March 1, 2013
Biological product and process characterization are not new to this quality by design (QbD) and process analytical technology (PAT) era. In the 1990s we saw the FDA introduce the concept of well-characterized biologics: an acknowledgment that analytical technology had advanced to the point where the bioprocess did not necessarily (or not fully, anyway) define a biopharmaceutical product. That ultimately led to the regulation of some types of products within the United States moving from the purview of FDA’s Center for Biologics Evaluation and Research (CBER) to its Center for Drug Evaluation and Research (CDER). Now some biopharmaceuticals go to market through the biologics license application (BLA) process whereas others use the new drug application (NDA) instead. While the regulators were adjusting to this new scientific reality, suppliers of analytical equipment continued to improve their instruments, making it ever more possible for bioprocess engineers to build quality into their manufacturing processes by way of PAT and design space definition — as well as ever more precision in product characterization.
I have described chromatography’s role in biopharmaceutical analysis elsewhere (1). But in my anniversary-issue discussions with our advisors last year, I learned just how important mass spectrometry (MS) has become as a complementary technology (2). Adriana Manzi elaborated in a sidebar that I felt was worth reprinting here (see the “Advances in MS” box). MS is one of several related analytical methods that include ion-mobility, Rutherford backscattering, neutron triple-axis, and optical spectrometry. For biopharmaceutical laboratories, however, MS is most important.
By measuring the mass/charge ratio of ionized particles, MS can be used to determine their masses, elemental composition, and chemical structures. Typically, a loaded sample undergoes vaporization, then ionization by one of several methods. The resulting ions are separated according to their mass/charge ratio and then detected. The resulting signal is processed into a mass spectrum for analysis. MS instruments are available from companies such as Agilent Technology, Bruker Corporation, Hitachi Instruments, MDS Inc., Perkin Elmer (PE Biosystems), Shimadzu Scientific Instruments, Thermo Scientific, Varian Inc., and Waters Corporation. Most of those companies also provide high-performance liquid chromatography (HPLC) instrumentation — often combining both technologies into one system.
A Brief History
Early “mass spectrograph” devices measured the mass/charge ratio of ions by recording a spectrum of mass values on a photographic plate. Later “mass spectroscopes” focused their ion beams onto phosphor screens. Modern MS can be traced to 1897 cathode-ray tube experiments by J.J. Thomson in Manchester, England (3). In 1886, Polish physicist Eugen Goldstein had observed in work at his Berlin University laboratory that positively charged rays traveled away from an anode and through channels in a perforated cathode with low-pressure gas discharges. Goldstein called them Kanalstrahlen, “canal rays” (4). About a decade later German physicist Wilhelm Wien found that strong electric or magnetic fields deflected those rays. He invented a way to separate them according to the charge/mass ratio (Q/m). Thomson later improved on that work to create a mass spectrograph.
MS was first applied to analyzing amino acids and peptides in the 1950s, especially in Germany. Wolfgang Paul’s 1953 invention of the quadrupole ion trap ultimately earned him the Nobel Prize in physics for 1989, shared with Hans Dehmelt, who developed the technique further in the 1960s. American chemist Malcolm Dole’s development of electrospray ionization (ESI) was largely ignored in 1968 but has since become very useful. At first, creating an aerosol in a vacuum produced a vapor that was considered impractical because of the 100×–1,000× volume increase (4). But Marvin Vestal and Cal Blakely’s 1983 MS work with liquid-stream heating (thermospray) made today’s commercial instruments possible. And Dole was finally vindicated when American chemist John Fenn’s follow-up ESI work earned a Nobel Prize in chemistry for 2002. He shared that honor with Japanese chemist Koichi Tanaka (who developed soft-laser desorption, SLD) for their applications related to biological macromolecules, especially proteins (5). About the same time, German scientists Franz Hillenkamp and Michael Karas developed a method that they named matrix-assisted laser desorption/ionization (MALDI), which was later used to ionize proteins.
Advances in Mass Spectrometry by Adriana Manzi
When I think of the past decade in analytical chemistry, I think about how advances in MS — not one instrument or one method, but multiple aspects of technology in combination with innovation — have had a great impact not only for researchers, but also for the pharma and biopharma sectors overall. The advent and development of soft ionization techniques enable extension of MS methods to large molecules and molecular complexes. Advances include
high mass-resolving power and the ability to carry out several stages of mass selection and activation
use of tandem MS, which implies that the activation of ions is distinct from the ionization step and that the precursor and product ions are both characterized independently by their mass/charge ratios
the advent of time-of-flight (TOF), tandem quadrupole, and ion-trapping instruments
collisional activation with the impact of precursor ions on solid surfaces, surface-induced dissociation (SID), and electron-capture dissociation (ECD), enabling analysis of unique fragmentation mechanisms of multiply charged species to study noncovalent interactions and gain sequence information in proteomics applications
trapping instruments, such as quadrupole ion traps and Fourier-transform ion-cyclotron resonance (FT-ICR) MS FT-ICR instruments
imaging MS (IMS) with its unparalleled capabilities to provide chemical analysis of intact tissue.
