Special focus issue y Protein therapeutics & target quantification by LC–MS

Editorial

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Progress in high-sensitivity hybrid LC–MS/MS

methods for the bioanalysis of protein drugs
and performance tests for their validation
Jeffrey X Duggan*,1
1
JXD Bioanalytics, Southbury, CT 06488, USA
*Author for correspondence: dugganjx@gmail.com

First draft submitted: 3 March 2018; Accepted for publication: 12 March 2018; Published online:
11 July 2018

Keywords: assay matrix effects • bioanalytical selectivity • bioanalytical validation • digestion efficiency • GXP
bioanalysis • hybrid bioanalysis • immunocapture • LC–MS bioanalysis of protein drugs • protein bioanalysis •
protein drug validation • regulated bioanalysis • sequential protein peptide immunocapture

Continuing evolution of protein LC–MS/MS bioanalytical methods

Modern protein therapeutics are being developed at a rapid rate. These drugs range from traditional monoclonal
antibodies to complicated multisubunit protein constructs. Many of the newer protein drugs exhibit increased
specificity and potency, thus requiring bioanalytical methods that are ever more selective and sensitive. In recent
years, there has been a large increase in the use of LC–MS/MS methods, where the drug is first subjected
to proteolytic digestion then quantified via LC–MS/MS of a unique surrogate peptide (PrD-LC–MS). Several
improved types of PrD-LC–MS methods have been developed over the past decade to address the bioanalytical
challenges presented by modern protein therapeutics.
As these methods have come online for regulated studies, they have been accompanied by new tests for validation
and performance verification. In this editorial, we will briefly discuss the evolution of some newer high-performance
PrD-LC–MS assay types and describe some of the validation techniques that have been or may potentially be applied
to them.

Reagent-free PrD-LC–MS methods

The first of these ‘reagent-free’ methods were discovered and implemented more than 10 years ago [1] and they
employ the direct proteolytic digestion of biotherapeutics in matrix followed by LC–MS/MS of the crude digest.
They have the advantage that they can be very quickly developed without the time-consuming production and
screening of the antibody capture and detection reagents required for ligand-binding assay (LBA) analysis. A further
advantage of these methods is that they are not highly susceptible to interference by antidrug antibodies (ADAs),
circulating ligands or other specific matrix elements that can interfere with either LBAs or hybrid LC–MS assays
utilizing immunocapture reagents. Reagent-free methods are also easily transferred between animal and human
matrices and they are generally considered to reflect the total drug concentration; but, with LLOQs typically
in the microgram per milliliter range, they often lack the sensitivity needed for many applications, particularly
high-sensitivity bioanalysis in clinical studies [1].

Hybrid PrD-LC–MS assays

Recently, PrD-LC–MS technology has been advanced by highly sensitive hybrid assays employing immunoaffinity
methods to capture the target protein therapeutic from the matrix and purify it prior to digestion and LC–
MS/MS quantification (IC PrD-LC–MS) [2] and IC PrD-LC–MS methods which are often called ‘hybrid’ LC–MS
assays because they share the capture mechanisms of an LBA method coupled to the detection technology of an
LC–MS/MS assay.

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Editorial Duggan

Combination Hybrid assays with sequential protein & peptide immunocapture

PrD- LC–MS methods of very high sensitivity and selectivity have been developed utilizing SISCAPA (Stable Isotope
Standards and Capture by Anti-Peptide Antibodies) [3] both with and without predigestion immunocapture [4,5].
In these methods antibody reagents are developed against the target peptide, and immunocapture of this peptide
is performed after proteolytic digestion, purifying it away from other components in the digest. Generally, post-
digestion peptide immunocapture methods with pre-digestion immunocapture (IC PrD-LC–MS SC) have been
found to provide very high selectivity and sensitivity with LLOQs sometimes in the pg range [3,4]. On the other
hand, methods utilizing peptide capture alone (PrD-LC–MS SC) have also been shown to be quite sensitive; and
they are considered indicative of total drug concentration, but with reported sensitivity similar to those of the
corresponding IC PrD-LC–MS assays [4].

