Analytical support for protein formulation development

Physical and chemical instabilities are the most challenging tasks in the development of protein therapeutics.  The molecular structure of protein is delicate in nature compared to small molecule drugs and is highly sensitive to environmental changes and stresses.  Ensuring native-like higher order structure in a biologic drug is essential because the overall conformation not only defines the stability and biological activity, but also the efficacy and safety.

SSCI offers analytical services in support of protein drug development at different stages and provides data to meet the expectations outlined in the ICH Q6B specifications.  A number of techniques (e.g., freeze-drying and spray drying) are utilized for solid state protein formulation development.

Pre-formulation screen

SSCI evaluates the physical stability of protein in various formulation excipients such as ionic strength, buffers, pH, surfactants, sugars, salts, antioxidants, and amino acids on the physicochemical properties of the protein.

  • Gel permeation chromatography (GPC) and size exclusion chromatography (SEC) coupled with a static-light-scattering (SLS) detector are commonly used techniques for detecting and quantifying protein aggregation.
  • Nano-DSC is a powerful analytical technique that is used to monitor conformational stability of biologics in their formulations.  Structural alterations (unfolding/aggregation) in a biologic can be detected in the form of a Tm (denaturation temperature) shift or a change in the shape of the endothermic peak (∆H and ∆Hv for domain and subunit organization)
  • Dynamic light scattering (DLS) or quasi elastic light scattering (QELS) is used to evaluate the aggregation state.  DLS is a non-destructive technique for the determination of size distribution of particles in the diameter range of 1 nm to 2 µm

Lyophilization process development and optimization

SSCI offers small scale lyophilization services to screen common excipients for protein stability.  Testing is tailored to each client’s individual requirements.

  • Formulate the protein in compatible excipients based on the physicochemical properties of the protein during preformulation screen.
  • Determine the glass transition temperature of the formulation in the frozen state (Tg’) by temperature modulated differential scanning calorimetry and the collapse temperature by freeze-drying microscopy.
  • Determine the optimal shelf temperatures and chamber pressures for primary and secondary drying.
  • Characterize the finished lyophilized product using a variety of analytical techniques such as X-ray powder diffraction (XRPD) and polarized light microscopy (PLM) to assess crystallinity, thermogravimetry (TG) and DSC for thermal properties, scanning electron microscopy (SEM) for particle morphology, Karl Fischer titration for water content, dynamic vapor sorption/desorption for hygroscopicity testing; solid state NMR, FT-IR, and Raman spectroscopy for chemical and physical finger printing.
  • Evaluate physical stability (unfolding/aggregation) of protein upon lyophilization by SEC, nano-DSC, DLS, CD, and HPLC.

Real time and accelerated stability studies

Storage stability of proteins shows a reasonably good correlation with the degree of retention of native structure of proteins and the level of hydration during drying.  Other environmental conditions, including temperature, pH, ionic strength, oxygen, and protease content can cause denaturation, aggregation, degradation, and chemical modification (oxidation and deamidation).  SSCI offers advanced analytical methodologies for stability evaluation.

  • Physical stability assessment: Aggregation state evaluation by SEC and DLS, conformational change by nano-DSC, and solid state characterization by various analytical techniques including but not limited to XRPD, TG, DSC, SEM, SSNMR, FT-IR, and Raman.
  • Chemical stability assessment: With the ultra-high resolution Q-TOF mass spectrometer and solids/liquids NMR spectrometer in conjunction with other techniques such as peptide mapping, MALDI-TOF MS, amino acid analysis, and Edman N-terminal sequencing, SSCI is capable of providing accurate mass and primary sequence of proteins, peptides, to ensure structural integrity and physicochemical identity.