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Cambridge Healthtech Institute’s Third Annual
Higher-Order Protein Structure
Characterization, Prediction, Comparability and Biosimilars
Part of CHI’s 6th Annual The Bioprocessing Summit

August 21-22, 2014 | Renaissance Waterfront Hotel | Boston, Massachusetts

Day 1 | Day 2 | Short Courses | Download Brochure | Speaker Bios 

Friday, August 22

8:00 am Registration and Morning Coffee


8:25 Chairperson’s Remarks

JenniferLaurenceJennifer S. Laurence, Ph.D., Associate Professor, Department of Pharmaceutical Chemistry, University of Kansas


8:30 Advantages of Hydrogen Deuterium Exchange Mass Spectrometry in Understanding Multi-Domain Proteins

ThomasWalesThomas E. Wales, Ph.D., Research Assistant Professor, Department of Chemistry and Chemical Biology, The Barnett Institute of Chemical and Biological Analysis, Northeastern University

Intramolecular interactions in multi-domain proteins may play a major role in protein function. Hydrogen deuterium exchange mass spectrometry (HDX MS) can be used to investigate how domains influence one another, and in particular how domain interactions can influence activity from a distance. The application of HDX MS for this purpose will be described for several proteins of varying size and domain architecture.

9:00 NMR Fingerprinting the Higher-Order Structure of Biosimilars: A High Resolution Comparability Tool

YvesAubinYves Aubin, Ph.D., Research Scientist, Protein Structure and Analysis Laboratories, Regulatory Research Division, Centre for Biologics Evaluation, Health Canada

Filgrastim is the generic name for recombinant methionyl human granulocyte colony-stimulating factor (r-metHuG-CSF). It is used for the treatment of neutropenia and it is marketed under the brand name Neupogen® by Amgen. Here we show that a simple NMR fingerprint assay can be used to assess the three-dimensional structure of the active ingredient in the formulated product at high-resolution. In addition, the results of an inter-laboratory NMR study of Neupogen® and biosimilars from the market place will be presented to demonstrate the robustness and precision of the method.

9:30 Adopting Imaging and Other Techniques to Study Novel Therapeutic Modes Such as the DVD-IgTM Molecule

IvanCorreiaIvan R. Correia, MBA, Ph.D., Senior Principal Research Scientist, Protein Analytics, Process Sciences, AbbVie Bioresearch Center

The architecture and dynamics of a DVD-Ig™ molecule and its parental mAbs was examined using single particle electron microscopy. Hinge angles measured for the DVD-Ig™ molecule were similar to the inner antigen parental mAb. The outer binding domain of the DVD-Ig™ molecule was highly mobile and three-dimensional (3D) analysis showed binding of inner antigen caused the outer domain to fold out of the plane with a major morphological change. Docking high-resolution X-ray structures into 3D electron microscopy map supports the extraordinary domain flexibility observed in the DVD-Ig™ molecule allowing antigen binding with minimal steric hindrance.

10:00 Mid-Morning Snack in the Exhibit Hall with Poster Viewing

10:45 Application of High Resolution UPLC-MS in Drug Product Comparability Studies

Yimin Hua, Ph.D., Quality Control Scientist I, Genzyme Corporation, a Sanofi Company

The comparability study will involve not only demonstration of analytical equivalence for protein structures, it also verify that the products have similar quality attributes and equivalent functionality. The product biological activities as well as safety are examined, including host cell DNA and proteins, degradants, aggregates of proteins, etc. The analytical demonstration in similarity commonly involves the use of forced degradation methods such that both structure quality attributes as well as product degradation pathways are also compared. This talk will present comparability studies utilizing the state-of-the-art technology with high resolution UPLC-MS technique

11:15 Advanced Mass Spectrometry for the Characterization of Biopharmaceutical Post-Translational Modifications

AngeloPalmeseAngelo Palmese, Ph.D., Junior Researcher, Structural Characterization, Analytical Development Biotech Products, Merck Serono (Italy)

A detailed knowledge of the protein structure is a prerequisite for the development of biopharmaceuticals. The conformation of a protein determines its function and is largely defined through its primary structure, although it can also be significantly influenced by post-translational modifications (PTMs). In this talk, a case study will be presented in which the intact molecule analysis, by means of mass spectrometry techniques, allowed explaining differences in CDC (Complement-dependent Cytotoxicity) among samples manufactured by two different processes.

