High Resolution Capillary Isoelectric Focusing Mass Spectrometry Analysis of Peptides, Proteins, And Monoclonal Antibodies with a Flow-through Microvial Interface

Wang, Lingyu; Tao, Bo; Zhang, Zhengxiang; Wang, Guanbo; Chen, David D. Y.

doi:10.1021/acs.analchem.8b02175

Cited by 54 publications

(57 citation statements)

References 42 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Two basic variants which should originate from C‐terminal lysine clippings were well resolved and easily discriminated, with the mass shifts of 128.1 ± 0.2 Da (K1–K0) and 129.7 ± 1.5 Da (K2–K1). The cIEF–MS results were in appropriate accordance with the data reported previously . IEF separation of the deamidated infliximab variant was slightly better than the cIEF–MS method using a pulled‐glass interface .…”

Section: Resultssupporting

confidence: 90%

“…IEF separation of the deamidated infliximab variant was slightly better than the cIEF–MS method using a pulled‐glass interface . The intact mass of the most abundant mAb variant (G0F/G0F, K0) was identical to our previous result . Based on BPCs of two consecutive analyses, the reproducibility was evaluated.…”

Section: Resultssupporting

confidence: 61%

“…A sandwich configuration containing the catholyte segment, the sample segment containing analyte and carrier ampholytes, and the anolyte segment in the IEF capillary was used to avoid the exchange step and achieve full automation . Direct online cIEF–MS analysis of intact mAbs have been reported using either a stainless‐steel flow‐through microvial interface , or a pulled‐glass electrokinetically pumped sheath‐flow interface . The reported total ion chromatograms (TIC) containing structural information demonstrated high similarity to imaging IEF traces of identical mAbs .…”

Section: Introductionmentioning

confidence: 99%

“…The reported total ion chromatograms (TIC) containing structural information demonstrated high similarity to imaging IEF traces of identical mAbs . Tryptic peptides , IdeS‐digested mAb (Cetuximab) , intact proteins/mAbs , and acidic oligosaccharides have been analyzed by direct online cIEF–MS.…”

Section: Introductionmentioning

confidence: 99%

“…With a flow‐through microvial interface , online cIEF–MS protocols can be established using a sandwich configuration. It has a sample segment located in the middle of the separation capillary and a small basic segment in the capillary distal end serving as the anolyte . To mitigate the diffusion of focused zones and prevent protein precipitation, 20% v/v of glycerol was added .…”

Section: Introductionmentioning

confidence: 99%

See 4 more Smart Citations

Analysis of four therapeutic monoclonal antibodies by online capillary isoelectric focusing directly coupled to quadrupole time‐of‐flight mass spectrometry

Wang

Chen

2019

Electrophoresis

Self Cite

View full text Add to dashboard Cite

Analysis of four therapeutic monoclonal antibodies by online capillary isoelectric focusing directly coupled to quadrupole time-of-flight mass spectrometryCapillary isoelectric focusing (cIEF) was online coupled to a Q-TOF MS by a flow-through microvial interface for the analysis of therapeutic mAb. Intact molecular weights obtained from the mass spectrum deconvolution of separated charge variants provided information on the structural heterogeneity of therapeutic mAbs. A sandwich cIEF-MS configuration composed of anolyte, sample, and catholyte segments sequentially injected into a neutrally coated capillary was used for the charge heterogeneity separation of four mAbs. Acetic acid and ammonium hydroxide were used in places of the non-volatile acids and bases commonly used for IEF but are incompatible with online MS detection. Glycerol was added as the anti-convective reagent. A chemical modifier was mixed with the cIEF effluent in the flow-throw microvial to maintain the ESI stability and to mitigate ion suppression from the co-eluted carrier ampholytes and glycerol. Analysis of mAb samples have shown relative populations of two basic variants originating from C-terminal lysine process and acidic variant of deamidation. The lysine clippings, deamidation, and sialic acid modification in oligosaccharide chains were revealed in infliximab. Two lysine clipping variants and a deamidated variant were observed in adalimumab. The duplicate analyses of a reference mAb demonstrated five charge variants separated by cIEF due to some unidentified modifications, as their mass spectra shared close similarities. The mAb analyses demonstrated the feasibility of the cIEF-MS method, and they demonstrated how charge and structural variants and minor differences in therapeutic mAbs are observed with this technology. Online cIEF-MS is an information rich technology with high throughput, demonstrated by the initial data presented here.

