Application of Natural Isotopic Abundance 1H–13C- and 1H–15N-Correlated Two-Dimensional NMR for Evaluation of the Structure of Protein Therapeutics

Arbogast, Luke W.; Brinson, Robert G.; Marino, John P.

doi:10.1016/bs.mie.2015.09.037

Cited by 33 publications

(68 citation statements)

References 39 publications

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“…9,10 However, we also demonstrated the susceptibility of this method to experimental noise and resolution, and results from the study suggested a high limit of detection (LOD) for discrimination between similar structural species, thus bringing into question the suitability of the method to therapeutic mAb spectral analysis. Here we present a multivariate principal component analysis (PCA) method applied directly to the matrix of spectral intensities, which in comparison, we demonstrate can readily discriminate mAb species that manifest both large and subtle spectral differences relative the reference mAb spectrum which arise from enzymatic deglycosylation or degalactosylation.…”

Section: Introductionmentioning

confidence: 95%

“…Nuclear Magnetic Resonance (NMR) spectroscopy is being increasingly recognized as a potent tool for the HOS assessment of protein therapeutics, including mAbs. 1–6 Indeed, we have recently demonstrated the feasibility of recording two-dimensional (2D) heteronuclear 1 H- 15 N and 1 H- 13 C correlation spectra on mAbs, 7–10 however, questions remain as to best practices for spectral analysis in connection with determination of structural comparability of HOS. 5 …”

Section: Introductionmentioning

confidence: 99%

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Multivariate Analysis of Two-Dimensional ¹H, ¹³C Methyl NMR Spectra of Monoclonal Antibody Therapeutics To Facilitate Assessment of Higher Order Structure

et al. 2017

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Two-dimensional (2D) 1H-13C methyl NMR provides a powerful tool to probe the higher order structure (HOS) of monoclonal antibodies (mAbs), since spectra can readily be acquired on intact mAbs at natural isotopic abundance, and small changes in chemical environment and structure give rise to observable changes in corresponding spectra, which can be interpreted at atomic resolution. This makes it possible to apply 2D NMR spectral fingerprinting approaches directly to drug products in order to systematically characterize structure and excipient effects. Systematic collections of NMR spectra are often analyzed in terms of the changes in specifically identified peak positions, as well as changes in peak height and linewidths. A complementary approach is to apply Principal Component Analysis (PCA) directly to the matrix of spectral data, correlating spectra according to similarities and differences in their overall shapes, rather than according to parameters of individually identified peaks. This is particularly well-suited for spectra of mAbs, where some of the individual peaks might not be well resolved. Here we demonstrate the performance of the PCA method for discriminating structural variation among systematic sets of 2D NMR fingerprint spectra using the NISTmAb and illustrate how spectral variability identified by PCA may be correlated to structure.

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Section: Introductionmentioning

confidence: 95%

Section: Introductionmentioning

confidence: 99%

Multivariate Analysis of Two-Dimensional ¹H, ¹³C Methyl NMR Spectra of Monoclonal Antibody Therapeutics To Facilitate Assessment of Higher Order Structure

et al. 2017

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“…A more in-depth comparison by direct point-to-point linear regression analysis of the spectral intensity matrices 6,21 between the three NS0-produced antibodies gave a pairwise correlation coefficient range of 0.960–0.984. The correlation coefficient range of the three NS0- produced antibodies compared to PS 8670 was found to be on the same order, but slightly lower (0.949–0.973).…”

Section: Resultsmentioning

confidence: 99%

“…It has been previously demonstrated that acquiring high-resolution 1 H- 13 C methyl NMR spectra on intact antibodies is feasible using PS 8670 20,21 . The RM 8671 is formulated at 10 mg/mL, a concentration that is too low for NMR analysis; therefore, spectral comparison was conducted versus PS 8670, which is available in higher concentrations.…”

