Editing Circular dichroism (section)

== Higher order structure comparisons ==
Modern protein CD spectroscopy is most powerful for comparative analysis of secondary and tertiary structure, commonly referred to as higher order structure (HOS) comparisons. Examples include assessing batch-to-batch consistency in biotherapeutics, evaluating the effects of mutations, and studying the properties of charge variants or glycovariants to a parent protein. HOS analysis is also highly effective for studying the impact of formulation excipients, pH, and ionic strength on protein conformation.

HOS comparisons have a wide range of applications and play a critical role at every stage of biotherapeutic development—from early-stage analytical assessments to late-stage quality control; from formulation development to forced degradation shelf stability assessments.

Higher order structure comparisons leverage both far- and near-UV wavelength ranges to enable statistically robust, reproducible, and quantifiable data analysis. An HOS comparison quantitatively evaluates whether any two spectra differ and, by extension, whether their underlying structural information is different. This is often difficult, if not impossible, to do by visual inspection. Detecting these subtle differences requires multiple independent spectra and robust statistical analysis, as many variations are too small to be discernible by eye.
[[File:HOS Comparisons.png|thumb|Weighted Spectral Difference (WSD) plot comparing Innovator versus Biosimilar sample lots. If the WSD values for enough sample replicates fall within the quality range, the sample is considered similar to the reference; otherwise, it is deemed different.]]
There are various approaches to HOS comparisons and numerous spectral comparison methods are described in the literature, however, regardless of the HOS comparison method or application, all methods are mathematical approaches that have the same goal of yielding a single value as a measure of similarity for pair-wise spectral comparisons. One such method is the Weighted Spectral Difference (WSD) method, an HOS comparison method favored by many biopharmaceutical companies and regulatory authorities.<ref>{{Cite journal |last1=Teska |first1=Brandon M. |last2=Li |first2=Cynthia |last3=Winn |first3=Bradley C. |last4=Arthur |first4=Kelly K. |last5=Jiang |first5=Yijia |last6=Gabrielson |first6=John P. |date=2013-03-01 |title=Comparison of quantitative spectral similarity analysis methods for protein higher-order structure confirmation |url=https://pubmed.ncbi.nlm.nih.gov/23219560/ |journal=Analytical Biochemistry |volume=434 |issue=1 |pages=153–165 |doi=10.1016/j.ab.2012.11.018 |issn=1096-0309 |pmid=23219560}}</ref><ref>{{Cite journal |last1=Dinh |first1=Nikita N. |last2=Winn |first2=Bradley C. |last3=Arthur |first3=Kelly K. |last4=Gabrielson |first4=John P. |date=2014-11-01 |title=Quantitative spectral comparison by weighted spectral difference for protein higher order structure confirmation |url=https://pubmed.ncbi.nlm.nih.gov/25051254/ |journal=Analytical Biochemistry |volume=464 |pages=60–62 |doi=10.1016/j.ab.2014.07.011 |issn=1096-0309 |pmid=25051254}}</ref>

Over the last decade, HOS comparisons have been established as an essential tool to consolidate CD data in submissions for new biologics drug applications. Particularly in biosimilars development, HOS comparisons are indispensable to meet requirements by the FDA, EMA, and other regulatory bodies. The rationale for this guidance is to regulate and control the safety, quality, and efficacy of therapeutic agents available to the public.<ref>{{Cite web |last=Research |first=Center for Drug Evaluation and |date=2022-06-15 |title=Development of Therapeutic Protein Biosimilars: Comparative Analytical Assessment and Other Quality-Related Considerations Guidance for Industry |url=https://www.fda.gov/regulatory-information/search-fda-guidance-documents/development-therapeutic-protein-biosimilars-comparative-analytical-assessment-and-other-quality |access-date=2025-02-17 |website=www.fda.gov |language=en}}</ref>