show abstract

Section: Resultssupporting

confidence: 90%

Section: Resultssupporting

confidence: 61%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Analysis of four therapeutic monoclonal antibodies by online capillary isoelectric focusing directly coupled to quadrupole time‐of‐flight mass spectrometry

Wang

Chen

2019

Electrophoresis

Self Cite

View full text Add to dashboard Cite

show abstract

A novel microchip‐based imaged CIEF‐MS system for comprehensive characterization and identification of biopharmaceutical charge variants

Mack¹,

Arnold²,

Bogdan³

et al. 2019

Electrophoresis

View full text Add to dashboard Cite

A microfluidic system has been designed that integrates both imaged capillary isoelectric focusing (iCIEF) separations and downstream MS detection into a single assay. Along with the construction of novel instrumentation and an innovative microfluidic chip, conversion to MS‐compatible separation reagents has also been established. Incorporation of 280 nm absorbance iCIEF‐MS analysis not only permits photometric quantitation of separated charge isoforms but also facilitates the direct monitoring of analyte focusing and mobilization in real‐time. The outcome of this effort is a device with the unique ability to allow for both the characterization and identification of protein charge and mass isoforms in under 15 min. Acquisition, quantitation, and identification of highly resolved intact mAb charge isoforms along with their critical N‐linked glycan pairs clearly demonstrate analytical utility of our innovative system. In total, 33 separate molecular features were characterized by the iCIEF‐MS system representing a dramatic increase in the ability to monitor multiple intact mAb critical quality attributes in a single comprehensive assay. Unlike previously reported CIEF‐MS results, relatively high ampholyte concentrations, of up to 4% v/v, were employed without impacting MS sensitivity, observed to be on the order of 1% composition.

show abstract

A robust sheath‐flow CE‐MS interface for hyphenation with Orbitrap MS

2020

View full text Add to dashboard Cite

The hyphenation of capillary electrophoresis with high‐resolution mass spectrometry, such as Orbitrap MS, is of broad interest for the unambiguous and exceptionally sensitive identification of compounds. However, the coupling of these techniques requires a robust ionization interface that does not influence the stability of the separation voltage while coping with oxidation of the emitter tip at large ionization voltages. Herein, we present the design of a sheath‐flow CE‐ESI‐MS interface which combines a robust and easy to operate set‐up with high‐resolution Orbitrap MS detection. The sheath liquid interface is equipped with a gold coated electrospray emitter which increases the stability and overall lifetime of the system. For the characterization of the interface, the spray stability and durability were investigated in dependence of the sheath‐flow rate, electrospray voltage, and additional gold coating. The optimized conditions were applied to a separation of angiotensin II and neurotensin resulting in LODs of 2.4 and 3.5 ng/mL.

show abstract

High Resolution Capillary Isoelectric Focusing Mass Spectrometry Analysis of Peptides, Proteins, And Monoclonal Antibodies with a Flow-through Microvial Interface

Cited by 54 publications

References 42 publications

Analysis of four therapeutic monoclonal antibodies by online capillary isoelectric focusing directly coupled to quadrupole time‐of‐flight mass spectrometry

Analysis of four therapeutic monoclonal antibodies by online capillary isoelectric focusing directly coupled to quadrupole time‐of‐flight mass spectrometry

A novel microchip‐based imaged CIEF‐MS system for comprehensive characterization and identification of biopharmaceutical charge variants

A robust sheath‐flow CE‐MS interface for hyphenation with Orbitrap MS

Contact Info

Product

Resources

About