Co-reporter:Ronald T. Toth IV, Brittney J. Mills, Sangeeta B. Joshi, Reza Esfandiary, Steven M. Bishop, C. Russell Middaugh, David B. Volkin, and David D. Weis
Analytical Chemistry September 5, 2017 Volume 89(Issue 17) pp:8931-8931
Publication Date(Web):July 28, 2017
DOI:10.1021/acs.analchem.7b01396
A barrier to the use of hydrogen exchange-mass spectrometry (HX-MS) in many contexts, especially analytical characterization of various protein therapeutic candidates, is that differences in temperature, pH, ionic strength, buffering agent, or other additives can alter chemical exchange rates, making HX data gathered under differing solution conditions difficult to compare. Here, we present data demonstrating that HX chemical exchange rates can be substantially altered not only by the well-established variables of temperature and pH but also by additives including arginine, guanidine, methionine, and thiocyanate. To compensate for these additive effects, we have developed an empirical method to correct the hydrogen-exchange data for these differences. First, differences in chemical exchange rates are measured by use of an unstructured reporter peptide, YPI. An empirical chemical exchange correction factor, determined by use of the HX data from the reporter peptide, is then applied to the HX measurements obtained from a protein of interest under different solution conditions. We demonstrate that the correction is experimentally sound through simulation and in a proof-of-concept experiment using unstructured peptides under slow-exchange conditions (pD 4.5 at ambient temperature). To illustrate its utility, we applied the correction to HX-MS excipient screening data collected for a pharmaceutically relevant IgG4 mAb being characterized to determine the effects of different formulations on backbone dynamics.
Co-reporter:Mohammed A. Al-Naqshabandi and David D. Weis
Biochemistry August 8, 2017 Volume 56(Issue 31) pp:4064-4064
Publication Date(Web):July 4, 2017
DOI:10.1021/acs.biochem.6b01312
The extent and location of transient structure in intrinsically disordered proteins (IDPs) provide valuable insights into their conformational ensembles and can lead to a better understanding of coupled binding and folding. Millisecond amide hydrogen exchange (HX) can provide such information, but it is difficult to quantify the degree of transient structuring. One reason is that transiently disordered proteins undergo HX at rates only slightly slower than the rate of amide HX by an unstructured random coil, the chemical HX rate. In this work, we evaluate several different methods of obtaining an accurate model for the chemical HX rate suitable for millisecond hydrogen exchange mass spectrometry (HX-MS) analysis of disordered proteins: (1) calculations using the method of Englander [Bai, Y., et al. (1993) Proteins 17, 75–86], (2) measurement of HX in the presence of 6 M urea or 3 M guanidinium chloride, and (3) measurement of HX by peptide fragments derived directly from the proteins of interest. First, using unstructured model peptides and disordered domains of the activator for thyroid and retinoid receptors and the CREB binding protein as the model IDPs, we show that the Englander method has slight inaccuracies that lead to underestimation of the chemical exchange rate. Second, HX-MS measurements of model peptides show that HX rates are changed dramatically by high concentrations of the denaturant. Third, we find that measurements of HX by reference peptides from the proteins of interest provide the most accurate approach for quantifying the extent of transient structure in disordered proteins by millisecond HX-MS.
