Preformulation characterization informs the formulation constraints for the development of the drug product based on the physicochemical properties of the drug substance and its performance in the various conditions likely to be encountered during formulation, process development and on storage. Our development site performs preformulation characterization in parallel with the development of the analytical methods, as the quality and meaningfulness of the data are directly dependent on the availability and utilization of relevant methods, while the samples necessary for development of stability-indicating analytical methods are generated during preformulation studies.
Developability Assessment Through Primary Sequence Analysis
A thorough characterization of the primary amino acid sequence of biologics informs formulation development, enables identification of stability concerns and establishes the baseline for future comparison of samples generated during development. Depending on the presence, number and location of particular residues, specific chemical and physical degradation can be anticipated, such as deamidation of asparagine, oxidation of methionine and sequence-dependent cleavage o the amide bond. Our development site determines primary sequence of peptides/proteins, as well as sequence confirmation where a putative primary sequence is known. Potential to develop a protein is also assessed using primary sequence and any known higher order structural information. This informs the content of hydrophobic amino acids, theoretical isoelectric point, charge distribution and presence of sequences prone to post translational modification or chemical transformation.
Analyzing Chemical Transformations
Having a biologic characterized with numerous orthogonal analytical methods is to key ensuring overall product quality to achieve development milestones. As an integrated research, development and testing CRO, analytical methods are developed to be stage appropriate and fit the needed specification requirements for the intended test/release.
Any change in the drug development process, from the expression system to process of manufacture, can result in alterations at the amino acid level, thereby impacting the conformational stability and/or activity of the biologic. As the impact of changes to upstream processes on downstream development is often not readily apparent and is difficult to predict, characterization of amino acid modifications coupled with accelerated stability studies can significantly contribute to de-risking of the development program. We utilize state-of-the-art equipment and experienced analytical development scientists to develop robust analytical methods to de-risk your therapeutic program.
Analyzing Aggregation
The propensity of the protein for self-association is a critical parameter to assess early in the drug development process, as formation of aggregates is among the more common degradation mechanisms for biologics. Aggregation can lead to loss of activity of the drug substance and may cause immunogenicity on administration. Appearance of aggregates, reversible or irreversible, is routinely monitored during formulation and process development. While reversible association is generally less worrisome than irreversible association, high protein concentration and formulation conditions such as pH and ionic strength can alter the equilibrium toward formation of irreversible aggregates. Early assessment of the propensity of a protein to form reversible and irreversible aggregates can significantly contribute to efficient and effective formulation development and enable the development of the appropriate analytical techniques in advance.
Analytical Methods
Chromatography – identity and purity methods to assess chemical and physical stability
- Reversed Phase, IP-RP, HIC
- HILIC
- IEX
- SEC, AF4– high molecular weight species
- UV/FLR/ELSD
- RI/MALS
- MS (Triple Quad and High Res)
- cIEF – isoelectric point and charge distribution
- CE-SDS – chain integrity
- Intact, Reduced, Deglycosylated Mass Determination
- Sequencing (Peptide Mapping, Oligonucleotides)
- Disulfide Bonding/Structural Elucidation
- Glycosylation (N- and O-linked)
- Carbohydrate Analysis (Released Glycans, Monosaccharides, Sialic Acid)
- Chemical & Post-Translational Modifications
- Identification of Process Impurities/ Related Substances
- Extractables & Leachables
- HCP Identification
- Bioanalytical Quantitation
Solubility Assessments
Pace assesses the effect of pH and ionic strength on the solubility of to inform the experimental design for formulation development and enable development high concentration formulations. The pH solubility profile, in combination with the pH stability profile, serves to inform suitable formulation approaches, as well as the expected in vivo performance.
pH Stability Profile
Integrating the pH stability profile is used for understanding how the compound behaves in different pH environments and informs formulation development, process development, drug product stability and administration of the molecule. The goal of the pH stability profile is to identify the pH of maximal stability. Our development site can develop mechanism-based approaches to stabilize the molecule with knowledge of the degradation pathways at different pH values.
Thermal Stability
Exposure to different temperatures during storage and the manufacturing process presents a direct liability to the physical stability of proteins. Protein denaturation and aggregation can occur as a result of elevated temperatures as well as on freezing or with freeze/thaw. Aside from the obvious logistical need to prevent degradation of the drug substance material, instability caused by storage or process conditions can lead to inaccurate interpretation of studies during development.
Stability to Shear
Drug substances are routinely subjected to various mechanical stresses during sample preparation and manufacturing, and for biologics, these stresses may induce conformational changes that may lead to loss of activity, as well as adsorption, aggregation and precipitation. Understanding which stressors cause instability directly affects sample handling, formulation and process development.
Determination of Concentration and Molar Absorption Coefficient
As concentration values throughout the development process of a protein are based almost exclusively on the molar absorption coefficient (ε), it is important that an accurate value for ε be used. PLS determines this value experimentally as early as possible in the development program to satisfy regulatory guidance and ensure the accuracy of experimental and clinical results.