Complex Hybrid PrD- LC–MS methods require specific validation techniques

All bioanalytical methods must be validated prior to use in regulated GxP studies, and the current US FDA or
EMA guidances have not yet provided specific references to PrD-LC–MS bioanalysis [6,7]; however, in the past 5
years, one major White Paper [8] and additional papers [9–12] have been published for the purpose of proposing
validation methodology and criteria for PrD-LC–MS methods. Because of the added complexity of PrD-LC–MS
methods, these papers recommended using the ‘20/25’ quantitative validation criteria now used for LBA assays
(where validation QCs and standards are acceptable if they are within 20% RSE and 20% CSV, with 25% for
these criteria at the LLOQ) rather than the ‘15/20’ rules currently set for small molecule validations. In addition
to the basic validation guidelines set out by the regulators, several other PrD-LC–MS-specific procedures have been
recommended in the recent White Paper and related articles [8–10]. Several of these are listed below.

Pre-validaton & validation evaluations

• Chromatographic selectivity experiments where both blanks and blanks spiked at the LLOQ for at least 10
individual matrices, patient and disease state matrices are evaluated where indicated. Acceptable selectivity
results are indicated by 8/10 samples quantifying within 25% of the nominal LLOQ concentration;
• Determination of the proteolytic digestion efficiency both for quantitative yield and for reproducibility;
• Selection of a second peptide to be monitored during the study to aid with in-study assay verification and in
some cases, to help verify the molecular integrity of the analyte protein.

Ongoing evaluations during sample GLP or GCP sample analysis

• Continual evaluation of critical reagents;
• Evaluation of assay performance in patient matrix;
• ISR evaluation of study samples using 2/3, 25% acceptance criteria.

Additional validation steps recommended for immunocapture & sequential immunocapture

PrD-LC–MS methods
The above validation and maintenance steps can help to ensure reliability for hybrid methods; but depending upon
the application, additional tests may be necessary to focus on critical parts of the assay. This can be especially
important in assays where there are sequential immunocapture assays, because these have two different critical
ligand-binding interactions. The most important of these will be discussed below.

Protein immunocapture methods

Hybrid methods, where a single immunocapture step is used to purify the analyte from matrix (IC PrD-LC–
MS), typically show increased sensitivity, but they are subject to some of the same potential matrix interferences
found with LBA methods. For these methods, conventional recovery and digestion efficiency experiments using
individual matrices and disease state matrix, where available, provide essential validation information. Extensive
selectivity/specificity testing using individual blanks is also recommended, using unspiked blanks paired with blanks
spiked at the LLOQ. This is a very sensitive way to detect inhibition of the assay, and it provides a chromatographic
record that may reveal any close-eluting isobaric MS/MS interferences [9–11]. These selectivity tests can also help to
detect matrix-related inhibition of the assay by comparing study matrices (disease state vs healthy), but they cannot
determine the underlying cause with certainty.

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 Progress in hybrid LC–MS/MS methods for protein drug bioanalytical methods & their validation Editorial

Recent studies have demonstrated that specific spike recovery experiments can help elucidate the nature of these
interferences [11,12]. One such experiment, called the immunocapture efficiency (ICE) experiment has been applied
to a IC PrD-LC–MS assay to help determine the effects of disease state vs normal matrices on the immunocapture
portion of the assay, comparing the relative amount of analyte measured after capture by the immunoaffinity
step as compared with that from a blank sample processed the same way but spiked with analyte downstream
from the immunocapture step [11]. In cases where competing ligands or other matrix components inhibit analyte
immunocapture, there will be a decrease in ICE. For a clinical study, ICE testing can be expanded to include disease
state individuals, pre-dose samples and blanks containing ADA if available [11].

