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European Biopharmaceutical Review

Protein Production

Biopharmaceuticals are a unique class of medicines due to their extreme structural complexity. Mass spectrometry (MS) offers a variety of approaches to study their structure and behaviour, and has become a default tool for characterising the convalent structure of protein therapeutics.

The selectivity and precision with which biopharmaceuticals interact with their therapeutic targets are possible due to their unique three-dimensional architecture or conformation. The elaborate higher order structure of protein therapeutics makes this class of medicines distinct from small molecule drugs, where covalent structure alone determines the physical shape (and therefore, the ability to interact with therapeutic targets). In contrast, the large physical size of proteins makes the multitude of non-covalent contacts not only inevitable, but in fact the defining element of their three-dimensional structure. Unfortunately, these sophisticated webs of non-covalent interactions may also become a liability under certain circumstances, trapping an entangled protein in a misfolded state.

Misfolding Proteins

Failure to fold or maintain the native conformation has a negative impact on the efficacy of the protein drug, since the ability to interact with physiological targets requires that the native conformation be maintained throughout the lifecycle of a protein molecule. Proteins that are not folded properly are prone to aggregation both in vitro and in vivo, and are targeted by proteases that affect the bioavailability of the protein drug. However, the most feared consequence of misfolding and aggregation of the protein drug is the immune response, which adversely affects its safety profile. Critical dependence of the protein drug potency, stability and safety on conformation places a premium on the ability to characterise it throughout the various stages of the drug development process, from design to manufacturing to post-approval monitoring.

Although protein conformation and dynamics can be probed at high resolution by X-ray crystallography and nuclear magnetic resonance (NMR), inherent limitations of these techniques often make it difficult to carry out the analyses of biopharmaceutical products under relevant production/ storage conditions (for example, protein drug substance, product or dosing solution), or physiological conditions (for example, mimicking the environment encountered by the protein post-administration). In most cases, the routine analyses of conformation and stability of protein drugs still rely on classical biophysical methods, such as optical spectroscopy (circular dichroism, fluorescence, UVabsorption and FTIR spectroscopy), light scattering, calorimetry, as well as analytical centrifugation and size exclusion chromatography. While these techniques are capable of probing conformational properties of protein drugs in relevant environments, they are typically focused on one particular aspect of higher order structure – for example, cumulative content of secondary structure, exposure of aromatic residues to solvent, and so on – and fail to characterise protein conformation in sufficient detail. Furthermore, these analytical methods provide characterisation across the entire ensemble of proteins and frequently fail to detect the presence of small populations of misfolded species on the background of natively folded biomolecules.

Mass Spectrometry-based Tools

Despite being a relatively recent addition to the biopharmaceutical analysis toolkit, biological MS has already become a default method for characterising the covalent structure of protein therapeutics (1,2). However, modern biological MS is capable of characterising structural properties of proteins well beyond sequencing and mapping post-translational modifications (PTM). Adaptation of MSbased techniques developed to probe protein conformation and dynamics to the specific needs of biopharmaceutical analysis will be a boon for the characterisation of biopharmaceutical products, hence the explosive growth of interest in using MS to probe conformation and dynamics of protein drugs in recent years.

Electrospray Ionisation Mass Spectrometry

Two MS-based techniques show particular promise as a means of probing conformational properties of protein therapeutics. These are hydrogen/deuterium exchange (HDX) with MS detection, and a suite of methods involving direct electrospray ionisation (ESI) MS analysis of biopolymers and their complexes in solution (3,4). The latter group of methods relies on the ability of ESI to transfer intact protein assemblies from solution to the gas phase, where their masses could be measured, revealing quaternary organisation of multi-unit proteins, presence of small-molecule ligands and co-factors, as well as the ability of proteins to interact with their receptors and other physiological partners or therapeutic targets. Another unique feature of ESI MS that is very useful in characterisation of biopolymers is multiple charging, which is sensitive to the biomolecule compactness in solution. Natively folded proteins undergo ESI to produce ions carrying a relatively small number of charges, because their compact shapes do not allow a significant number of charges to be accommodated on their surface. Conformers lacking a native structure have larger solvent-exposed surfaces and, therefore, give rise to ions carrying a significantly larger number of charges. Co-existence of native and non-native conformers results in bimodal distributions of protein ion charge in ESI MS. Dramatic changes of charge-state distributions in ESI MS signal the occurrence of large-scale conformational changes in protein drugs that may be triggered by extrinsic factors, such as solution composition, or non-enzymatic post-translational modifications, which may occur under stress conditions (see Figure 1A and Figure 1B, respectively).