Biological applications of mass spectrometry have grown exponentially since the discovery of matrix-assisted laser-desorption ionization (MALDI) and electrospray ionization techniques. Single-cell analysis is gaining popularity in the MS field as a method for analyzing protein and peptide content in cells. The spatial resolution of MALDI MS imaging is largely limited by the laser-focal diameter and displacement of analytes during matrix deposition. Because of recent advancements in both laser optics and matrix deposition methods, spatial resolution of a single eukaryotic cell is now achievable.
MALDI imaging MS (IMS) can measure the distribution of hundreds of analytes at once and has great potential in the field of tissue-based research. An ambitious goal is to monitor the level and modification of all proteins and metabolites in a biological sample (e.g., plasma), and MS is poised to enable this dream in the next few years.
Adriana Manzi
is managing director of Atheln, Inc., 6051 Scripps Street, San Diego, CA 92122; 1-858-554-0636, fax 1-858-405-4584; [email protected]. She is also a member of BPI’s editorial advisory board.
How MS Works: As implied above, a mass spectrometer’s ionizer determines the kind of work it can do. Electron and chemical ionization are used for gases and vapors. ESI and MALDI techniques are commonly used for liquid and solid biological samples. Inductively coupled plasma (ICP) sources are used for cation analysis. Mass analyzers separate the resulting ions according to their mass/charge ratio using static or dynamic, magnetic or electrical fields. Each analyzer type has its own strengths and weaknesses, and some modern mass spectrometers address those by using two or more analyzers in tandem (MS/MS). Here are some familiar combinations used in biopharmaceutical laboratories:
MALDI-TOF MS combines a MALDI ionizer with a time-of-flight (TOF) analyzer that electrically accelerates ions and measures the time they take to reach a detector. If the particles all have the same charge, their kinetic energies will be identical so that their velocity depends on mass alone (lighter ions reach a detector first).
LC–MS/MS systems combine high-performance liquid chromatography (HPLC) with tandem-quadrupole mass analysis. HPLC separates compounds chromatographically before they are introduced to a mass spectrometer, often by way of an ESI source. In the triple-quadrupole variation, one quadrupole acts as a mass filter that transmits incoming ions to a second quadrupole, which breaks them into fragments, and the third filters those fragments to a detector. Quadrupole ion traps work similarly but trap the ions and sequentially eject them — allowing multiple rounds of measurement with only one analyzer. Tandem mass spectrometers use multiple analyzers to do so.
Some Spectrometry from the BPI Archives at www.bioprocessintl.com
BPSA Extractables and Leachables Subcommittee. Recommendations for Extractables and Leachables Testing. BioProcess Int. 6(1) 2008: 44–52.
Mørtz E, et al. Proteomics Technology Applied to Upstream and Downstream Process Development of a Protein Vaccine. BioProcess Int. 6(1) 2008: 36–43.
Whitford W, Julien C. The Biopharmaceutical Industry’s New Operating Paradigm. BioProcess Int. 6(3) 2008: S6–S14.
Whitford W, Julien C. Appendix 1: Designing for Process Robustness. BioProcess Int. 6(3) 2008: S34–S44.
Seamans TC. Cell Cultivation Process Transfer and Scale-Up. BioProcess Int. 6(3–4) 2008: 26–36; 34–42.
Aschermann K, Lutter P, Wattenberg A. Current Status of Protein Quantification Technologies. BioProcess Int. 6(4) 2008: 44–53.
Krishnamurthy R, et al. Emerging Analytical Technologies for Biotherapeutics Development. BioProcess Int. 6(5) 2008: 32–42.
BPSA Extractables and Leachables Subcommittee. Recommendations for Extractables and Leachables Testing. BioProcess Int. 6(5) 2008: S28–S39.
Coco-Martin JM, Harmsen MM. A Review of Therapeutic Protein Expression By Mammalian Cells. BioProcess Int. 6(6) 2008: S28–S33.
Manzi AE. Carbohydrates and Their Analysis, Part Three. BioProcess Int. 6(6) 2008: 54–65.
Chakraborty AB. Rapid Profiling of Reduced and Intact Monoclonal Antibodies By LC/ESI-TOF MS. BioProcess Int. 6(7) 2008: 128.
Baker K. Purification of a Monoclonal Antibody Using a Combination of UNOsphere™ Rapid S and UNOsphere Q Media Ion Exchange Chromatography After Protein A Capture. BioProcess Int. 6(7) 2008: 88–89.