Section: Resultsmentioning

confidence: 99%

Heterologous recombinant expression of non-originator NISTmAb

Kashi

Yandrofski

Preston

et al. 2018

mAbs

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The successful development and regulatory approval of originator and biosimilar therapeutic proteins requires a systems approach to upstream and downstream processing as well as product characterization and quality control. Innovation in process design and control, product characterization strategies, and data integration represent an ecosystem whose concerted advancement may reduce time-to-market and further improve comparability and biosimilarity programs. The biopharmaceutical community has made great strides to this end, yet there currently exists no pre-competitive monoclonal antibody (mAb) expression platform for open innovation. Here, we describe the development and initial expression of an intended copy of the NISTmAb using three non-originator murine cell lines. It was found that, without optimization and in culture flasks, all three cell lines produce approximately 100 mg mAb per liter of culture. Sodium dodecyl sulfate polyacrylamide gel electrophoresis, size-exclusion chromatography, nuclear magnetic resonance spectroscopy, intact mass spectrometry, and surface plasmon resonance were used to demonstrate that the products of all three cell lines embody quality attributes with a sufficient degree of sameness to the NISTmAb Reference Material 8671 to warrant further bioreactor studies, process improvements and optimization. The implications of the work with regard to pre-competitive innovation to support process design and feedback control, comparability and biosimilarity assessments, and process analytical technologies are discussed.

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“…36,47–49 More recently, the 2D-NMR method is becoming more established as a tool for assessing mAb HOS. 33,35,50,51 However, in the absence of resonance assignments 2D-NMR spectra can only be used as a composite high resolution structural fingerprint. The E. coli expression described herein provides an assessable platform to label a mAb with stable isotopes.…”

Section: Discussionmentioning

confidence: 99%

Platform development for expression and purification of stable isotope labeled monoclonal antibodies inEscherichia coli

Reddy

Brinson

Hoopes

et al. 2018

mAbs

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The widespread use of monoclonal antibodies (mAbs) as a platform for therapeutic drug development in the pharmaceutical industry has led to an increased interest in robust experimental approaches for assessment of mAb structure, stability and dynamics. The ability to enrich proteins with stable isotopes is a prerequisite for the in-depth application of many structural and biophysical methods, including nuclear magnetic resonance (NMR), small angle neutron scattering, neutron reflectometry, and quantitative mass spectrometry. While mAbs can typically be produced with very high yields using mammalian cell expression, stable isotope labeling using cell culture is expensive and often impractical. The most common and cost-efficient approach to label proteins is to express proteins in Escherichia coli grown in minimal media; however, such methods for mAbs have not been reported to date. Here we present, for the first time, the expression and purification of a stable isotope labeled mAb from a genetically engineered E. coli strain capable of forming disulfide bonds in its cytoplasm. It is shown using two-dimensional NMR spectral fingerprinting that the unlabeled mAb and the mAb singly or triply labeled with 13C, 15N, 2H are well folded, with only minor structural differences relative to the mammalian cell-produced mAb that are attributed to the lack of glycosylation in the Fc domain. This advancement of an E. coli-based mAb expression platform will facilitate the production of mAbs for in-depth structural characterization, including the high resolution investigation of mechanisms of action.

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Application of Natural Isotopic Abundance 1H–13C- and 1H–15N-Correlated Two-Dimensional NMR for Evaluation of the Structure of Protein Therapeutics

Cited by 33 publications

References 39 publications

Multivariate Analysis of Two-Dimensional ¹H, ¹³C Methyl NMR Spectra of Monoclonal Antibody Therapeutics To Facilitate Assessment of Higher Order Structure

Multivariate Analysis of Two-Dimensional ¹H, ¹³C Methyl NMR Spectra of Monoclonal Antibody Therapeutics To Facilitate Assessment of Higher Order Structure

Heterologous recombinant expression of non-originator NISTmAb

Platform development for expression and purification of stable isotope labeled monoclonal antibodies inEscherichia coli

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Application of Natural Isotopic Abundance 1H–13C- and 1H–15N-Correlated Two-Dimensional NMR for Evaluation of the Structure of Protein Therapeutics

Cited by 33 publications

References 39 publications

Multivariate Analysis of Two-Dimensional 1H, 13C Methyl NMR Spectra of Monoclonal Antibody Therapeutics To Facilitate Assessment of Higher Order Structure

Multivariate Analysis of Two-Dimensional 1H, 13C Methyl NMR Spectra of Monoclonal Antibody Therapeutics To Facilitate Assessment of Higher Order Structure

Heterologous recombinant expression of non-originator NISTmAb

Platform development for expression and purification of stable isotope labeled monoclonal antibodies inEscherichia coli

Contact Info

Product

Resources

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Multivariate Analysis of Two-Dimensional ¹H, ¹³C Methyl NMR Spectra of Monoclonal Antibody Therapeutics To Facilitate Assessment of Higher Order Structure

Multivariate Analysis of Two-Dimensional ¹H, ¹³C Methyl NMR Spectra of Monoclonal Antibody Therapeutics To Facilitate Assessment of Higher Order Structure