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. Pace Life Sciences Boston, MA 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 the analytical methods are generated during preformulation studies. Pace typically characterizes primary sequence; modifications to amino acids; propensity for self-association; molar extinction coefficient; isoelectric point; and effects of pH, excipients and ionic strength on solubility and stability, as well as on thermal and freeze/thaw stability.
Primary Sequence Analysis
A thorough characterization of the primary amino acid sequence of biologics informs formulation development, enables identification of stability issues 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 and oxidation of methionine. Pace Life Sciences Boston, MA development site determines primary sequence of peptides/proteins, as well as sequence confirmation where a putative primary sequence is known.
Analysis of Modifications to Amino Acids
Chemical modifications to individual amino acid residues may occur in native peptides/proteins during expression; therefore, characterization of these modifications is essential to establish the baseline profile of the drug substance. Any change in the drug development process, from the expression cell line through to process of manufacture, can result in changes in the amino acid modifications, 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 can significantly contribute to de-risking of the development program.
Pace performs the analyses for determination of amino acid modifications summarized in the following table.
Summary of analysis of amino acid modifications performed by Pace Life Sciences
RP-HPLC, IE-HPLC or peptide mapping (LC-MS/MS).
RP-HPLC, IE-HPLC or peptide mapping (LC-MS/MS).
SEC-HPLC or peptide mapping (LC-MS/MS)
Identification of glycosylation sites
Deglycosylation followed by peptide mapping (LC-MS/MS)
Oligosaccharide population – N-linked and O-linkedsed by enzymatic digest followed by HPLC-MS/MS
Glycan linkage analysis (including antennary profile)
Exoglycosidase digestion + HPLC-MS/MS
Characterization of Propensity for Self-Association
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 biological therapeutics. 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 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.
Determination of 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. Pace Life Sciences Boston, MA 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.
Pace assesses the effect of pH and ionic strength on the solubility of biologics as part of preformulation studies in order to inform the experimental design for formulation development. The pH solubility profile is an essential tool for successful development of biologics and, in combination with the pH stability profile, serves to inform suitable formulation approaches, as well as the expected in vivo performance.
pH Stability Profile
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. The Pace Life Sciences Boston, MA development site can develop mechanism-based approaches to stabilize the molecule with knowledge of the degradation pathways at different pH values.
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.