Co-reporter:Farai I. Rusinga and David D. Weis
Analytical Chemistry 2017 Volume 89(Issue 2) pp:
Publication Date(Web):December 12, 2016
DOI:10.1021/acs.analchem.6b04057
Measuring amide hydrogen exchange (HX) of intrinsically disordered proteins (IDPs) in solutions containing high concentrations of macromolecular crowding agents would give new insights into the structure and dynamics of these proteins under crowded conditions. High concentrations of artificial crowders, required to simulate cellular crowding, introduce overwhelming interferences to mass spectrometry (MS) analysis. We have developed a fully automated, dual-stage online cleanup that uses strong cation-exchange (SCX) followed by reversed-phase desalting to remove Ficoll, a synthetic polymer, for HX-MS analysis of proteins under crowded conditions. We tested the efficiency of our method by measuring the HX-MS signal intensities of myoglobin peptides from crowded samples containing 300 g L–1 Ficoll and from uncrowded samples. Although there was loss of abundance relative to uncrowded myoglobin analyzed using conventional HX-MS, 97% coverage of the myoglobin sequence was still obtained. Control HX-MS experiments using unstructured peptides labeled at pD 4.0 under crowded and uncrowded conditions confirmed that Ficoll does not alter chemical exchange and that the same extent of HX is achieved in uncrowded solutions as in solutions containing 300 g L–1 of predeuterated Ficoll. We validated our method by measuring HX of CBP, the intrinsically disordered nuclear coactivator binding domain of CREB binding protein (UniProt CBP_MOUSE P45481), residues 2059–2117, at pD 6.5 under crowded and uncrowded conditions. Ficoll induced both protection and deprotection from HX in different regions of CBP, with the greatest deprotection occurring at the edges of helices. These results are consistent with previous observation of IDPs under the influence of synthetic polymers.
Co-reporter:Jayant Arora, Sangeeta B. Joshi, C. Russell Middaugh, David D. Weis, David B. Volkin
Journal of Pharmaceutical Sciences 2017 Volume 106, Issue 6(Volume 106, Issue 6) pp:
Publication Date(Web):1 June 2017
DOI:10.1016/j.xphs.2017.02.007
Multidose formulations of biotherapeutics, which offer better dosage management and reduced production costs, require the addition of antimicrobial preservatives (APs). APs have been shown, however, to decrease protein stability in solution and cause protein aggregation. In this report, the effect of 4 APs, m-cresol, phenol, phenoxyethanol, and benzyl alcohol on conformational stability, aggregation propensity, and backbone flexibility of an IgG1 mAb, mAb-4, is investigated. Compared with no preservative control, each of the APs decreased the conformational stability of mAb-4 as measured by differential scanning calorimetry and extrinsic fluorescence spectroscopy. The addition of APs resulted in increased monomer loss and aggregate accumulation at 50°C over 28 days, as monitored by size-exclusion chromatography. The extent of conformational destabilization and protein aggregation of mAb-4 induced by APs followed their calculated octanol–water partition coefficients. Increases in backbone flexibility, as measured by hydrogen exchange, of a region located in the CH2 domain of the mAb (heavy chain 237-254) in the presence of APs also correlated with hydrophobicity. Based on these results, the destabilizing effect of APs on mAb-4 correlates with the increased hydrophobicity of the APs and their ability to enhance the local backbone flexibility of an aggregation hot spot within the CH2 domain of the mAb.
Co-reporter:Walid S. Maaty
Journal of the American Chemical Society 2016 Volume 138(Issue 4) pp:1335-1343
Publication Date(Web):January 7, 2016
DOI:10.1021/jacs.5b11742
There is considerable interest in the discovery of peptide ligands that bind to protein targets. Discovery of such ligands is usually approached by screening large peptide libraries. However, the individual peptides must be tethered to a tag that preserves their individual identities (e.g., phage display or one-bead one-compound). To overcome this limitation, we have developed a method for screening libraries of label-free peptides for binding to a protein target in solution as a single batch. The screening is based on decreased amide hydrogen exchange by peptides that bind to the target. Hydrogen exchange is measured by mass spectrometry. We demonstrate the approach using a peptide library derived from the Escherichia coli proteome that contained 6664 identifiable features. The library was spiked separately with a peptide spanning the calmodulin binding domain of endothelial nitric oxide synthase (eNOS, 494–513) and a peptide spanning the N-terminal 20 residues of bovine ribonuclease A (S peptide). Human calmodulin and bovine ribonuclease S (RNase S) were screened against the library. Using a novel data analysis workflow, we identified the eNOS peptide as the only calmodulin binding peptide and S peptide as the only ribonuclease S binding peptide in the library.
