Genetic Engineering & Biotechnology News

MAY15 2018

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20 | MAY 15, 2018 | GENengnews.com | Genetic Engineering & Biotechnology News Doug Marshall, Ph.D. Circular dichroism (CD) spectroscopy has long been regarded as a low-resolution tech- nique used to obtain an estimate of the a-he- lix and b-sheet content of proteins. This view of CD, though widely held, is no longer ac- curate and is being replaced by the realization that modern CD spectroscopy systems offer enhanced resolution and thereby provide critical information regarding changes in both secondary and tertiary structural elements. The potential for relatively minor changes in these higher order structure (HOS) ele- ments to have a significant effect on biologi- cal activity, interactions, and stability, or on the efficacy and safety of a biotherapeutic, is well recognized. A reliable method for HOS comparison is an essential tool to ensure comprehensive biophysical characterization. Setting Standards for HOS Comparisons To provide scientists with a standard suit- able for use with commonly used biophysical characterization techniques, the National In- stitute of Standards and Technology (NIST) at the U.S. Department of Commerce offers a specifically selected monoclonal antibody, NISTmAb (www.nist.gov/programs-proj ects/nist-monoclonal-antibody-reference- material-8671). Selection of a mAb as a biological standard reflects the importance of these proteins in both research and biotherapeutic development. Figure 1 shows the secondary structure of the NISTmAb, as revealed by analysis in the far-UV. This data contributed to the ini- tial characterization of the NIST antibody standard. Meeting Changes to Regulatory Demands During biotherapeutic development, many biophysical characterization techniques are required to support informed decision mak- ing and contribute to the totality of evidence in regulatory submissions. As an essential part of this toolset, CD has been referred to in more than 95% of biosimilar applications involving mAbs and other biotherapeutics. This figure was cited by Maria Teresa Guti- errez Lugo, Ph.D., Office of Biotechnology Products, Center for Drug Evaluation and Research (CDER), Food and Drug Adminis- tration (FDA), at the Fifth International Sym- posium on Higher Order Structure of Protein Therapeutics, in a talk entitled "Regulatory Consideration for the Characterization of HOS in Biotechnology Products." However, regulatory authorities are in- creasing their demand for state-of-the-art techniques that provide "statistically validat- able data." Until now, obtaining such results for HOS comparisons has presented chal- lenges in terms of data acquisition and suit- ability of statistical methods. With the introduction of an automated and integrated solution for CD analysis that eliminates the errors associated with manual sample handling, and thereby ensures the reproducibility essential for multiple repeat analyses, high-quality CD data has become amenable to statistical interrogation. This allows for a more rigorous, objective assess- ment when looking for even minor changes in the HOS of complex biomolecules. HOS Comparisons During Biotherapeutic Development To ensure an effective, efficient workflow, detecting and confirming minor differences upstream is as important as proving similar- ity downstream. Consequently, an ability to detect minor changes in HOS is crucial throughout development and scale-up of in- novator drugs or potential biosimilars. The secondary and tertiary structures of commercially available innovator lots were compared with test batches of a potential biosimilar. Analysis in the near-UV (Figure 2) revealed the possible presence of minor differences in tertiary structure. However, any conclusions drawn by visual comparison of the CD spec- tra are likely to be subjective. To facilitate an objective conclusion, CD spectral data were converted into a numer- Provides Key Information on Changes in Secondary and Tertiary Structure of Proteins Optimizing Circular Dichroism Spectroscopy Bioprocessing Doug Marshall, Ph.D. (doug.marshall@ photophysics.com), is Chirascan product manager at Applied Photophysics. Website: www.photophyics.com. Tutorial CD is the difference in absorption of left-handed circularly polarized light and right-handed circularly polar- ized light that occurs when a mol- ecule contains one or more light- absorbing groups (chromophores) that are chiral or in a chiral environ- ment. The difference in absorption generates a CD spectrum. Secondary structure elements generate a CD spectrum due to the chirality of the peptide backbone when scanning in the far-UV. Tertiary structure elements are revealed when scanning in the near-UV as CD signals are gener- ated predominantly from aromatic side chains (tryptophan, tyrosine, and phenylalanine). n Circular Dichroism— A Brief Reminder Figure 2. CD analysis on a Chirascan Q100 system comparing innovator and biosimilar lots. Figure 1. CD spectrum of NISTmAb—standard sample for biophysical characterization— reveals a predominantly β-sheet structure typical of many mAbs. Analysis on a Chirascan Q100 system includes simultaneous absorbance for accurate normalization of CD data. Figure 3. Tier 2 quality range test with ±2 SD acceptance criteria as recommended by the Office of Biostatistics and the Office of Biotechnology Products, CDER/FDA, based on weighted spectral difference analysis.

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