One common orthogonal technique used in biophysical characterization, in academia as well as in the biopharmaceutical industry, is CD spectroscopy. Used predominantly in the study of biomolecules (for example, proteins and peptides), CD spectroscopy is versatile and unique when compared to alternative methods used in similar applications, and provides information about both secondary and tertiary structure. Moreover, the technique can provide vital information regarding the stability of a protein, with minimal requirements for sample preparation.
A protein’s secondary structure describes the local structural elements that are stabilized primarily through short-range interactions within the protein backbone — usually hydrogen bonds that connect amino acids. These bonds are often in close proximity to the protein sequence (i.e. the primary structure). α‑helices are the most common among such elements, and are usually found in insulin, whereas β-strands that typically make up larger β‑sheets, are dominant elements in monoclonal antibodies. However, there are other secondary structures, such as different types of helices, turns and β‑sheets, which can be pleated or twisted. Many of these sub-elements can be found even within a fairly small protein.
The overall 3-dimensional arrangement of a protein is known as its tertiary structure, which is stabilized by long-range interactions such as electrostatic and hydrophobic interactions, salt bridges and disulfide bonds. These interactions are formed among amino acids’ side chains, which can be far apart from one another in the sequence but spatially close.
Peptide bonds, aromatic amino acids and disulfide bonds (Figure 1) are the principle chromophores in proteins that interact with the circularly polarized light used in CD spectroscopy (Section 5). The α-carbon atoms in amino acids are the chiral centers of the protein backbone. Moreover, when peptide bonds absorb circularly polarized light, it gives rise to typical CD signals at low wavelengths in the range of 180 and 250 nm.
This wavelength range contains information about the secondary structure and is therefore termed far‑UV (since it is distant from the electromagnetic spectrum’s visible range). In addition, the side chains of the aromatic amino acids are chromophores that produce CD signals, despite the fact that they are considered non-chiral. This results from the way in which aromatic amino acids are affected by their chiral environment. The corresponding CD signals arise in a range of wavelengths between 250 and 350 nm, while the near‑UV (close to the visible spectrum) contains information pertaining to the tertiary structure. Finally, disulfide bonds can contribute to both the far- and near-UV.
Spectral bands in the far-UV are characteristic for secondary structure elements that form similar arrangements in different proteins - for example, the presence of a high content of α‑helices encourages a positive peak at about 190 nm and two negative peaks at about 208 and 222 nm. In contrast, a near-UV CD spectrum comprises what could be considered a fingerprint region, since its profile predominantly depends on the type and number of aromatic amino acids and their environment. This explains why there are substantial differences between individual proteins.
Figure 1: Spectral ranges of protein CD spectroscopy and the information they provide.
Two separate measurements are usually required to obtain information about both secondary and tertiary structure by CD. This is due to the fact that aromatic amino acids are scarcer in a protein than in peptide bonds. Thus a significantly higher concentration of protein (under otherwise identical experimental conditions) is required for an adequate signal in the near‑UV rather than the far‑UV.
Proteins such as myoglobin contain prosthetic groups that absorb in the visible range of the electromagnetic spectrum, resulting in CD data that can be procured beyond the far- and near-UV. In certain conditions, chromophores can emit a CD signal at substantially higher wavelengths in the near infrared (IR).
The principal application of protein CD spectroscopy is partially dictated by the spectral range under examination. The fundamental assumption is that typical spectral profiles in the far-UV display a correlation with the relative content of the most common secondary structure elements. For this reason, the spotlight has traditionally been on the far‑UV to compute the fractions of α‑helices and β‑strands present in proteins. However, it is less reliable to determine the secondary structure by CD than commonly perceived (Section 6).
The most powerful method of secondary and tertiary structure (also known as higher order structure) analysis is modern protein CD spectroscopy, which utilizes both far- and near‑UV.
The first step involves comparing one macromolecule with another: for example, wild-type versus mutant; different variants, isoforms, homologs; or biotherapeutics from different lots or batches. Moreover, the same molecule can be compared across environments to understand the impact on structure and the stability of changes in buffer, excipients, pH, ionic strength, ligands, stress conditions, or the changes over time that result from prolonged storage.
Case Study: Structural Characterization of NISTmAb
The characterization of biotherapeutics in general (and monoclonal antibody (mAb) therapeutics, in particular), is a complex task that involves many functional and biophysical techniques. Until recently, it has been challenging to put different mAb studies into perspective, as common standards and procedures to assess these analytical techniques were missing. This is despite the fact that mAbs are recognized as one of the most prevalent classes of biotherapeutics—since the beginning of this decade, global annual sales revenue for mAb products has continued to total more than half of all biopharmaceutical products combined .
