Publications by authors named "George M Bou-Assaf"

12 Publications

  • Page 1 of 1

A Multicompany Assessment of Submicron Particle Levels by NTA and RMM in a Wide Range of Late-Phase Clinical and Commercial Biotechnology-Derived Protein Products.

J Pharm Sci 2020 01 21;109(1):830-844. Epub 2019 Oct 21.

Process Development, Amgen, Thousand Oaks, California 91320.

One of the major product quality challenges for injectable biologics is controlling the amount of protein aggregates and particles present in the final drug product. This article focuses on particles in the submicron range (<2 μm). A cross-industry collaboration was undertaken to address some of the analytical gaps in measuring submicron particles (SMPs), developing best practices, and surveying the concentration of these particles present in 52 unique clinical and commercial protein therapeutics covering 62 dosage forms. Measured particle concentrations spanned a range of 4 orders of magnitude for nanoparticle tracking analysis and 3 orders of magnitude for resonant mass measurement. The particle concentrations determined by the 2 techniques differed significantly for both control and actual product. In addition, results suggest that these techniques exhibit higher variability compared to well-established subvisible particle characterization techniques (e.g., flow-imaging or light obscuration). Therefore, in their current states, nanoparticle tracking analysis and resonant mass measurement-based techniques can be used during product and process characterization, contributing information on the nature and propensity for formation of submicron particles and what is normal for the product, but may not be suitable for release or quality control testing. Evaluating the level of SMPs to which humans have been routinely exposed during the administration of several commercial and late-phase clinical products adds critical knowledge to our understanding of SMP levels that may be considered acceptable from a safety point of view. This article also discusses dependence of submicron particle size and concentration on the dosage form attributes such as physical state, primary packaging, dose strength, etc. To the best of our knowledge, this is the largest study ever conducted to characterize SMPs in late-phase and commercial products.
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http://dx.doi.org/10.1016/j.xphs.2019.10.025DOI Listing
January 2020

Recommendations for performing, interpreting and reporting hydrogen deuterium exchange mass spectrometry (HDX-MS) experiments.

Nat Methods 2019 07 27;16(7):595-602. Epub 2019 Jun 27.

Roy J. Carver Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, IA, USA.

Hydrogen deuterium exchange mass spectrometry (HDX-MS) is a powerful biophysical technique being increasingly applied to a wide variety of problems. As the HDX-MS community continues to grow, adoption of best practices in data collection, analysis, presentation and interpretation will greatly enhance the accessibility of this technique to nonspecialists. Here we provide recommendations arising from community discussions emerging out of the first International Conference on Hydrogen-Exchange Mass Spectrometry (IC-HDX; 2017). It is meant to represent both a consensus viewpoint and an opportunity to stimulate further additions and refinements as the field advances.
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http://dx.doi.org/10.1038/s41592-019-0459-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6614034PMC
July 2019

Interlaboratory Comparison of Hydrogen-Deuterium Exchange Mass Spectrometry Measurements of the Fab Fragment of NISTmAb.

Anal Chem 2019 06 14;91(11):7336-7345. Epub 2019 May 14.

Department of Chemistry , University of Kansas , 1567 Irving Hill Road , Lawrence , Kansas 66045 , United States.

Hydrogen-deuterium exchange mass spectrometry (HDX-MS) is an established, powerful tool for investigating protein-ligand interactions, protein folding, and protein dynamics. However, HDX-MS is still an emergent tool for quality control of biopharmaceuticals and for establishing dynamic similarity between a biosimilar and an innovator therapeutic. Because industry will conduct quality control and similarity measurements over a product lifetime and in multiple locations, an understanding of HDX-MS reproducibility is critical. To determine the reproducibility of continuous-labeling, bottom-up HDX-MS measurements, the present interlaboratory comparison project evaluated deuterium uptake data from the Fab fragment of NISTmAb reference material (PDB: 5K8A ) from 15 laboratories. Laboratories reported ∼89 800 centroid measurements for 430 proteolytic peptide sequences of the Fab fragment (∼78 900 centroids), giving ∼100% coverage, and ∼10 900 centroid measurements for 77 peptide sequences of the Fc fragment. Nearly half of peptide sequences are unique to the reporting laboratory, and only two sequences are reported by all laboratories. The majority of the laboratories (87%) exhibited centroid mass laboratory repeatability precisions of ⟨ s⟩ ≤ (0.15 ± 0.01) Da (1σ). All laboratories achieved ⟨s⟩ ≤ 0.4 Da. For immersions of protein at T = (3.6 to 25) °C and for DO exchange times of t = (30 s to 4 h) the reproducibility of back-exchange corrected, deuterium uptake measurements for the 15 laboratories is σ( t) = (9.0 ± 0.9) % (1σ). A nine laboratory cohort that immersed samples at T = 25 °C exhibited reproducibility of σ( t) = (6.5 ± 0.6) % for back-exchange corrected, deuterium uptake measurements.
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http://dx.doi.org/10.1021/acs.analchem.9b01100DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6745711PMC
June 2019

