Secondary, Tertiary and Higher Order Structure of Proteins

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.

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.

Various extrinsic fluorescent dyes offer additional possibilities for protein characterization. Extrinsic dyes can be covalently attached to proteins, e.g. via the ɛ-amino group of lysine, the α-amino group of the N-terminus, or the thiol group of cysteine. More interesting for the analysis of pharmaceutical formulations are extrinsic dyes that interact noncovalently with proteins and protein degradation products, e.g. via hydrophobic or electrostatic interactions.

The ability of extrinsic dyes to assess surface hydrophobicity or posttranslational modifications can be beneficial for the characterization of hydrophobic recombinant proteins in particular, as surface hydrophobicity can be relevant for the activity, aggregation and adsorption properties of the protein.

BioPharmaSpec recommends NMR analysis of proteins for a high-end assessment of higher order structure. The US FDA has stated in numerous presentations that NMR is used in their laboratories to provide a detailed assessment of higher order structure. NMR is an orthogonal assessment, alongside CD, FT-IR and Fluorescence 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 (normally¹H – 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.

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) in their respective formulation buffers.

SV-AUC is a column-free method for assessing the aggregation state of a biopharmaceutical. The regulators request both a column method (such as SEC-MALS) and a column-free method be used to assess aggregation until the manufacturing process is shown to be under control.