The multi-attribute
method (MAM) is a liquid chromatography–mass
spectrometry based method that is used to directly characterize and
monitor many product quality attributes and impurities on biotherapeutics,
most commonly at the peptide level. It utilizes high-resolution accurate
mass spectral data which are analyzed in an automated fashion. MAM
is a promising approach that is intended to replace or supplement
several conventional assays with a single LC-MS analysis and can be
implemented in a Current Good Manufacturing Practice environment.
MAM provides accurate site-specific quantitation information on targeted
attributes and the nontargeted new peak detection function allows
to detect new peaks as impurities, modifications, or sequence variants
when comparing to a reference sample. The high resolution MAM workflow
was applied here for three independent case studies. First, to monitor
the behavior of monoclonal antibody product quality attributes over
the course of a 12-day cell culture experiment providing an insight
into the behavior and dynamics of product attributes throughout the
process. Second, the workflow was applied to test the purity and identity
of a product through analysis of samples spiked with host cell proteins.
Third, through the comparison of a drug product and a biosimilar with
known sequence variants. The three case studies presented here, clearly
demonstrate the robustness and accuracy of the MAM workflow that implies
suitability for deployment in the regulated environment.
Charge
sensitive separation methods such as ion exchange chromatography
(CEX) and capillary electrophoresis (CE) have recently been coupled
to mass spectrometry to facilitate high resolution profiling of proteoforms
present within the charge variant profile of complex biopharmaceuticals.
Here we apply pH gradient cation exchange chromatography and microfluidic
capillary electrophoresis using the ZipChip platform for comparative
characterization of the monoclonal antibody Cetuximab. Cetuximab harbors
four glycans per molecule, two on each heavy chain, of which the Fab
glycans have been reported to be complex and multiply sialylated.
The presence of these extra glycosylation sites in the variable region
of the molecule causes significant charge variant and glycan heterogeneity,
which makes comprehensive analysis on the intact protein level challenging.
Both pH gradient CEX-MS and CE-MS were found to be powerful for the
separation of Cetuximab charge variants with eight major peaks being
baseline resolved using both separation platforms. Informative native-like
mass spectra were collected for each charge variant peak using both
platforms that facilitated deconvolution and further analysis. The
total proteoform coverage was exceptionally high with >100 isoforms
identified and relatively quantified with CEX-MS, in case of CE-MS
the proteoform coverage was >200. A deep insight into the heterogeneity
of Cetuximab was unveiled, the high level of sensitivity achievable
enables the implementation of the presented technologies even at early
stages of the biopharmaceutical development platform, such as in developability
assessment, process development and also for monitoring process consistency.
Peptide mapping analysis is a regulatory expectation to verify the primary structure of a recombinant product sequence and to monitor post-translational modifications (PTMs). Although proteolytic digestion has been used for decades, it remains a labour-intensive procedure that can be challenging to accurately reproduce. Here, we describe a fast and reproducible protocol for protease digestion that is automated using immobilised trypsin on magnetic beads, which has been incorporated into an optimised peptide mapping workflow to show method transferability across laboratories. The complete workflow has the potential for use within a multi-attribute method (MAM) approach in drug development, production and QC laboratories. The sample preparation workflow is simple, ideally suited to inexperienced operators and has been extensively studied to show global applicability and robustness for mAbs by performing sample digestion and LC-MS analysis at four independent sites in Europe. LC-MS/MS along with database searching was used to characterise the protein and determine relevant product quality attributes (PQAs) for further testing. A list of relevant critical quality attributes (CQAs) was then established by creating a peptide workbook containing the specific mass-to-charge (m/z) ratios of the modified and unmodified peptides of the selected CQAs, to be monitored in a subsequent test using LC-MS analysis. Data is provided that shows robust digestion efficiency and low levels of protocol induced PTMs.
Monoclonal antibodies
(mAbs) and related products undergo a wide
range of modifications, many of which can often be directly associated
to culture conditions during upstream processing. Ideally, such conditions
should be monitored and fine-tuned based on real-time or close to
real-time information obtained by the assessment of the product quality
attribute (PQA) profile of the biopharmaceutical produced, which is
the fundamental idea of process analytical technology. Therefore,
methods that are simple, quick and robust, but sufficiently powerful,
to allow for the generation of a comprehensive picture of the PQA
profile of the protein of interest are required. A major obstacle
for the analysis of proteins directly from cultures is the presence
of impurities such as cell debris, host cell DNA, proteins and small-molecule
compounds, which usually requires a series of capture and polishing
steps using affinity and ion-exchange chromatography before characterization
can be attempted. In the current study, we demonstrate direct coupling
of protein A affinity chromatography with native mass spectrometry
(ProA-MS) for development of a robust method that can be used to generate
information on the PQA profile of mAbs and related products in as
little as 5 min. The developed method was applied to several samples
ranging in complexity and stability, such as simple and more complex
monoclonal antibodies, as well as cysteine-conjugated antibody–drug
conjugate mimics. Moreover, the method demonstrated suitability for
the analysis of protein amounts of <1 μg, which suggests
applicability during early-stage development activities.
Complete inline reduction of NISTmAb disulfide bonds using an electrochemical cell coupled to LC-MS analysis, reduction is carried out without the use of traditional reducing agents or enzymes.
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