Richert S, et al. “Combine All Files” Maps. BioProcess Int. 6(9) 2008: 62–71.
Ding W, Martin J. Implementing Single-Use Technology in Biopharmaceutical Manufacturing. BioProcess Int. 6(9) 2008: 34–42.
Schmidt J, et al. Monitoring ATP Status in the Metabolism of Production Cell Lines. BioProcess Int. 6(11) 2008: 46–54.
Bestwick D, Colton R. Extractables and Leachables from Single-Use Disposables. BioProcess Int. 7(2) 2009: S88–S94.
Viebahn M. Encyclopedia of Rapid Microbiological Methods. BioProcess Int. 7(4) 2009: 60.
Ding W, Martin J. Implementation of Single-Use Technology in Biopharmaceutical Manufacturing. BioProcess Int. 7(5) 2009: 46–51.
Wanninger I, Artz C. A New Solution for Endotoxin Removal in Biomanufacturing Processes. BioProcess Int. 7(7) 2009: 87.
Fenge C. A One-Stop, Full-Service Provider. BioProcess Int. 7(7) 2009: 21.
Kauffman JS. Analytical Strategies for Monitoring Residual Impurities Encountered in Bioprocessing. BioProcess Int. 7(7) 2009: 40–42.
Paul WC, et al. Maintaining Product Titer While Replacing Undefined Components in a CHO Culture System. BioProcess Int. 7(8) 2009: 30–38.
McLeod LD. Manufacturing Efficiency and Supply Chain Security. BioProcess Int. 7(8) 2009: S10–S19.
Mücke M, Ostendorp R, Leonhartsberger S. E. coli Secretion Technologies Enable Production of High Yields of Active Human Antibody Fragments. BioProcess Int. 7(8) 2009: 40–47.
Scott C, McLeod LD, Montgomery SA. Putting All the Pieces Together. BioProcess Int. 7(9) 2009: S36–S44.
Lye G, et al. Shrinking the Costs of Bioprocess Development. BioProcess Int. 7(9) 2009: S18–S22.
Scott C, McLeod LD. Managing the Product Pipeline. BioProcess Int. 8(3) 2010: S6–S23.
Nims RW, Meyers E. Contractee Responsibilities in Outsourced Pharmaceutical Quality Control Testing. BioProcess Int. 8(5) 2010: 26–33
Schwertner D, Kirchner M. Are Generic HCP Assays Outdated? BioProcess Int. 8(5) 2010: 56–62.
Lehman T. Lancaster Labs Develops Extractables Database for LC/MS Analysis. BioProcess Int. 8(7) 2010: 46.
Clarke H. Rapid Cell Line Development with Integrated Protein Analysis. BioProcess Int. 8(7) 2010: 36–37.
Strahlendorf K, Harper K. Mixing in Small-Scale Single-Use Systems. BioProcess Int. 8(10) 2010: 42–49.
Ding W, Martin J. Implementation of Single-Use Technology in Biopharmaceutical Manufacturing. BioProcess Int. 8(10) 2010: 52–61.
Scott C. Protein Conjugates. BioProcess Int. 8(10) 2010: 28–37.
Hoffman K. Letter to the Editor. BioProcess Int. 8(11) 2010: 8–10.
Wei Z, et al. The Role of Higher-Order Structure in Defining Biopharmaceutical Quality. BioProcess Int. 9(4) 2011: 58–66.
Scott C. Quality By Design and the New Process Validation Guidance. BioProcess Int. 9(5) 2011: 14–21.
Siemiatkoski J, et al. Glycosylation of Therapeutic Proteins. BioProcess Int. 9(6) 2011: 48–53.
Bunch RP. Increase Efficiency and Accelerate Protein Workflows with the LabChip GXII. BioProcess Int. 9(7) 2011: 140–142.
Clarke H. Rapid Cell Line Development with Integrated Protein Analysis. BioProcess Int. 9(7) 2011: 38–39.
Squires CH. Pseudomonas fluorescens Expression Technology for Subunit Vaccine Production and Development. BioProcess Int. 9(8) 2011: S36–S39.
Harris J, et al. Uniting Small Molecule and Biologic Drug Perspectives. BioProcess Int. 9(8) 2011: 12–22.
Dimasi L. Meeting Increased Demands on Cell-Based Processes By Using Defined Media Supplements. BioProcess Int. 9(8) 2011: 48–58.
Michels DA, Salas-Solano O, Felten C. Imaged Capillary Isoelectric Focusing for Charge-Variant Analysis of Biopharmaceuticals. BioProcess Int. 9(10) 2011: 48–54.
Liu N, et al. Host Cellular Protein Quantification. BioProc
ess Int. 10(2) 2012: 44–50.
