John Marino

Associate Director, IBBR and Research Chemist

NIST

Contact

Email: john.marino@nist.gov

Call: (240) 314-6361

Education

  • Postdoctoral Fellowship, Geothe Universität, Frankfurt, Germany, 1995 – 1997
  • Ph.D., Chemistry, Yale University, 1995      
  • A.B., Chemistry, Princeton University, 1989

Profile

Dr. John Marino’s research is focused on developing nuclear magnetic resonance (NMR) and other biophysical measurements to accurately and precisely define the conformational structure, stability, and dynamics of biomolecules and their interactions at a molecular level.

In addition to enabling fundamental insights into biomolecular structure and function, Dr. Marino’s work provides innovative, yet practical methods that can form the basis for a robust measurement infrastructure that supports biopharmaceutical development and regulation.

CURRENT RESEARCH

Biopharmaceuticals represent over 40% of the medicines in development today, making this industry a valuable and important sector of the US economy. A key focus is on the development of advanced techniques for accurate and precise characterization of higher-order-structure (HOS) in biotherapeutics, particularly monoclonal antibodies, which are sought by industry and regulators to establish consistency in drug manufacturing, detect process-related drug-product variations and compare biosimilars to innovator reference products. 

(Left) Representative 2D NMR methyl spectral fingerprint of a Fab fragment from a mAb. (Right) Structure of the Fab with methyl groups highlighted by pink balls.
mAbs are highly flexible, as seen in a TEM image of NISTmAb using negative stain enhancing contrast agents. Cartoon models shown below TEM images.

2D NMR for Biopharmaceutical Structure Assessment

Correct folding of biopharmaceuticals is critical for drug efficacy, with misfolding also impacting drug safety by eliciting unwanted immune and/or other off-target responses. High-resolution measurements of the structure(s) of protein therapeutics provide important tools for establishing consistency in drug manufacturing and detection of drug product changes resulting from modifications in the manufacturing process. NMR, a high-resolution and information-rich technique, is being applied as a tool for structural assessment of monoclonal antibodies (mAbs) biopharmaceuticals. This method is highly sensitive to molecular structure alterations that result from mutations, misfoldings, or contaminants. Experimental approaches are being developed to obtain structural 'fingerprints' of the bioactive form of protein therapeutics, as well as detection of variants, at atomic resolution. In parallel, computational alternatives (e.g., chemometrics and machine learning) to interactive analysis of spectral features are being explored. 

Measurements of Biopharmaceuticals using Cryogenic Electron Microscopy (cryo-EM)

In biopharmaceutical applications, analysis of small, asymmetric molecules, which exhibit inherent flexibility, is required. Cryo-EM approaches are being explored that will enable high-resolution (better than 0.3 nm) structural characterization of flexible, protein-based drugs that are below a molecular weight limit of ~200 kDa. To address current limitations of low contrast and the challenges with alignment and averaging of small, flexible biomolecules, new strategies for bioengineering sample platforms, image analysis, and mathematical modeling are being pursued.

Publications
2021
Excipient Innovation Through Precompetitive Research.
Highly conserved s2m element of SARS-CoV-2 dimerizes via a kissing complex and interacts with host miRNA-1307-3p.
The emerging landscape of single-molecule protein sequencing technologies.
Grand Challenges in Pharmaceutical Research Series: Ridding the Cold Chain for Biologics.
2020
Principal component analysis for automated classification of 2D spectra and interferograms of protein therapeutics: influence of noise, reconstruction details, and data preparation.
Leveraging nature's biomolecular designs in next-generation protein sequencing reagent development.
Assessment of the Higher-Order Structure of Formulated Monoclonal Antibody Therapeutics by 2D Methyl Correlated NMR and Principal Component Analysis.
Comparative Analysis of One-Dimensional Protein Fingerprint by Line Shape Enhancement and Two-Dimensional 1H,13C Methyl NMR Methods for Characterization of the Higher Order Structure of IgG1 Monoclonal Antibodies.
Best Practices in Utilization of 2D-NMR Spectral Data as the Input for Chemometric Analysis in Biopharmaceutical Applications.
Chemometric Outlier Classification of 2D-NMR Spectra to Enable Higher Order Structure Characterization of Protein Therapeutics.
2019
Structure and Dynamics of a Site-Specific Labeled Fc Fragment with Altered Effector Functions.
2D J-correlated proton NMR experiments for structural fingerprinting of biotherapeutics.
Single-Molecule 3D Images of "Hole-Hole" IgG1 Homodimers by Individual-Particle Electron Tomography.
Engineering ClpS for selective and enhanced N-terminal amino acid binding.
2018
Enabling adoption of 2D-NMR for the higher order structure assessment of monoclonal antibody therapeutics.
Heterologous recombinant expression of non-originator NISTmAb.
Non-Uniform Sampling for All: More NMR Spectral Quality, Less Measurement Time.
2017
Multivariate Analysis of Two-Dimensional 1H, 13C Methyl NMR Spectra of Monoclonal Antibody Therapeutics To Facilitate Assessment of Higher Order Structure.
Application of 2D-NMR with room temperature NMR probes for the assessment of the higher order structure of filgrastim.
2016
A small protein inhibits proliferating cell nuclear antigen by breaking the DNA clamp.