Deciphering the Biophysical Effects of Oxidizing Sulfur-Containing Amino Acids in Interferon-beta-1a using MS and HDX-MS.

J Am Soc Mass Spectrom 2017 05 13;28(5):840-849. Epub 2017 Feb 13.

Technical Development, Biogen, 225 Binney St., Cambridge, MA, 02142, USA.

Introduction of a chemical change to one or more amino acids in a protein's polypeptide chain can result in various effects on its higher-order structure (HOS) and biophysical behavior (or properties). These effects range from no detectable change to significant structural or conformational alteration that can greatly affect the protein's biophysical properties and its resulting biological function. The ability to reliably detect the absence or presence of such changes is essential to understanding the structure-function relationship in a protein and in the successful commercial development of protein-based drugs (biopharmaceuticals). In this paper, we focus our attention on the latter by specifically elucidating the impact of oxidation on the HOS, structural dynamics, and biophysical properties of interferon beta-1a (IFNβ-1a). Oxidation is a common biochemical modification that occurs in many biopharmaceuticals, specifically in two naturally-occurring sulfur-containing amino acids, methionine and cysteine. To carry out this work, we used combinations of hydrogen peroxide and pH to differentially oxidize IFNβ-1a (to focus on only methionine oxidation versus methionine and cysteine oxidation). We then employed several analytical and biophysical techniques to acquire information about the differential impact of these two oxidation scenarios on IFNβ-1a. In particular, the use of MS-based techniques, especially HDX-MS, play a dominant role in revealing the differential effects. Graphical Abstract ᅟ.
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http://dx.doi.org/10.1007/s13361-016-1585-8DOI Listing
May 2017

Conformational Analysis of Proteins in Highly Concentrated Solutions by Dialysis-Coupled Hydrogen/Deuterium Exchange Mass Spectrometry.

J Am Soc Mass Spectrom 2016 Apr 9;27(4):669-76. Epub 2016 Feb 9.

Department of Pharmacy, University of Copenhagen, Copenhagen, Denmark.

When highly concentrated, an antibody solution can exhibit unusual behaviors, which can lead to unwanted properties, such as increased levels of protein aggregation and unusually high viscosity. Molecular modeling, along with many indirect biophysical measurements, has suggested that the cause for these phenomena can be due to short range electrostatic and/or hydrophobic protein-protein interactions. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) is a useful tool for investigating protein conformation, dynamics, and interactions. However, "traditional" continuous dilution labeling HDX-MS experiments have limited utility for the direct analysis of solutions with high concentrations of protein. Here, we present a dialysis-based HDX-MS (di-HDX-MS) method as an alternative HDX-MS labeling format, which takes advantage of passive dialysis rather than the classic dilution workflow. We applied this approach to a highly concentrated antibody solution without dilution or significant sample manipulation, prior to analysis. Such a method could pave the way for a deeper understanding of the unusual behavior of proteins at high concentrations, which is highly relevant for development of biopharmaceuticals in industry. Graphical Abstract ᅟ.
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http://dx.doi.org/10.1007/s13361-015-1331-7DOI Listing
April 2016

Technical Decision-Making with Higher Order Structure Data: Detecting Reversible Concentration-Dependent Self-Association in a Monoclonal Antibody and a Preliminary Investigation to Eliminate It.

J Pharm Sci 2015 Nov 26;104(11):3984-3989. Epub 2015 Aug 26.

