Qualitative and quantitative characterization of protein biotherapeutics with liquid chromatography mass spectrometry

Qu, Miao; An, Bo; Shen, Shichen; Zhang, Ming; Shen, Xiaomeng; Duan, Xiaotao; Balthasar, Joseph P.; Qu, Jun

doi:10.1002/mas.21500

Cited by 58 publications

(54 citation statements)

References 214 publications

(341 reference statements)

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“…Currently, an enzyme-linked immunosorbent assay (ELISA) is general method that has been extensively applied for measuring blood concentrations of therapeutic mAbs. Recently, numerous efforts have been made to develop another quantification method of mAbs using LC/MS/MS [3, 4]. On the other hand, a robust method to assess structures of therapeutic mAbs in the body has not been developed to date in spite of their structural heterogeneities [5].…”

Section: Introductionmentioning

confidence: 99%

Time-Dependent Structural Alteration of Rituximab Analyzed by LC/TOF-MS after a Systemic Administration to Rats

Otani¹,

Yonezawa²,

Tsuda³

et al. 2017

PLoS ONE

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Therapeutic monoclonal antibodies (mAbs) have heterogeneities in their structures. Multiple studies have reported that the variety of post-translational modifications could affect the pharmacokinetic profiles or pharmacological potencies of therapeutic mAbs. Taking into the account that the structural modification of mAbs would affect the efficacy, it is worth investigating the structural alteration of therapeutic mAbs in the blood and the relationship between their structures and pharmacological effects. Herein, we have developed the method to isolate rituximab from plasma in which endogenous IgGs interfere the detection of rituximab, and successfully developed the analytical method with a liquid chromatograph time-of-flight mass spectrometer to detect the structure of rituximab in plasma with errors less than 30 parts per millions. Eight types of carbohydrate chains in rituximab were detected by this method. Interestingly, time-dependent changes in carbohydrate chains such as AAF (G2F) and GnGn (G0) were observed in rats, although the amino acids were stable. Additionally, these structural changes were observed via incubation in plasma as in the rat experiment, suggesting that a certain type of enzyme in plasma caused the alterations of the carbohydrate chains. The present analytical methods could clarify the actual pharmacokinetics of therapeutic mAbs, and help to evaluate the interindividual variations in pharmacokinetics and efficacy.

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Section: Introductionmentioning

confidence: 99%

Time-Dependent Structural Alteration of Rituximab Analyzed by LC/TOF-MS after a Systemic Administration to Rats

Otani¹,

Yonezawa²,

Tsuda³

et al. 2017

PLoS ONE

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“…Sensitivity is the primary concern because of the large molecular weights of proteins, the high protein contents in most biomatrices, and importantly the high chemical noises. 5 High-flow LC-MS is commonly employed in pharmaceutical and clinical applications as it affords short cycles and acceptable robustness. However, it often leads to compromised sensitivity and thus becomes less applicable to quantify biomarkers or protein biotherapeutics at low (and biologically relevant) concentrations.…”

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confidence: 99%

“…6 To address this limitation, nano-LC-MS-based methods have been developed, 3,7–10 but several disadvantages have been reported. 5,11,12 First, the loading capacity for nano-LC-MS is low, leading to compromised concentration sensitivity (i.e., sensitivity expressed in concentration) in biological matrices, despite its high mass sensitivity. 5,12,13 As a result, enrichment and fractionation procedures (e.g., gel or immunoaffinity separation to reduce matrix components) are often employed prior to nano-LC-MS. Second, its relatively poor operational robustness, the extra efforts required for its maintenance, and its relatively low analytical throughput (e.g., each run is >40 min unless a highly selective enrichment is used 3 ) limit its applications in large-cohort analyses.…”

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confidence: 99%

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Sensitive, High-Throughput, and Robust Trapping-Micro-LC-MS Strategy for the Quantification of Biomarkers and Antibody Biotherapeutics

Zhang

et al. 2018

Anal. Chem.

