Developing the formulation of a biopharmaceutical can be challenging and a solid understanding of the structure and conformation of your product is essential. Different formulations may alter the secondary and tertiary structure of a biopharmaceutical, which in turn can affect protein activity. At BioPharmaSpec, we provide analytical methods to analyze the structure and stability of your biopharmaceutical in its formulation.
Circular Dichroism (CD)
The two main types of secondary structure to consider are the α-helix and the ß-sheet. The α-helix is a right-handed coiled strand, with hydrogen bonding between the coil making the structure very stable. ß-sheets are made up of inter-strand hydrogen bonding, with pairs of bonded strands lying side-by-side.
CD measures differences in the absorption of left- and right-handed circularly polarized light. α-helices and β-sheets have specific CD profiles and so we can use changes in these spectra to assess samples in different formulations (e.g. different buffers) to determine if there are any structural changes.
Fluorescence utilizes the natural intrinsic fluorescence of Tyrosine (Tyr) and Tryptophan (Trp) residues and provides information on the local environments around these residues. Fluorescence is an excellent comparative tool and is complementary to CD and FT-IR.
The below image shows the relative intrinsic fluorescence of Trp and Tyr residues for a mAb product in its formulation buffer (Left: 280nm excitation. Right: 295nm excitation)
Protein Nuclear Magnetic Resonance (NMR)
NMR is available as an orthogonal assessment, alongside CD analysis, for secondary and tertiary structure determination. The technique is particularly useful in the early stages of biopharmaceutical development and for initial comparability assessments.
Both 1-D and 2-D NMR can be used to assess whether a product is folding as expected and also to assess the secondary and tertiary structure of the biopharmaceutical. Specific structural assessments can be made for products of defined tertiary structure. Significant instruments (800MHz and above) are needed to provide the resolution required for these assessments.
Differential Scanning Calorimetry (DSC)
BioPharmaSpec uses DSC to assess thermal transitions of biopharmaceuticals, such as unfolding. This technique can be utilized to assess conformational stability and also provide the melting temperature of the protein. DSC is particularly useful for assessing the comparability of thermal stability of Biosimilar relative to Innovator / Reference Medicinal Products (RMPs).
Fourier Transform InfraRed Spectroscopy (FT-IR)
InfraRed (IR) spectra provide qualitative and quantitative information on the secondary structure of proteins such as α helices, β sheets, β turns and disordered structures.
The most informative IR bands for protein analysis are amide I (1620-1700 cm-1), amide II (1520-1580 cm-1) and amide III (1220-1350 cm-1).
Amide I is the most intense absorption band in proteins and consists of stretching vibration of the C=O (70-85% and C-N groups (10-20%).
Amide II is governed by in-plane N-H bending (40-60%), C-N (18-40%) and C-C (10%) stretching vibrations.
FT-IR provides an orthogonal assessment of secondary structure to Far UV CD analysis. It is often considered more useful than CD for products with high levels of α helices and β sheets because, unlike Far UV CD, FT-IR does not show a disproportionately high response to α helix.
The profiles obtained and the fitting data can be used to assess the comparability of the secondary structure of different batches or formulations, and between originator and biosimilar samples.
Overlay of FT-IR spectra at 2200-1000cm-1