Protein Pharmaceutical Development, Biogen Inc., Cambridge, Massachusetts. Electronic address:

Protein self-association or aggregation is a property of significant concern for biopharmaceutical products due to the potential ability of aggregates to cause adverse toxicological and immunological effects. Thus, during the development of a protein biopharmaceutical, it is important to detect and quantify the level and nature of aggregate species as early as possible in order to make well-informed decisions and to mitigate and control potential risks. Although a deeper understanding of the mechanism of aggregation (i.e., protein-protein interactions) is desirable, such detailed assessment is not always necessary from a biopharmaceutical process development point of view. In fact, the scope of characterization efforts is often focused on achieving a well-controlled process, which generates a product that reliably meets established acceptance criteria for safety and efficacy. In this brief note, we evaluated the utility of size-exclusion chromatography, dynamic light scattering, and analytical ultracentrifugation in their simplest forms, to effectively reveal and confirm the presence of concentration-dependent reversible self-association (RSA) in a monoclonal antibody in the early stages of formulation development. Using these techniques, we also initiated preliminary work aimed at reducing the occurrence of this RSA behavior by varying the pH of the formulation buffer.
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http://dx.doi.org/10.1002/jps.24616DOI Listing
November 2015

Mapping the interaction between factor VIII and von Willebrand factor by electron microscopy and mass spectrometry.

Blood 2015 Aug 11;126(8):935-8. Epub 2015 Jun 11.

Department of Cell Biology, Harvard Medical School, Boston, MA; Howard Hughes Medical Institute, Harvard Medical School, Boston, MA.

Association with the D'D3 domain of von Willebrand factor (VWF) stabilizes factor VIII (FVIII) in the circulation and maintains it at a level sufficient to prevent spontaneous bleeding. We used negative-stain electron microscopy (EM) to visualize complexes of FVIII with dimeric and monomeric forms of the D'D3 domain. The EM averages show that FVIII interacts with the D'D3 domain primarily through its C1 domain, with the C2 domain providing a secondary attachment site. Hydrogen-deuterium exchange mass spectrometry corroborated the importance of the C1 domain in D'D3 binding and implicates additional surface regions on FVIII in the interaction. Together, our results establish that the C1 domain is the major binding site on FVIII for VWF, reiterate the importance of the a3 acidic peptide in VWF binding, and suggest that the A3 and C2 domains play ancillary roles in this interaction.
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http://dx.doi.org/10.1182/blood-2015-04-641688DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4543227PMC
August 2015

Investigating monoclonal antibody aggregation using a combination of H/DX-MS and other biophysical measurements.

J Pharm Sci 2013 Dec 17;102(12):4315-29. Epub 2013 Oct 17.

Department of Chemistry and Chemical Biology, Northeastern University, Boston, Massachusetts, 02115.

To determine how structural changes in antibodies are connected with aggregation, the structural areas of an antibody prone to and/or impacted by aggregation must be identified. In this work, the higher-order structure and biophysical properties of two different monoclonal antibody (mAb) monomers were compared with their simplest aggregated form, that is, dimers that naturally occurred during normal production and storage conditions. A combination of hydrogen/deuterium exchange mass spectrometry and other biophysical measurements was used to make the comparison. The results show that the dimerization process for one of the mAb monomers (mAb1) displayed no differences in its deuterium uptake between monomer and dimer forms. However, the other mAb monomer (mAb2) showed subtle changes in hydrogen/deuterium exchange as compared with its dimer form. In this case, differences observed were located in specific functional regions of the CH 2 domain and the hinge region between CH 1 and CH 2 domains. The importance and the implications of these changes on the antibody structure and mechanism of aggregation are discussed.
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http://dx.doi.org/10.1002/jps.23754DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3970822PMC
December 2013

Improved sequence resolution by global analysis of overlapped peptides in hydrogen/deuterium exchange mass spectrometry.

J Am Soc Mass Spectrom 2012 Jul 24;23(7):1202-8. Epub 2012 Apr 24.

Institute of Molecular Biophysics, Biological Sciences Department, Florida State University, Tallahassee, FL 32306, USA.

Management of the enormous amount of data produced during solution-phase hydrogen/deuterium exchange monitored by mass spectrometry has stimulated software analysis development. The proteolysis step of the experiment generates multiple peptide fragments, most of which overlap. Prior automated data reduction algorithms extract the deuteration level for individual peptides, but do not exploit the additional information arising from fragment overlap. Here, we describe an algorithm that determines discrete rate constant values to each of the amide hydrogens in overlapped fragments. By considering all of the overlapped peptide segments simultaneously, sequence resolution can be improved significantly, sometimes to the individual amino acid level. We have validated the method with simulated deuterium uptake data for seven overlapped fragments of a poly-Ala nonapeptide, and then applied it to extract rate constant values for the first 29 N-terminal amino acids of C22A FK506-binding protein.
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http://dx.doi.org/10.1007/s13361-012-0373-3DOI Listing
July 2012

Complexation and Calcium-Induced Conformational Changes in the Cardiac Troponin Complex Monitored by Hydrogen/Deuterium Exchange and FT-ICR Mass Spectrometry.