Co-reporter:Ranajoy Majumdar;C. Russell Middaugh;David B. Volkin
Journal of Pharmaceutical Sciences 2015 Volume 104( Issue 2) pp:327-345
Publication Date(Web):
DOI:10.1002/jps.24224
The dynamic nature of the structure of monoclonal antibodies (mAbs) can be probed at a resolution of 5–20 residues using hydrogen–deuterium exchange mass spectrometry (H/D-MS). Recent studies using H/D-MS have shown that distinct regions of IgG1 mAbs experience significant changes in backbone dynamics in response to specific physicochemical alterations, varying solution conditions, or exposure to different environmental stresses. Tracking such changes in local dynamics may therefore serve as a key analytical tool, not only to monitor stability changes, but also to design improved, and more stable formulations of therapeutic mAbs in pharmaceutical dosage forms. This review article describes the H/D-MS method as applied to the analysis of formulations containing mAbs and summarizes recent studies monitoring changes in mAb local dynamics in response to chemical modifications, physical degradation, and presence of stabilizing and destabilizing excipients. Furthermore, the nature of the local dynamics of a highly conserved peptide segment in the CH2 domain of IgG1 mAbs is reviewed, and the results are correlated with decreased pharmaceutical stability, supporting the identification of a common aggregation hotspot sequence in the Fc region of human IgG1 mAbs. In addition, unresolved challenges (and opportunities) in applying H/D-MS technology for stabilization and formulation development of mAbs are discussed. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 104:327–345, 2015
Co-reporter:Theodore R. Keppel
Journal of The American Society for Mass Spectrometry 2015 Volume 26( Issue 4) pp:547-554
Publication Date(Web):2015 April
DOI:10.1007/s13361-014-1033-6
Measurement of residual structure in intrinsically disordered proteins can provide insights into the mechanisms by which such proteins undergo coupled binding and folding. The present work describes an approach to measure residual structure in disordered proteins using millisecond hydrogen/deuterium (H/D) exchange in a conventional bottom-up peptide-based workflow. We used the exchange mid-point, relative to a totally deuterated control, to quantify the rate of H/D exchange in each peptide. A weighted residue-by-residue average of these midpoints was used to map the extent of residual structure at near single-residue resolution. We validated this approach both by simulating a disordered protein and experimentally using the p300 binding domain of ACTR, a model disordered protein already well-characterized by other approaches. Secondary structure elements mapped in the present work are in good agreement with prior nuclear magnetic resonance measurements. The new approach was somewhat limited by a loss of spatial resolution and subject to artifacts because of heterogeneities in intrinsic exchange. Approaches to correct these limitations are discussed.
Co-reporter:Ranajoy Majumdar, Prakash Manikwar, John M. Hickey, Hardeep S. Samra, Hasige A. Sathish, Steven M. Bishop, C. Russell Middaugh, David B. Volkin, and David D. Weis
Biochemistry 2013 Volume 52(Issue 19) pp:
Publication Date(Web):April 17, 2013
DOI:10.1021/bi400232p
This work examines the effect of three anions from the Hofmeister series (sulfate, chloride, and thiocyanate) on the conformational stability and aggregation rate of an IgG1 monoclonal antibody (mAb) and corresponding changes in the mAb’s backbone flexibility (at pH 6 and 25 °C). Compared to a 0.1 M NaCl control, thiocyanate (0.5 M) decreased the melting temperatures (Tm) for three observed conformational transitions within the mAb by 6–9 °C, as measured by differential scanning calorimetry. Thiocyanate also accelerated the rate of monomer loss at 40 °C over 12 months, as monitored by size exclusion chromatography. Backbone flexibility, as measured via H/D exchange mass spectrometry, increased in two segments in the CH2 domain with more subtle changes across several additional regions. Chloride (0.5 M) caused slight increases in the Tm values, small changes in aggregation rate, and minimal yet consistent decreases in flexibility across various domains with larger effects noted within the VL, CH1, and CH3 domains. In contrast, 0.5 M sulfate increased Tm values, had small stabilizing influences on aggregate formation over time, yet substantially increased the flexibility of two specific regions in the CH1 and VL domains. While thiocyanate-induced conformational destabilization of the mAb correlated with increased local flexibility of specific regions in the CH2 domain (especially residues 241–251 in the heavy chain), the stabilizing anion sulfate did not affect these CH2 regions.
