Secondary, Tertiary and Higher Order Protein Structure

Protein structure can be characterized into different levels:

  • Primary Order Structure – The sequence of amino acids in the polypeptide chain held together by peptide bonds. The primary sequence of a protein is unique to that protein, and defines the structure and function of the protein.
  • Secondary Order Structure – Localized structures that form based on interactions within the protein backbone. The most common types of secondary structures are the α helix and the β pleated sheet. Both structures are held in shape by hydrogen bonds, which form between the carbonyl and amino group of the peptide bond.
  • Tertiary Order Structure – The three-dimensional (3D) shape of a protein. Interactions of the amino acid side chains within a particular protein determine its tertiary structure.
  • Quaternary Order Structure – The structure of a multi-protein complex such as dimer, trimer and more complex multi-protein subunit systems.

Secondary, tertiary and quaternary structure is often collectively termed as the higher order structure (HOS) of a protein. HOS is responsible for the correct folding and three-dimensional shape of a biopharmaceutical. This can be affected by different formulations, which in turn can affect protein activity. The folding and shape of the protein impacts directly on the functionality of the drug and this correlation is often termed as the “structure function relationship”.

Incorrect higher order structure can also raise safety concerns. If the overall folding and therefore 3D shape of a protein is wrong, receptor binding can be inhibited, immunogenic epitopes can be exposed and aggregation can occur. It is therefore crucial to employ a thorough characterization of the higher order structure of the protein. Biophysical characterization methods should be performed alongside functional analyses and primary structure characterization to allow a full understanding of the overall protein structure.

BioPharmaSpec offers a comprehensive range of biophysical techniques to fully characterize the higher order structure and stability of your protein.

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.

Circular Dichroism measures differences in the absorption of left- and right-handed circularly polarized light. BioPharmaSpec scientists make use of the fact that α-helices and β-sheets have specific CD profiles and use changes in these spectra to assess samples and determine if there are any structural changes.

Secondary structure can be determined by CD spectroscopy in the “far-UV” spectral region (190-250 nm). At these wavelengths the chromophore is the peptide bond, and the signal arises when it is located in a regular, folded environment. α-helix, β-sheet and random coil structures each give rise to a characteristic CD spectrum. The approximate fraction of each secondary structure type that is present in any protein can thus be determined by deconvoluting the data and comparing to a protein structure database using algorithms such as CDsstr.

Measuring CD over the “near-UV” range (>250 nm) generates a CD fingerprint of the tertiary structure of biomolecules. The fingerprint is influenced by the nature of the surrounding environment of aromatic side chains of the amino-acids tryptophan, phenylalanine and tyrosine. The presence of disulfide bonds may also influence the final fingerprint.

Sample Relative percentage of α-helix Relative percentage of other helix Relative percentage of β-sheet Relative percentage of turns Relative percentage of other structures
RMP 1 11 33 19 36
Biosimilar 0 10 34 20 37

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

Intrinsic Fluorescence

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)

Extrinsic Fluorescence

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.

Protein Nuclear Magnetic Resonance (NMR)

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.

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) in their respective formulation buffers.

Sedimentation Velocity Analytical UltraCentrifugation (SV-AUC)

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.