THE DELAWARE VALLEY DRUG METABOLISM DISCUSSION GROUP
A Symposium on Mass Spectrometric Analysis of Protein Drugs (18April2012)
Presentation Abstracts
Ian Blair
Relative Quantification of Serum Proteins in Pancreatic Cancer by Stable Isotope
Dilution LC-MS
Many methods have been developed to characterize differential proteome expression.
Application of these methods has generated vast lists of candidate biomarker proteins
which await quantification in human samples for validation. Enzyme-linked
immunosorbent assay is the most common method used for the quantification of proteins
in serum due to its high sensitivity and specificity. However, this method becomes
expensive and labor intensive when applied to a large number of proteins and samples. An
innovative multiplexed liquid chromatography-multiple reaction monitoring/mass
spectrometry (LC-MRM/MS)-based assay has been developed for rapidly measuring a
large number of disease specific protein biomarkers in human serum. Furthermore, this
approach uses stable isotope dilution methodology to reliably quantify candidate protein
biomarkers. Human serum was diluted using a stable isotope labeled proteome (SILAP)
standard prepared from the secretome of pancreatic cell lines, subjected to immunoaffinity
removal of the most highly abundant proteins, trypsin digested, and analyzed by
LC-MRM/MS. The method was found to be precise, linear, and specific for the relative
quantification of 72 proteins when analyte response was normalized to the relevant internal
standard (IS) from the SILAP. The method made it possible to determine statistically
different concentrations for three proteins (cystatin M, IGF binding protein 7, and villin 2)
in control and pancreatic cancer patient samples. This method proves the feasibility of
using a SILAP standard in combination with stable isotope dilution LC-MRM/MS analysis
of tryptic peptides to compare changes in the concentration of candidate protein
biomarkers in human serum. Supported by NIH grants R25CA101871, P30ES013508.
Daryl Davis
Mass Spectrometry in a Biopharmaceutical Setting; Qualitative and Quantitative
Case Studies of the Application of Bioanalytical MS
Mass spectrometry as a characterization tool has matured alongside the biotechnology
industry. The mass spectrometry techniques in use today are able to tell us much about the
proteins we make but there are still gaps in our knowledge. This presentation focuses on
detailing past, current and proposed future MS techniques for characterizing proteins as
applied in a production setting.
The aim is to answer the following: Why do we perform the assays we do? What are the
ideal attributes for a protein characterization method? Does it matter when and where the
characterization occurs? What is the smallest number of assays one could use to monitor
quality? Do we have enough orthogonality built in to our testing scheme?
A Retrospective of 15 years of Using Mass Spectrometry to Answer Questions of a
Biological Nature.
Steve Dueker
Quantitative Proteomics Using 14C-Accelerator Mass Spectrometry
Accelerator Mass Spectrometry (AMS) is a mass spectrometric method that quantifies 14C
for high specificity but uses mass spectrometry for high sensitivity. Originally applied to
geochronology, it is now a common tool for human microdosing, low radioactive human
(and animal) ADME, and the tracing of endogenous molecules in biochemistry, nutrition,
and pediatric research. More recently, it has found application to the very difficult
bioanalytical area of protein and 'biologics' quantitation. The AMS measurement is wellsuited for this task as the measurement process differs from other analytical methods in
very important ways: 1) it is independent of molecular structure; 2) it is not susceptible to
matrix interferences (non-specific binding in ligand binding assays, ion suppression in
mass spectrometry); and 3) quantitation is absolute, not comparative, and proceeds without
resorting to compound-specific internal or external standards. The merging of Mass
Spectrometry for identification and AMS for quantitation will enable studies into the tissue
binding, fate and disposition of biologics in ways not easily achieved by other techniques.
This talk will present both the challenges as well as opportunities for AMS as applied to
Biologics research. A short background on the principles of AMS operation, sample
preparation, and appropriate isotopic labeling methods will begin the presentation.
Mohammed Jemal
Universal Surrogate Peptide Concept and Other Recent Insights in Therapeutic
Protein Quantitative Bioanalysis by LC-MS/MS
The presentation will deal with the identification and evaluation of a universal surrogate
peptide found in the Fc region of human monoclonal antibody or Fc-fusion protein drug
candidates. Accordingly, the universal peptide can be used for the LC-MS/MS bioanalysis
of a variety of such protein drug candidates in animal pharmacokinetic studies. The
presentation will also include the concepts of a dual universal peptide and the concept of a
universal reagent for immunoaffinity LC-MS/MS. Other recent insights will also be
discussed, including the technique of pellet digestion, trypsin digestion optimization,
process facilitation and cost reduction.