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For LC-MS-based targeted quantification of biotherapeutics and biomarkers in clinical and pharmaceutical environments, high sensitivity, high throughput, and excellent robustness are all essential but remain challenging. For example, though nano-LC-MS has been employed to enhance analytical sensitivity, it falls short because of its low loading capacity, poor throughput, and low operational robustness. Furthermore, high chemical noise in protein bioanalysis typically limits the sensitivity. Here we describe a novel trapping-micro-LC-MS (T-μLC-MS) strategy for targeted protein bioanalysis, which achieves high sensitivity with exceptional robustness and high throughput. A rapid, high-capacity trapping of biological samples is followed by μLC-MS analysis; dynamic sample trapping and cleanup are performed using pH, column chemistry, and fluid mechanics separate from the μLC-MS analysis, enabling orthogonality, which contributes to the reduction of chemical noise and thus results in improved sensitivity. Typically, the selective-trapping and -delivery approach strategically removes >85% of the matrix peptides and detrimental components, markedly enhancing sensitivity, throughput, and operational robustness, and narrow-window-isolation selected-reaction monitoring further improves the signal-to-noise ratio. In addition, unique LC-hardware setups and flow approaches eliminate gradient shock and achieve effective peak compression, enabling highly sensitive analyses of plasma or tissue samples without band broadening. In this study, the quantification of 10 biotherapeutics and biomarkers in plasma and tissues was employed for method development. As observed, a significant sensitivity gain (up to 25-fold) compared with that of conventional LC-MS was achieved, although the average run time was only 8 min/sample. No appreciable peak deterioration or loss of sensitivity was observed after >1500 injections of tissue and plasma samples. The developed method enabled, for the first time, ultrasensitive LC-MS quantification of low levels of a monoclonal antibody and antigen in a tumor and cardiac troponin I in plasma after brief cardiac ischemia. This strategy is valuable when highly sensitive protein quantification in large sample sets is required, as is often the case in typical biomarker validation and pharmaceutical investigations of antibody therapeutics.

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“…LC-MS/MS has become an attractive technique to measure protein biomarkers in biological samples due to its superior selectivity and specificity [26][27][28][29][30][31][32][33]. The LC-MS/MS approach quantifies a protein by selectively analyzing an appropriate surrogate peptide (SP) derived from the target analyte using multiple reaction monitoring (MRM).…”

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confidence: 99%

2D-LC–MS/MS to Measure Cleaved High-Molecular-Weight Kininogen in Human Plasma as A Biomarker for C1-Inh-Hae

et al. 2017

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Aim: C1-INH-HAE is caused by activation of plasma kallikrein which subsequently cleaves high-molecular-weight kininogen (HMWK) to generate bradykinin and cHMWK. Materials & methods: A novel ion-pair 2D LC-MS/MS assay was developed to measure the 46 kDa cHMWK in plasma as a biomarker for C1-INH-HAE. The sample preparation included sodium dodecyl sulfate denaturation, methanol crash, chymotryptic digestion and peptide enrichment by solid phase extraction. Results: The LLOQ was 200 ng/ml. The overall cHMWK recovery combining crash and digestion was 57.5%. The precision of the method was ≤12.7% and accuracy ≤-13.8%. Conclusion: A reagent-free LC-MS assay has been developed for the quantitation of 46 kDa cHMWK, which was shown to be elevated in plasma of C1-INH-HAE patients due to C1-INH deficiency relative to that of healthy subjects.

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Qualitative and quantitative characterization of protein biotherapeutics with liquid chromatography mass spectrometry

Cited by 58 publications

References 214 publications

Time-Dependent Structural Alteration of Rituximab Analyzed by LC/TOF-MS after a Systemic Administration to Rats

Time-Dependent Structural Alteration of Rituximab Analyzed by LC/TOF-MS after a Systemic Administration to Rats

Sensitive, High-Throughput, and Robust Trapping-Micro-LC-MS Strategy for the Quantification of Biomarkers and Antibody Biotherapeutics

2D-LC–MS/MS to Measure Cleaved High-Molecular-Weight Kininogen in Human Plasma as A Biomarker for C1-Inh-Hae

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