Aranha H. Current Issues in Assuring Virological Safety of Biopharmaceuticals. BioProcess Int. 10(3) 2012: 12–17.
Scott C. A Decade of Characterization. BioProcess Int. 10(6) 2012: S58–S61.
Scott C. A Decade of Formulation. BioProcess Int. 10(6) 2012: S48–S53.
Mire-Sluis A, Kutza J, Frazier-Jessen M. Rapid Pharmaceutical Product Development. BioProcess Int. 10(11) 2012: 12–21.
Applications
Tandem mass spectrometry enables many experimental strategies that increase the specificity of detection for known and unknown molecules. Using HPLC as a form of “sample preparation” makes the mass resolving and determining capabilities of MS even more powerful. The LC–MS combination is used to study the physical, chemical, and biological properties of biomolecules, raw materials, and process intermediates. In addition to proteomics and metabolomics, it has utility throughout biopharmaceutical development: peptide and glycoprotein mapping; bioaffinity screening; in vivo drug screening and metabolic stability studies; identification of metabolites, impurities, and degradants; quantitative bioanalysis; product characterization; and quality control (6).
Pharmacokinetics studies use MS to make sense of complex samples (e.g., blood and urine) with high sensitivity for drug dosage and clearance information. HPLC–MS/MS is most commonly used in this application with triple-quadrupole mass spectrometers (4). Some scientists are beginning to use high-sensitivity MS for microdosing studies as an alternative to testing on animals.
Protein Characterization: MS is a powerful method for characterizing and sequencing proteins, typically using ESI and MALDI ionizers. Intact proteins can be ionized by either method and then introduced to a mass analyzer (a “top-down” strategy) or enzymatically digested first using proteases such as trypsin or pepsin (in a “bottom-up” approach). The resulting peptides are then loaded onto a mass analyzer, and a characteristic pattern of them can be used to identify proteins in peptide mass fingerprinting (PMF).
Glycan Analysis: Because it works with small-volume samples and offers high sensitivity, MS is used for characterizing and elucidating the glycan structures of glycosylated proteins, especially with the help of HPLC. Intact glycans may be detected directly as singly charged ions by MALDI. Permethylation or peracetylation can fragment them for fast-atom bombardment (FAB-MS). ESI works well for smaller glycans.
Getting Together
If you want to meet with other users to discuss the finer points of MS analysis, the first place to go is the website of the American Society for Mass Spectrometry (ASMS): www.asms.org. In addition to online forums and special committees for the membership, this organization offers many in-person meeting formats. Its 61st annual conference will be in Minneapolis, MN, 9–13 June 2013.
The international separation science society known as CASSS (formerly the California Separation Science Society) has an annual “Mass Spec” event as well, which will be 23–26 September 2013 in Boston, MA. Find more information online here: https://m360.casss.org/event.aspx?eventID=60846.
Outside the United States, you might check out “Mass Spec 2013: New Horizons in MS Hyphenated Techniques and Analysis” at the Biopolis in Singapore (6–7 March 2013) or “Inn Mass Spec 2013” in St. Petersburg, Russia (14–18 July 2013). More properly the Conference on Innovations in Mass Spectrometry Instrumentation, the latter is primarily for developers of the MS technology itself (www.sepscience.com/Information/Events/Conferences/Mass-Spec-2013), whereas the former is more about its applications (www.sepscience.com/Information/Events/Conferences/Mass-Spec-2013).
MS technology supplier AB Sciex of Framingham, MA, offers seminars and webinars. Check out the 8–9 April 2013 LC/MS/MS Symposium in Berlin (www.absciex.com/events/conferences/5-berliner-lcmsms-symposium). And many other events are less specific to MS but will feature it heavily. Examples include HPLC-2013 in Amsterdam, The Netherlands, on 16–20 June 2013 (www.hplc2013.org/Introduction.html) and the 40th International Symposium on High Performance Liquid Phase Separations and Related Topics in Hobart, Australia, on 18–21 November 2013.
About the Author
Author Details
Cheryl Scott is cofounder and senior technical editor of BioProcess International, 1574 Coburg Road #242, Eugene, OR 97401; 1-646-957-8879; [email protected].
1.) Scott, C. 2005. Chapter 2: Chromatography’s Role in Biotech Analysis. BioProcess Int. 3:S18-S28.
2.) Scott, C. 2012. A Decade of Characterization. BioProcess Int. 10:S58-S61.
3.) 2013. MS: Mass Spectrometry, Waters Corporation, Milford.
4.) Mass Spectrometry 2013. Wikipedia.
5.) 2012. An Overview of MS History, Scripps Center for Metabolomics and Mass Spectrometry, La Jolla.
6.) Liquid Chromatography–Mass Spectrometry 2012. Wikipedia.
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