Co-reporter:Prakash Manikwar;Ranajoy Majumdar;John M. Hickey;Santosh V. Thakkar;Hardeep S. Samra;Hasige A. Sathish;Steven M. Bishop;C. Russell Middaugh;David B. Volkin
Journal of Pharmaceutical Sciences 2013 Volume 102( Issue 7) pp:2136-2151
Publication Date(Web):
DOI:10.1002/jps.23543
Abstract
The effects of sucrose and arginine on the conformational and storage stability of an IgG1 monoclonal antibody (mAb) were monitored by differential scanning calorimetry (DSC) and size-exclusion chromatography (SEC), respectively. Excipient effects on protein physical stability were then compared with their effects on the local flexibility of the mAb in solution at pH 6, 25°C using hydrogen/deuterium-exchange mass spectrometry (H/D-MS). Compared with a 0.1 M NaCl control, sucrose (0.5 M) increased conformational stability (Tm values), slowed the rate of monomer loss, reduced the formation of insoluble aggregates, and resulted in a global trend of small decreases in local flexibility across most regions of the mAb. In contrast, the addition of arginine (0.5 M) decreased the mAb's conformational stability, increased the rate of loss of monomer with elevated levels of soluble and insoluble aggregates, and led to significant increases in the local flexibility in specific regions of the mAb, most notably within the constant domain 2 of the heavy chain (CH2). These results provide new insights into the effect of sucrose and arginine on the local dynamics of IgG1 domains as well as preliminary correlations between local flexibility within specific segments of the CH2 domain (notably heavy chain 241–251) and the mAb's overall physical stability. © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 102:2136–2151, 2013
Co-reporter:Ranajoy Majumdar;Prakash Manikwar
Journal of The American Society for Mass Spectrometry 2012 Volume 23( Issue 12) pp:2140-2148
Publication Date(Web):2012 December
DOI:10.1007/s13361-012-0485-9
Chromatographic carry-over can severely distort measurements of amide H/D exchange in proteins analyzed by LC/MS. In this work, we explored the origin of carry-over in the online digestion of an IgG1 monoclonal antibody using an immobilized pepsin column under quenched H/D exchange conditions (pH 2.5, 0 °C). From a consensus list of 169 different peptides consistently detected during digestion of this large, ~150 kDa protein, approximately 30 % of the peptic peptides exhibited carry-over. The majority of carry-over originates from the online digestion. Carry-over can be substantially decreased by washing the online digestion flow-path and pepsin column with two wash cocktails: [acetonitrile (5 %)/ isopropanol (5 %)/ acetic acid (20 %) in water] and [2 M guanidine hydrochloride in 100 mM phosphate buffer pH 2.5]. Extended use of this two-step washing procedure does not adversely affect the specificity or activity of the immobilized pepsin column. The results suggest that although the mechanism of carry-over appears to be chemical in nature, and not hydrodynamic, carry-over cannot be attributed to a single factor such as mass, abundance, pI, or hydrophobicity of the peptides.