Int J Mass Spectrom 2011 Apr;302(1-3):116-124

Department of Chemistry and Biochemistry, Florida State University, Tallahassee, FL 32306.

Cardiac muscle contraction is regulated by the heterotrimeric complex: troponin. We apply solution-phase hydrogen/deuterium exchange monitored by FT-ICR mass spectrometry to study the structural dynamics and the Ca-induced conformational changes of the cardiac isoform of troponin, by comparing H/D exchange rate constants for TnC alone, the binary TnC:TnI complex, and the ternary TnC:TnI:TnT complex for Ca-free and Ca-saturated states. The wide range of exchange rate constants indicates that the complexes possess both highly flexible and very rigid domains. Fast exchange rates were observed for the N-terminal extension of TnI (specific to the cardiac isoform), the DE linker in TnC alone, and the mobile domain of TnI. The slowest rates were for the IT coiled-coil that grants stability and stiffness to the complex. Ca(2+) binding to site II of the N-lobe of TnC induces short-range allosteric effects, mainly protection for the C-lobe of TnC that transmits long-range conformational changes that reach the IT coiled-coil and even TnT1. The present results corroborate prior X-ray crystallography and NMR interpretations and also illuminate domains that were not resolved or truncated in those experiments.
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http://dx.doi.org/10.1016/j.ijms.2010.08.023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3134279PMC
April 2011

Advantages of isotopic depletion of proteins for hydrogen/deuterium exchange experiments monitored by mass spectrometry.

Anal Chem 2010 Apr;82(8):3293-9

Department of Chemistry and Biochemistry, Florida State University, Tallahassee, Florida 32306, USA.

Solution-phase hydrogen/deuterium exchange (HDX) monitored by mass spectrometry is an excellent tool to study protein-protein interactions and conformational changes in biological systems, especially when traditional methods such as X-ray crystallography or nuclear magnetic resonance are not feasible. Peak overlap among the dozens of proteolytic fragments (including those from autolysis of the protease) can be severe, due to high protein molecular weight(s) and the broad isotopic distributions due to multiple deuterations of many peptides. In addition, different subunits of a protein complex can yield isomeric proteolytic fragments. Here, we show that depletion of (13)C and/or (15)N for one or more protein subunits of a complex can greatly simplify the mass spectra, increase the signal-to-noise ratio of the depleted fragment ions, and remove ambiguity in assignment of the m/z values to the correct isomeric peptides. Specifically, it becomes possible to monitor the exchange progress for two isobaric fragments originating from two or more different subunits within the complex, without having to resort to tandem mass spectrometry techniques that can lead to deuterium scrambling in the gas phase. Finally, because the isotopic distribution for a small to medium-size peptide is essentially just the monoisotopic species ((12)C(c)(1)H(h)(14)N(n)(16)O(o)(32)S(s)), it is not necessary to deconvolve the natural abundance distribution for each partially deuterated peptide during HDX data reduction.
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http://dx.doi.org/10.1021/ac100079zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2878611PMC
April 2010

Fast reversed-phase liquid chromatography to reduce back exchange and increase throughput in H/D exchange monitored by FT-ICR mass spectrometry.

J Am Soc Mass Spectrom 2009 Mar 21;20(3):520-4. Epub 2008 Nov 21.

National High Magnetic Field Laboratory, Florida State University, Tallahassee, Florida 32310-4005, USA.

In solution-phase hydrogen/deuterium exchange (HDX), it is essential to minimize the back-exchange level of H for D after the exchange has been quenched, to accurately assign protein conformation and protein-protein or protein-ligand interactions. Reversed-phase HPLC is conducted at low pH and low temperature to desalt and separate proteolytic fragments. However, back exchange averages roughly 30% because of the long exposure to H(2)O in the mobile phase. In this report, we first show that there is no significant backbone amide hydrogen back exchange during quench and digestion; backbone exchange occurs primarily during subsequent liquid chromatography separation. We then show that a rapid reversed-phase separation reduces back exchange for HDX by at least 25%, resulting from the dramatically reduced retention time of the peptide fragments on the column. The influence of retention time on back exchange was also evaluated. The rapid separation coupled with high-resolution FT-ICR MS at 14.5 T provides high amino acid sequence coverage, high sample throughput, and high reproducibility and reliability.
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http://dx.doi.org/10.1016/j.jasms.2008.11.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2673454PMC
March 2009
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