11:45 Characterization of the NIST Standard Monoclonal Antibody by 2D NMR Fingerprinting Methodologies

Robert G. Brinson, Ph.D., Research Chemist, National Institute of Standards and Technology; Institute for Bioscience and Biotechnology Research

The development of advanced techniques, such as NMR spectroscopy, for the characterization of tertiary and higher order structure in protein therapeutics is emerging as a major priority in the pharmaceutical industry. To demonstrate the viability and applicability of NMR fingerprinting techniques, we have examined the IgG-based NIST standard monoclonal antibody and present its NMR amide and methyl fingerprint. We further demonstrate rapid acquisition techniques to afford a CH-methyl spectral fingerprint in less than one hour.

12:15 pm Site Directed Spin Labeling to Assess Higher Order Protein Structure

David E. BudilDavid E. Budil Ph.D., Associate Professor of Chemistry and Chemical Biology, Associate Dean for Research, College of Science, Northeastern University

The spin label method, combined with site-directedCys substitutions in proteins, has been shown to be of immense utility to protein structure determination where other methods fail. This methodology is highly suited for membrane proteins as crystallographic methods are often not possible in such cases.  The advantages are no requirement for optical transparency, molecular weight limits are not an issue, and measurements can be carried out in the solid state.

12:45 Enjoy Lunch on Your Own


1:25 Chairperson’s Remarks

Marina Kirkitadze, Ph.D., Deputy Director, Analytical Research & Development, Sanofi Pasteur, Canada

1:30 Qualification of Analytical Method Used for Characterization of Protein Higher Order Structure

MarinaKirkitadzeMarina Kirkitadze, Ph.D., Deputy Director, Analytical Research & Development, Sanofi Pasteur, Canada

The topic of this presentation is qualification of an analytical method. Qualification consists of a simplified evaluation of a subset of validation characteristics. There are no predefined acceptability criteria for evaluation of qualification characteristics, and the purpose to collect experimental data to demonstrate whether an analytical method is suitable for its intended use. Qualification of Differential Scanning Calorimetry (DSC) is shown as an example.

2:00 Evaluation of Vibrational Spectroscopic Techniques for Structural Characterization of a Therapeutic Monoclonal Antibody in Formulation Matrix

GeethaThiagarajanGeetha Thiagarajan, Ph.D., Senior Scientist, Sterile Product and Analytical Development, Merck & Co.

Structural complexity of biological drug products presents an analytical challenge in terms of early detection of aggregation and/or degradation. A set of spectroscopic and non-spectroscopic analytical tests (HP-SEC, SV-AUC, Raman, ROA, CD, Fluorescence, FTIR and DLS) were evaluated for their sensitivity to detect heat-induced molecular instability in a therapeutic monoclonal antibody present in its formulation matrix. The first signs of biophysical changes in the molecule were degradation involving exposure of hydrophobic patches due to partial unfolding, followed by aggregation. Sensitivity of the different assays was rank ordered.

2:30 Probing Higher-Order Structure in Protein Pharmaceuticals Using Infrared and Raman Vibrational Optical Activity

LaurenceNafieLaurence A. Nafie, Ph.D., Distinguished Professor Emeritus, Department of Chemistry, Syracuse University

Vibrational optical activity (VOA), comprised of infrared vibrational circular dichroism (VCD) and vibrational Raman optical activity (ROA) provides enhanced sensitivity to higher order structure (HOS) in proteins compared to their parent IR and Raman spectra, as well as other spectroscopic techniques. Examples of the sensitivity of VOA to both protein secondary structure and HOS in proteins will be presented as a sensitive new tool for evaluating structural differences between originator biopharmaceuticals and their bio-similars.

3:00 Refreshment Break

3:15 Comparison of Higher Order Structure of Soluble and Insoluble Protein

Kelly Neelon, Ph.D., Associate Director, Drug Product Formulation, Momenta Pharmaceuticals, Inc.

Characterization of higher order structure can be instrumental in determining the sensitivities of a molecule to processing conditions during manufacturing.  This talk will discuss utilization of chemical and biophysicaltechniques to compare the higher order structure of a protein in the soluble and insoluble state and feedback of this information to improve processing conditions.

3:45 Applications of Hydrogen Deuterium Exchange – Mass Spectrometry for Biopharmaceutical Development

Damian Houde, Ph.D., Scientist II, Protein Pharmaceutical Development, Biogen Idec; Adjunct Professor, Northeastern University

Protein biopharmaceuticals can exhibit unwanted properties when solution conditions are changed. Analytical tools capable of detecting changes in a protein rapidly, accurately, and with high sensitivity are therefore highly desirable to the biopharmaceutical industry. Hydrogen/deuterium exchange mass spectrometry (H/DX-MS) can be useful for investigating protein conformation, dynamics and interactions. In this presentation, specific applications of H/DX-MS will be discussed that include the characterization of protein biopharmaceuticals at high concentrations.

4:15 End of Conference

Day 1 | Day 2 | Short Courses | Download Brochure | Speaker Bios