Unexpected changes to the higher order structure of your biopharmaceutical can have dramatic effects on mechanism of action. If the overall 3D shape is wrong, receptor binding can be inhibited, immunogenic epitopes can be exposed and aggregation can occur. Therefore, a solid understanding of the structure and conformation of your product is essential. Different formulations may alter the secondary, tertiary and higher order 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)
BioPharmaSpec recommends NMR analysis of biopharmaceutical products for the high-end assessment of higher order structure (HOS). The US FDA has also stated in numerous presentations that NMR is used in their laboratories to provide a detailed assessment of HOS. NMR is 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.
BioPharmaSpec’s NMR service comprises 1D 1H-NMR for an initial assessment (and for analysis of products at too low a concentration for 2D-NMR) followed by 2D-NMR (normally 1H – 13C 2D NMR analysis). BioPharmaSpec recommends 1H – 13C 2D NMR analysis over 1H – 15N 2D NMR analysis because of the higher natural abundance of the 13C isotope which provides a more significant response, particularly for products larger than 50kDa.
During NMR analysis, samples are supplemented with D20 and 1D 1H spectra followed by 2D 1H-13C heteronuclear single quantum coherence (HSQC). NMR spectra are recorded at approximately 298K on a Bruker DRX800 equipped with a triple resonance cryoprobe.
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