Sequential protein & peptide immunocapture methods

A number of very sensitive methods have been recently documented wherein the protein analyte is first purified
from matrix by immunocapture then digested, and the resulting surrogate peptide is immunopurified from the
peptide digest using a solid-state technique (IC PrD-LC–MS SC) [3–5]. Because they utilize two different and very
specific immunocapture steps, these methods can have very high sensitivities, some reported in the picogram per ml
range [3]. For the peptide capture step, some of these methods use an immunoabsorptive column with immobilized
capture antibodies (SISCAPA, acronym defined above). After digestion, the sample is injected on to this column,
the column is washed to remove contaminants and then, using a valve switching system, the purified peptides are
eluted directly onto the analytical column for LC–MS/MS analysis [3].
A recent method by Chen et al. was developed for high-sensitivity measurement of an ocular protein drug in
plasma [4]. This assay differs from other IC PrD-LC–MS SC methods because it performs both the protein level and
the post digestion peptide immunocapture steps in 96-well plates using immunocapture reagents bound to magnetic
beads [4]. The processing of the magnetic beads for both stages of the assay is automated using a robotic bead-
handling system to increase precision and throughput. These authors have separately evaluated the ’sub component
assays’ of their sequential immunocapture assay: Protein level immunocapture only (IC PrD-LC–MS), peptide level
immunocapture only (PrD-LC–MS SC) and sequential protein level/peptide immunocapture (IC PrD-LC–MS
SC). They found that the sequential immunocapture assay, with an LLOQ of 50 pg/ml, was 100× more sensitive
than either the protein or peptide single immunocapture assays [4]. IC PrD-LC–MS SC assays with this level of
complexity require extensive characterization and thorough validation to be used reliably in regulated environments,
particularly clinical GCP studies. Much of the additional validation testing is the same as that for IC PrD-LC–
MS assays, such as extensive selectivity testing with individual matrix lots and immunocapture efficiency testing
with control and study-based matrices, as mentioned above. There are also spike-recovery experiments that can
potentially be applied to the peptide immunocapture step of these assays which are analogous to immunocapture
efficiency (ICE) for protein immunocapture [11]. These tests utilize post digestion peptide-spiked blanks prepared
from processed control and test (i.e., disease state or patient) matrices. Addition of a stable isotope labeled peptide
internal standard would take place after the peptide immunocapture step. This type of validation experiment can
provide valuable information on the impact of the test matrix on peptide capture and it may also be used for trouble
shooting in-study issues.
These IC PrD-LC–MS SC methods, with two immunocapture steps, require more reagent maintenance than
protein level IC assays. While the assays themselves provide ongoing holistic indicators of performance (standard
curves, QC performance, LLOQ reproducibility), reagent life cycle management per best practices is important
for both the protein and peptide level the immunocapture reagents in the same way as for LBA assay critical
reagents. These practices can include: producing a C of A, monitoring storage conditions, monitoring stability and
documenting lot-to-lot variability [13].

Conclusion
Great advances have been made in protein LC–MS bioanalytical assays in recent years. Some newly-developed IC
PrD-LC–MS hybrid assays can quantify protein analytes at ng/ml levels and some of the recent PrD-LC–MS IC
SC assays can measure pg/ml concentrations in plasma. Along with these dramatic gains comes the potential for
ligand-binding interferences to impact the assay’s performance. Developers are employing newer procedures with
the ability to specifically probe immunocapture steps in the presence and absence of specific matrices or matrix
components that may inhibit analyte ligand binding. Special care must be taken with IC PrD-LC–MS SC assays as
they contain two distinct ligand-binding steps using two different immunocapture reagents, each with the potential
for some form of matrix interference. In addition to thorough testing and validation of these high sensitivity

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Editorial Duggan

methods, attention needs to be paid to life-cycle management of the critical immunocapture reagents for both IC
PrD-LC–MS and IC PrD-LC–MS SC methods, governing their storage, stability, testing and documentation.
As hybrid PrD-LC–MS assays continue to grow in sensitivity, selectivity and complexity; new validation tech-
niques will be co-developed to help ensure reliable in-study performance. It is also expected that the continued
proliferation of hybrid PrD-LC–MS assays with established reliability will also be accompanied by official validation
references in upcoming regulatory guidances.

Acknowledgements
The author acknowledges the efforts of Lin-Zhi Chen, David Roos, Elsy Philip, Shirin Pagels, Shan Jiang, John Yu, Yan Mao, Bailuo
Ren, Jennifer Bleecker, Meghan Mimnaugh, David Dube, Nicole Riolo and Helen Luo of my former lab at Boehringer-Ingelheim
Pharmaceuticals, Ridgefield, CT for their pioneering contributions in developing, validating and applying new PrD-LC-MS methods.

Financial & competing interests disclosure

The author has no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial
conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock
ownership or options, expert testimony, grants or patents received or pending, or royalties.
No writing assistance was utilized in the production of this manuscript.

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