Rapid characterization of <i>N</i>‐linked glycans from secreted and gel‐purified monoclonal antibodies using MALDI‐ToF mass spectrometry

Hansen, Rasmus; Dickson, Alan J.; Goodacre, Royston; Stephens, Gill; Sellick, Christopher A.

doi:10.1002/bit.22879

Cited by 16 publications

(12 citation statements)

References 26 publications

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“…The analysis of cleaning eluents by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) and 2D-PAGE can provide details of the chemical profile of the fouling contaminants 28 41 42 . Unfortunately, mobile phase analysis does not reveal bound fouling contaminants while preserving the resin intact.…”

mentioning

confidence: 99%

In-column ATR-FTIR spectroscopy to monitor affinity chromatography purification of monoclonal antibodies

Boulet-Audet

Kazarian

Byrne

2016

Sci Rep

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In recent years many monoclonal antibodies (mAb) have entered the biotherapeutics market, offering new treatments for chronic and life-threatening diseases. Protein A resin captures monoclonal antibody (mAb) effectively, but the binding capacity decays over repeated purification cycles. On an industrial scale, replacing fouled Protein A affinity chromatography resin accounts for a large proportion of the raw material cost. Cleaning-in-place (CIP) procedures were developed to extend Protein A resin lifespan, but chromatograms cannot reliably quantify any remaining contaminants over repeated cycles. To study resin fouling in situ, we coupled affinity chromatography and Fourier transform infrared (FTIR) spectroscopy for the first time, by embedding an attenuated total reflection (ATR) sensor inside a micro-scale column while measuring the UV 280 nm and conductivity. Our approach quantified the in-column protein concentration in the resin bed and determined protein conformation. Our results show that Protein A ligand leached during CIP. We also found that host cell proteins bound to the Protein A resin even more strongly than mAbs and that typical CIP conditions do not remove all fouling contaminants. The insights derived from in-column ATR-FTIR spectroscopic monitoring could contribute to mAb purification quality assurance as well as guide the development of more effective CIP conditions to optimise resin lifespan.

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mentioning

confidence: 99%

In-column ATR-FTIR spectroscopy to monitor affinity chromatography purification of monoclonal antibodies

Boulet-Audet

Kazarian

Byrne

2016

Sci Rep

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“…Similarly, specific PTM analysis may involve an enzymatic digestion that, in turn, may lead to the loss of PTMs (e.g., carbohydrates) prior to MS and MS/MS analysis. 5 Prolonged digestion at relatively high (37 °C) temperature, which is required for complete enzymatic action, can produce the artifacts and, particularly for IgGs, lead to an increased rate of deamidation and oxidation, preventing the correct identification of certain PTMs, such as Asp isomerization. 2 Top-down mass spectrometry refers to mass spectrometric monitoring of the gas-phase fragmentation of biomolecular ions of any size.…”

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confidence: 99%

Structural Analysis of Intact Monoclonal Antibodies by Electron Transfer Dissociation Mass Spectrometry

et al. 2011

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Improving qualitative and quantitative characterization of monoclonal antibodies is essential, because of their increasing popularity as therapeutic drug targets. Electron transfer dissociation (ETD)-based top-down mass spectrometry (MS) is the method of choice for in-depth characterization of post-translationally modified large peptides, small- and medium-sized proteins, and noncovalent protein complexes. Here, we describe the performance of ETD-based top-down mass spectrometry for structural analysis of intact 150 kDa monoclonal antibodies, immunoglobulins G (IgGs). Simultaneous mass analysis of intact IgGs as well as a complex mixture of ETD product ions at sufficiently high resolution and mass accuracy in a wide m/z range became possible because of recent advances in state-of-the-art time-of-flight (TOF) mass spectrometry. High-resolution ETD TOF MS performed on IgG1-kappa from murine myeloma cells and human anti-Rhesus D IgG1 resulted in extensive sequence coverage of both light and heavy chains of IgGs and revealed information on their variable domains. Results are superior and complementary to those previously generated by collision-induced dissociation. However, numerous disulfide bonds drastically reduce the efficiency of top-down ETD fragmentation within the protected sequence regions, leaving glycosylation uncharacterized. Further increases in the experiment sensitivity and improvement of ion activation before and after ETD reaction are needed to target S-S bond-protected sequence regions and post-translational modifications.

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“…The time required for all the aforementioned tools to measure the performance loss and the feasibility of its online monitoring is highlighted in Table . Among the different tools, fluorescence is the only one that can be used for online monitoring of resin fouling.…”

Section: Resultsmentioning

confidence: 99%

Analytical tools for monitoring changes in physical and chemical properties of chromatography resin upon reuse

et al. 2019

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Protein A resins are often reused for multiple cycles to improve process economy during mAb purification. Significant reduction in binding capacity and product recovery are typically observed due to the presence of unwanted materials (foulants) deposited on the resin upon reuse. In this paper, we have used a wide spectrum of qualitative and quantitative analytical tools (particle size analysis, HPLC, fluorescence, SEM, MS, and FTIR) to compare the strengths and shortcomings of different analytical tools in terms of their capability to detect the fouling of the resin and relate it to chromatographic cycle performance. While each tool offers an insight into this complex phenomena, fluorescence is the only one that can be used for real‐time monitoring of resin fouling. A correlation could be established between fluorescence intensity and the process performance attributes (like yield or binding capacity) impacted upon resin reuse. This demonstration of the application of fluorescence for real‐time monitoring correlated empirically with process performance attributes and the results support its use as a PAT tool as part of a process control strategy. While the focus of this paper is on fouling of protein A chromatography resin, the approach and strategy are pertinent to other modes of chromatography as well.

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Rapid characterization of N‐linked glycans from secreted and gel‐purified monoclonal antibodies using MALDI‐ToF mass spectrometry

Cited by 16 publications

References 26 publications

In-column ATR-FTIR spectroscopy to monitor affinity chromatography purification of monoclonal antibodies

In-column ATR-FTIR spectroscopy to monitor affinity chromatography purification of monoclonal antibodies

Structural Analysis of Intact Monoclonal Antibodies by Electron Transfer Dissociation Mass Spectrometry

Analytical tools for monitoring changes in physical and chemical properties of chromatography resin upon reuse

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