Hongyan Li
LC-MS/MS Method Approach to Quantify Therapeutic Monoclonal Antibodies
Using a Common Whole Antibody Internal Standard with Application to Preclinical
Studies (H. Li, R. Ortiz, L.T.B. Tran, C.A. James and J.W. Lee)
Monoclonal antibodies (mAbs) are now the leading class of biotherapeutics. High
throughput quantitative analysis of mAbs in biological fluids is crucial to provide
pharmacokinetic data for exposure/pharmacodynamic and toxicity relationships at early
stage of preclinical studies. Ligand binding assays (LBAs) such as enzyme-linked
immunosorbent assays (ELISA) are widely used for quantitative determination of mAbs in
biological matrices. Major limitations are long method development times, reagent
procurement, and matrix effects. LC-MS/MS methods using surrogate peptides are
emerging as an alternative approach to overcome those limitations, and when it is
implemented with immunoaffinity capture, it can provide quantitation limit similar to that
of ligand binding assays. However, multiple analytical steps are required in order to
produce appropriate surrogate peptides of the target mAb from biological sample prior to
LC-MS/MS quantification; thus, internal standard choice, surrogate peptide selection and
analytical workflows are critical for assay consistency and throughput. We have
developed an immunoaffinity-LC-MS/MS approach employing a whole antibody
uniformly labeled with [15N, 13C6]-Leu (~95% isotopic purity) as a common internal
standard. Efficient workflows using immunoaffinity capture for sample processing have
been developed and streamlined with automation. Method qualification of 4 IgG2 mAbs
and 4 IgG1 mAbs using common surrogate peptides from FC region of mAbs showed
lower limit of quantitation (LLOQ) of 0.1 µg/mL and linearity of 0.1-15 µg/mL; quality
control (QC) data of these 8 mAbs were accurate and precise. A multiplexed selected
reaction monitoring (SRM) method using unique surrogate peptides from CDRs of each
antibody was also developed and qualified to allow applications for cassette dosing in
preclinical species. The pharmacokinetic results of mAbs dosed in rats were comparable
to those of LBA.
Rand Jenkins
Hybrid Ligand Binding-LC/MS Bioanalytical Methods: How Well do They Work
and What are Some Considerations?
The importance of measuring macromolecule analytes, including potential therapeutic
drugs, receptor targets, and biomarkers, is rapidly growing within the pharmaceutical
industry. Large molecules as a category–primarily composed of polypeptides–are also
becoming increasingly diverse, ranging from simple peptides to complex conjugates and
fusion proteins. Bioanalysis of these compounds has traditionally been carried out using
indirect ligand-binding assays (LBAs), which rely on a sequence of specific protein
binding interactions with critical reagents to ultimately generate a measurable signal that is
proportional to the analyte concentration in a sample. While these approaches provide
very high sensitivity and good assay throughput, they are prone to various interference
issues that can impact assay accuracy and performance. Recent advances in LC-MS
instrumentation and applications such as proteomics are now being applied to supplement
and in some cases replace LBAs for bioanalysis of large molecules where a more direct
physicochemical-based detection approach offers significant advantages. As a general
rule, smaller macromolecules (up to ca. 10 kDa) may be analyzed intact, while larger
proteins are typically subjected to proteolysis (either before or after isolation) to produce
characteristic ‘signature’ peptide fragments that are more readily analyzed.
Depending on the assay needs, with respect to required sensitivity and specificity, LC-MSbased methods can employ a wide range of sample preparation techniques, including direct
matrix dilution, protein precipitation, SPE, and immunoprecipitation (IP). The use of IP
techniques for analyte purification can be especially powerful when the maximum assay
sensitivity and/or specificity are needed. With appropriate choice of reagents and
processing conditions, up to 1000-fold analyte enrichment is possible using relatively
simple and reproducible procedures. In addition, the use of IP procedures can eliminate
common matrix-related sources of ion suppression, such as salts and lipids, thereby
improving overall electrospray ionization MS sensitivity. Use of certain IP procedures
may also provide support that the measured analyte has retained its structure (e.g., IP
capture of a mAb based on Fc region binding and detection based on CDR region tryptic
peptides) and possibly even its actual biological activity.
Considerations and precautions to successfully develop and apply IP-based techniques will
be covered in this presentation, including:
 Specific vs. generic capture reagents (e.g. anti-idiotypic Abs, Protein A/G)
 Media and capacity (e.g. magnetic vs. agarose beads, plates)
 Binding conditions/time
 Potential interference issues
Kevin Meyer
Automation of Protein Sample Preparation for Mass Spectrometry
There are two driving forces behind the surge in mass spectrometry based protein
analyses: i) the time and cost associated with developing an enzyme linked immunoassay
and ii) increased awareness of the role isoform variations and post-translational
modifications play in protein function and activity. The utility of LC/MS is mitigated by
the reality that analysts are rarely presented with a sample in a suitable form for injection
and there are few robust alternatives to time consuming benchtop methods.
As an alternative to conventional methods of mass spec sample preparation this
presentation will teach the details of a high throughput, column based, automated
approach. The use of affinity selection, immobilized enzymes, on-line desalting and
integrated reverse phase separation will be described at length and their limitations
balanced in regards to their utility.