The simplicity of the practical implementation of this technique and the ease of data analysis makes the protein ion charge state distribution analysis in ESI MS very appealing as a means of monitoring protein behaviour in solution. However, its acceptance within the biopharmaceutical industry was somewhat limited until recently due to the need to carry out all measurements in the socalled ‘ESI-friendly’ solvent systems. Since the presence of non-volatile electrolytes (which are present in all biopharmaceutical formulations) has a detrimental effect on the quality of ESI MS data, the need to transfer the protein to a volatile electrolyte solution (typically ammonium acetate or ammonium bicarbonate) often raises concerns that the observed protein behaviour may be partially attributed to the influence of the ‘foreign’ environment, rather than the intrinsic features of the biopharmaceutical product itself. Having said that, there is growing evidence, at least in the case of stressed protein therapeutics, that the detection of unfolding events by direct ESI MS is not affected by the solvent exchange step (3). Another shortcoming of the protein ion charge state distribution analysis as a means of probing conformations of protein is its low resolution, as it does not localise the unfolding events within specific protein segments.

Hydrogen/Deuterium Exchange Mass Spectrometry

All these shortcomings are overcome by HDX MS; a reliable, robust and sensitive technique, capable not only of detecting the presence of misfolded proteins on the background of the natively folded species in highly complex matrices, but also localising the unstructured/ misfolded protein segments. HDX provides information on conformational properties of proteins in solution by monitoring the exchange of their labile hydrogen atoms with the solvent. The labile hydrogen atoms in proteins comprise two distinct groups: those on polar and ionic side chains; and those attached to backbone amide nitrogen atoms. Without any protection from the solvent, all labile hydrogen atoms exchange with the solvent protons. The rates of these chemical reactions are determined by solution temperature and pH; the slowest chemical exchange for the backbone amide protons occurs between pH 2.5 and 3.0 and temperature close to 0oC. Such conditions are frequently referred to as ‘quenching’, although this term relates to only the backbone amides (labile hydrogen atoms located on side chains continue to exchange relatively fast under these conditions). While the exchange kinetics in short unstructured polypeptides is determined solely by the chemical exchange rates, the presence of a higher order structure alters it in a very significant way. Protons that are involved in hydrogen bonding or sequestered from solvent in the protein interior are not able to exchange with the solvent unless they become exposed to it through a local conformational fluctuation or more global unfolding events. Therefore, protection of any labile hydrogen atom (deviation of its exchange rate from the value dictated by the chemical exchange) is an indicator of its involvement in the higher order structure.

Placing a protein in D2O-based solution followed by MS analysis provides a straightforward means to monitor the progress of the exchange reactions. Even though each individual HDX exchange event results in a very modest mass increase (1.01 Da), this difference can easily be detected by most modern MS instruments even in the context of large proteins. While this method can provide information on protection of all labile hydrogen atoms, the most useful information is deduced from protection patterns of the backbone amides due to their intimate involvement in H-bonding networks that maintain the integrity of secondary structural elements. Therefore, contribution of the side chains to the overall exchange is usually eliminated by introducing the quenching step (vide supra) prior to MS measurements (see Figure 2).