The solution to this problem was provided in the form of a comprehensively characterized and publicly available reference for the development of new mAb therapeutics. In 2016, the National Institute of Standards and Technology (NIST) in the USA undertook a holistic study using a humanized monoclonal antibody, the NISTmAb .
The study utilized the biophysical characterization techniques most commonly used in biotherapeutic development, and compiled the updated results into a mock Investigational New Drug (IND) filing . The NISTmAb is now available to biopharmaceutical companies as a representative test molecule to assist in method development, qualification, and in evaluating and comparing instrument performance and analytical techniques. Applied Photophysics was approached by NIST to contribute to this project—the case study featured here is based on this collaboration.
A primary standard of the NISTmAb (PS 8670) was compared with the NISTmAb reference material (RM 8671) that had been derived from the primary standard by homogenization, aliquoting and dilution. The secondary and tertiary structure of PS 8670 and RM 8671 was characterized using a Chirascan Q100 automated CD spectrometer (Applied Photophysics). Samples were dialyzed into a common preparation of phosphate-buffered saline and diluted to obtain suitable concentrations for far- and near‑UV measurements. Spectra were obtained for multiple independent replicates, corrected for the dialysate baseline and normalized by absorbance at 192 nm in the far-UV and 280 nm in the near‑UV (Figure 2).
As expected for a monoclonal antibody, the far-UV spectrum is characteristic of a protein with a high β‑sheet content, with a distinct peak at 202 nm and a negative peak at 218 nm. However, the far-UV spectra contain information beyond secondary structure elements—as highlighted in the inset plot, signals of lower amplitude around and above 230 nm arise from disulfide bonds and are expected to be sensitive to the dihedral angles of these bonds.
The profile of the near-UV spectra is much more complex, as it is composed of overlapping contributions by the aromatic amino acid side chains of 24 tryptophan, 52 tyrosine, 50 phenylalanine residues as well as 16 disulfide linkages.
Although the near-UV spectra appear to be superimposable by visual inspection, statistical analysis reveals that the tertiary structure of RM 8671 is significantly different to that of PS 8670, while this is not the case for the secondary structure (lower panels in Figure 2). Such small spectral differences in the near-UV can only be found with state-of-the-art CD instruments that provide the required sensitivity. Moreover, the automation offered by a Chirascan Q100 enabled the reproducible generation of a sufficient number of replicates as required for robust statistical analysis.
Figure 2: Characterization of secondary and tertiary structure of NISTmAb. Reference material RM 8671 was compared against primary standard PS 8670 and statistical significance of structural differences evaluated by means of Weighted Spectral Difference, applying an acceptance criterion of two standard deviations (±2SD).For details of statistical analysis, see Section 4. Data was recorded with a Chirascan Q100.
 D. M. Ecker, S. D. Jones, and H. L. Levine, “The therapeutic monoclonal antibody market,” MAbs, vol. 7, no. 1, pp. 9–14, Jan. 2015.
 NIST, “NIST Monoclonal Antibody Reference Material 8671,” 2018. [Online]. Available: https://www.nist.gov/programs-projects/nist-monoclonal-antibody-reference-material-8671. [Accessed: 27-Jun-2018].
 J. Schiel and C. Vessely, “NISTmAb Common Technical Document Case Study,” presented in workshop at the 6th International Symposium on Higher Order Structure of Protein Therapeutics (HOS 2017). [Online]. Available: https://www.casss.org/page/HOS1700. [Accessed: 27-Jun-2018].
About Applied Photophysics
Applied Photophysics is a leading provider of systems and accessories for the biophysical characterization of biomolecules. Headquartered in Leatherhead, Surrey, UK, the Company has been established for more than 40 years.
The SX-range of stopped-flow spectrometers, used to monitor changes in absorbance and fluorescence during fast biological reactions, is acknowledged globally as the gold standard for kinetic studies. In 2005, the Company introduced the first Chirascan™ system, using the phenomenon of circular dichroism (CD) to characterize changes in the higher order structure of proteins.
Since then, the company has continued to incorporate its in-depth knowledge and understanding of CD into a range of Chirascan products that are used in cutting-edge research and to support the development of innovator drugs and biosimilars in the biopharmaceutical industry. Compared to conventional CD instruments, the new generation of Chirascan systems ensures that every scientist gets the most from every CD analysis.
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