Co-reporter:Theodore R. Keppel, Brent A. Howard, and David D. Weis
Biochemistry 2011 Volume 50(Issue 40) pp:
Publication Date(Web):September 6, 2011
DOI:10.1021/bi200875p
Mapping the structured and disordered regions and identifying disorder-to-order transitions are essential to understanding intrinsically disordered proteins (IDPs). One technique that can provide such information is H/D exchange coupled with mass spectrometry (H/D-MS). To explore the feasibility of H/D-MS for mapping disordered and ordered regions in IDPs, we undertook a systematic evaluation of an unstructured protein, a molten globular protein, and the well-folded complex of the two proteins. Most segments of the unstructured protein, ACTR (activator of thyroid and retinoid receptors, NCOA3_HUMAN, residues 1018–1088), exchange at rates consistent with its assignment as an unstructured protein, but there is slight protection in regions that become helical in the ACTR–CBP complex. The molten globular protein, CBP (the nuclear coactivator binding domain of the CREB binding protein, CBP_MOUSE, residues 2059–2117), is moderately protected from exchange, and the protection is nearly uniform across the length of the protein. The uniformity arises because of rapid interconversion between an ensemble of folded conformers and an ensemble of unstructured conformers. Rapid interconversion causes the H/D exchange kinetics to be dominated by exchange by molecules in unstructured conformations. For the folded ACTR–CBP complex, the exchange data provide a qualitatively accurate description of the complex. Our results provide a useful framework to use in the interpretation of H/D-MS data of intrinsically disordered proteins.
Co-reporter:Prakash Manikwar, Ranajoy Majumdar, John M. Hickey, Santosh V. Thakkar, ... David B. Volkin
Journal of Pharmaceutical Sciences (July 2013) Volume 102(Issue 7) pp:2136-2151
Publication Date(Web):1 July 2013
DOI:10.1002/jps.23543
The effects of sucrose and arginine on the conformational and storage stability of an IgG1 monoclonal antibody (mAb) were monitored by differential scanning calorimetry (DSC) and size-exclusion chromatography (SEC), respectively. Excipient effects on protein physical stability were then compared with their effects on the local flexibility of the mAb in solution at pH 6, 25°C using hydrogen/deuterium-exchange mass spectrometry (H/D-MS). Compared with a 0.1 M NaCl control, sucrose (0.5 M) increased conformational stability (Tm values), slowed the rate of monomer loss, reduced the formation of insoluble aggregates, and resulted in a global trend of small decreases in local flexibility across most regions of the mAb. In contrast, the addition of arginine (0.5 M) decreased the mAb's conformational stability, increased the rate of loss of monomer with elevated levels of soluble and insoluble aggregates, and led to significant increases in the local flexibility in specific regions of the mAb, most notably within the constant domain 2 of the heavy chain (CH2). These results provide new insights into the effect of sucrose and arginine on the local dynamics of IgG1 domains as well as preliminary correlations between local flexibility within specific segments of the CH2 domain (notably heavy chain 241–251) and the mAb's overall physical stability. © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 102:2136–2151, 2013
Co-reporter:Ranajoy Majumdar, C.Russell Middaugh, David D. Weis, David B. Volkin
Journal of Pharmaceutical Sciences (February 2015) Volume 104(Issue 2) pp:327-345
Publication Date(Web):1 February 2015
DOI:10.1002/jps.24224
The dynamic nature of the structure of monoclonal antibodies (mAbs) can be probed at a resolution of 5-20 residues using hydrogen-deuterium exchange mass spectrometry (H/D-MS). Recent studies using H/D-MS have shown that distinct regions of IgG1 mAbs experience significant changes in backbone dynamics in response to specific physicochemical alterations, varying solution conditions, or exposure to different environmental stresses. Tracking such changes in local dynamics may therefore serve as a key analytical tool, not only to monitor stability changes, but also to design improved, and more stable formulations of therapeutic mAbs in pharmaceutical dosage forms. This review article describes the H/D-MS method as applied to the analysis of formulations containing mAbs and summarizes recent studies monitoring changes in mAb local dynamics in response to chemical modifications, physical degradation, and presence of stabilizing and destabilizing excipients. Furthermore, the nature of the local dynamics of a highly conserved peptide segment in the CH2 domain of IgG1 mAbs is reviewed, and the results are correlated with decreased pharmaceutical stability, supporting the identification of a common aggregation hotspot sequence in the Fc region of human IgG1 mAbs. In addition, unresolved challenges (and opportunities) in applying H/D-MS technology for stabilization and formulation development of mAbs are discussed. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 104:327-345, 2015
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