Dataset Information

Fast protein sequencing of monoclonal antibody by real-time digestion on emitter during nanoelectrospray.

ABSTRACT: Growth in the pharmaceutical industry has led to an increasing demand for rapid characterization of therapeutic monoclonal antibodies. The current methods for antibody sequence confirmation (e.g., N-terminal Edman sequencing and traditional peptide mapping methods) are not sufficient; thus, we developed a fast method for sequencing recombinant monoclonal antibodies using a novel digestion-on-emitter technology. Using this method, a monoclonal antibody can be denatured, reduced, digested, and sequenced in less than an hour. High throughput and satisfactory protein sequence coverage were achieved by using a non-specific protease from Aspergillus saitoi, protease XIII, to digest the denatured and reduced monoclonal antibody on an electrospray emitter, while electrospray high voltage was applied to the digestion mixture through the emitter. Tandem mass spectrometry data was acquired over the course of enzyme digestion, generating similar information compared to standard peptide mapping experiments in much less time. We demonstrated that this fast protein sequencing method provided sufficient sequence information for bovine serum albumin and two commercially available monoclonal antibodies, mouse IgG1 MOPC21 and humanized IgG1 NISTmAb. For two monoclonal antibodies, we obtained sequence coverage of 90.5-95.1% for the heavy chains and 98.6-99.1% for the light chains. We found that on-emitter digestion by protease XIII generated peptides of various lengths during the digestion process, which was critical for achieving sufficient sequence coverage. Moreover, we discovered that the enzyme-to-substrate ratio was an important parameter that affects protein sequence coverage. Due to its highly automatable and efficient design, our method offers a major advantage over N-terminal Edman sequencing and traditional peptide mapping methods in the identification of protein sequence, and is capable of meeting an ever-increasing demand for monoclonal antibody sequence confirmation in the biopharmaceutical industry.

SUBMITTER: Mao Y

PROVIDER: S-EPMC6601538 | biostudies-literature | 2019 May/Jun

REPOSITORIES: biostudies-literature

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Fast protein sequencing of monoclonal antibody by real-time digestion on emitter during nanoelectrospray.

Mao Yuan Y Zhang Lichao L Kleinberg Andrew A Xia Qiangwei Q Daly Thomas J TJ Li Ning N

mAbs 20190507 4

Growth in the pharmaceutical industry has led to an increasing demand for rapid characterization of therapeutic monoclonal antibodies. The current methods for antibody sequence confirmation (e.g., N-terminal Edman sequencing and traditional peptide mapping methods) are not sufficient; thus, we developed a fast method for sequencing recombinant monoclonal antibodies using a novel digestion-on-emitter technology. Using this method, a monoclonal antibody can be denatured, reduced, digested, and seq ...[more]

PMID: 30919719

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Project description:Photoimmunotherapy (PIT) is an emerging low side effect cancer therapy based on a monoclonal antibody (mAb) conjugated with a near-infrared (NIR) phthalocyanine dye IRDye 700DX. IR700 is fluorescent, can be used as an imaging agent, and also is phototoxic. It induces rapid cell death after exposure to NIR light. PIT induces highly selective cancer cell death, while leaving most of tumor blood vessels unharmed, leading to an effect called super-enhanced permeability and retention (SUPR). SUPR significantly improves the effectiveness of the anticancer drug. Currently, the therapeutic effects of PIT are monitored using the IR700 fluorescent signal based on macroscopic fluorescence reflectance imagery. This technique, however, lacks the resolution and depth information to reveal the intratumor heterogeneity of mAb-IR700 distribution. We applied a minimally invasive two-channel fluorescence fiber imaging system by combining the traditional fluorescence imaging microscope with two imaging fiber bundles (~0.85mm). This method monitored mAb-IR700 distribution and therapeutic effects during PIT at different intratumor locations (e.g., tumor surface vs. deep tumor) in situ and in real time simultaneously. This enabled evaluation of the therapeutic effects in vivo and treatment regimens. The average IR700 fluorescence intensity recovery after PIT to the tumor surface is 91.50%, while it is 100.63% in deep tumors. To verify the results, two-photon microscopy combined with a microprism was also used to record the mAb-IR700 distribution and fluorescence intensity of green fluorescent protein (GFP) at different tumor depths during PIT. After PIT treatment, there was significantly higher IR700 fluorescence recovery in deep tumor than in the tumor surface. This phenomenon can be explained by increased vascular permeability immediately after NIR-PIT. Fluorescence intensity of GFP at the tumor surface decreased significantly more compared to that of deep tumor and in controls (no PIT).

Dataset Information

Fast protein sequencing of monoclonal antibody by real-time digestion on emitter during nanoelectrospray.

Publications

Fast protein sequencing of monoclonal antibody by real-time digestion on emitter during nanoelectrospray.

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