, for analysis in formulation development, as well as for drug product release. Fluorescence measurements reflect the presence of aromatic amino acids and the effects of the surrounding environment on the fluorescence signal, which allows for evaluation of the conformational structure and stability of proteins. Fluorescence is very useful in assessment of solution conditions such as pH, temperature, etc., with regard to the unfolding of proteins.

Peptide Mapping

Peptide mapping is used in early development through commercialization to perform identification and purity testing of the drug substance, including determination of the primary amino acid sequence, intact parent masses of peptide fragments, as well as peptide fragmentation and identification of specific degradation sites. Wolfe Laboratories selects preliminary digest conditions based on predicted peptide digest results for multiple enzymes and subsequently optimizes, with evaluation of different enzymes, enzyme/protein ratios, concentrations and chromatography conditions.

Other Characterization Techniques

Isoelectric Focusing and Capillary Isoelectric Focusing

Isoelectric focusing (IEF) and capillary isoelectric focusing (cIEF) separates peptides and proteins based on their overall charge, specifically their isoelectric point, and is used for identity confirmation and as a highly sensitive stability indicating method.

SDS-PAGE

SDS-PAGE is an indispensable technique conventionally used to perform size-based analysis of peptides and proteins. SDS-PAGE is an efficient stability-indicating technique that offers high specificity; various degradants can be resolved in the same gel, and a differentiation can be made between covalent and non-covalent aggregates, as well as clipped species.

Thermal Analysis

Differential scanning calorimetry (DSC) is used to characterize the stability of proteins as well as inform the development and optimization of the lyophilization process. Thermal data obtained by DSC serve as an indicator of conformational stability. Specific thermal events are indicative of different modes of degradation; endothermic events indicate protein unfolding, while exothermic events indicate aggregation. Changes in the onset and the melting point indicate changes in the protein secondary and/or tertiary structure. Applied to lyophilization process development, Wolfe Laboratories uses DSC data to identify the optimal process parameters such as freezing and annealing temperatures, thus enabling development of a stable drug product with a pharmaceutically elegant appearance.

Particulate Matter Analysis

Particulate matter analysis is used to characterize the solution properties of the proteins and assess aggregation and association. Wolfe Laboratories uses two light scattering techniques, dynamic light scattering and zetasizing for particulate matter analysis, particle size measurement, aggregation assessment and particle count measurement.

Karl-Fischer Titration

Karl-Fischer titration (KF) is routinely used to determine the moisture (water) content of the samples. Wolfe Laboratories uses the data to inform lyophilization process development and ultimately determine the specifications for the release testing and stability testing of the final drug product.

Thermogravimetric Analysis

Thermogravimetric analysis (TGA) is another tool used to determine the moisture content of materials, and may be used to assess lyophilized drug products for process development and optimization. Unlike KF, which is specific for detection of water content in a formulation, TGA detects all volatiles present. Temperatures of the transitions observed on the resulting thermogram differentiate the volatile components.