 

Mass evolution of the intact protein undergoing HDX in solution provides information on its stability at the global level. At the same time, it is possible to obtain local information on the protein’s higher order structure and dynamics by carrying out proteolysis during the quenching step using acidic proteases, such as pepsin (see Figure 2). Several recent reports highlight the power of HDX MS as a tool providing detailed information on changes in conformation and dynamics of recombinant protein therapeutics caused by either non-enzymatic PTMs under stress conditions or engineered PTMs (5-8). In many cases, the analysis of HDX MS data not only allows the loss of conformational integrity within a pharmaceutical product to be easily detected and localised, but also the mechanism of ensuing loss of activity to be understood.

Conclusion

The MS-based methods of characterising protein conformation had been applied successfully in recent years to probe various aspects of higher order structure of large protein drugs; nevertheless, some challenges still remain. Perhaps the most formidable challenge is presented by the heterogeneity of many biopharmaceutical products (for example, due to extensive glycosylation or conjugation to a synthetic polymer). Ultimately, the success of MS-based tools as a means to probe conformation of recombinant therapeutic proteins will be determined by their adaptability to the specific needs of the biopharmaceutical industry. For example, reproducibility (both between laboratories and across the platforms) of these measurements is a critical issue that still needs to be addressed.

Although a significant amount of work will be required in the near future in order to address this and other questions, these efforts are well justified, since adoption of MS for the new role in the biopharmaceutical sector highlighted in this article will become a boon to the analytical characterisation and quality control. Furthermore, availability of these new tools will provide fresh impetus in other areas as well, for example by catalysing development of new proteinbased therapies.

References

  1. Zhang Z, Pan H and Chen X, Mass spectrometry for structural characterization of therapeutic antibodies, Mass Spectrom Rev 28(1): pp147-176, 2009
  2. Srebalus-Barnes CA and Lim A, Applications of mass spectrometry for the structural characterization of recombinant protein pharmaceuticals, Mass Spectrom Rev 26(3): pp370-388, 2007
  3. Kaltashov IA et al, Advances and challenges in analytical characterization of biotechnology products: Mass spectrometrybased approaches to study properties and behavior of protein therapeutics, Biotechnol Adv, in press, 2011
  4. Kaltashov IA et al, Conformation and dynamics of biopharmaceuticals: transition of mass spectrometrybased tools from academe to industry, J Am Soc Mass Spectrom 21(3): pp323-337, 2010
  5. Bobst CE et al, Impact of oxidation on protein therapeutics: conformational dynamics of intact and oxidized acid-β-glucocerebrosidase at nearphysiological pH, Protein Sci 19(12): pp2,366-2,378, 2010
  6. Bobst CE et al, Detection and characterization of altered conformations of protein pharmaceuticals using complementary mass spectrometry-based approaches, Anal Chem 80(19): pp7,473-7,481, 2008
  7. Burkitt W, Domann P and O’Connor G, Conformational changes in oxidatively stressed monoclonal antibodies studied by hydrogen exchange mass spectrometry, Protein Sci 19(4): pp826-835, 2010
  8. Houde D et al, Posttranslational modifications differentially affect IgG1 conformation and receptor binding, Mol Cel Proteom 9(8): pp1,716-1,728, 2010
  9. Bobst CE and Kaltashov IA, Advanced Mass Spectrometry-Based Methods for the Analysis of Conformational Integrity of Biopharmaceutical Products, Curr Pharm Biotechnol, in press, 2011

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Igor A Kaltashov received his undergraduate degree at Moscow Institute of Physics and Technology in 1989 and PhD from the University of Maryland, Baltimore in 1996. Following two years as a post-doctoral fellow at Johns Hopkins Medical School, he became a Director of the newly created Mass Spectrometry Center at the University of Massachusetts-Amherst in 1997. He was appointed an Assistant Professor in the Chemistry Department at UMass-Amherst in 2000, promoted to Associate Professor with tenure in 2006, and to Full Professor in 2011. He has co-authored over seventy papers and book chapters and a monograph entitled ‘Mass spectrometry in biophysics: conformation and dynamics of biomolecules’. Email: kaltashov@chem.umass.edu
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Igor A Kaltashov
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