Validation of a peptide mapping method for a therapeutic monoclonal antibody: what could we possibly learn about a method we have run 100 times?

Bongers, Jacob; Cummings, John J.; Ebert, Mary Beth; Federici, Marcia; Gledhill, Linden; Gulati, Dipti; Hilliard, George M.; Jones, Brian H.; Lee, Kwan R.; Mozdzanowski, Jacek; Naimoli, Michael; Burman, Sudhir

doi:10.1016/s0731-7085(99)00181-8

Cited by 95 publications

(48 citation statements)

References 34 publications

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“…Tryptic peptide mapping is commonly used to determine chemical modifications and sequence variants in proteins [22]. Peptide mapping relies on specific cleavage of the protein sequence with a proteolytic enzyme such as trypsin, giving rise to a known set of peptides.…”

Section: Lc/ms Analysis After Trypsin Digestion (Xic Method)mentioning

confidence: 99%

Comparison of LC and LC/MS methods for quantifying N-glycosylation in recombinant IgGs

Sinha

Pipes

Topp

et al. 2008

J. Am. Soc. Mass Spectrom.

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Section: Lc/ms Analysis After Trypsin Digestion (Xic Method)mentioning

confidence: 99%

Comparison of LC and LC/MS methods for quantifying N-glycosylation in recombinant IgGs

Sinha

Pipes

Topp

et al. 2008

J. Am. Soc. Mass Spectrom.

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“…Tryptic peptide mapping was performed using the reversephase HPLC (RP-HPLC) method based upon the work by Bongers et al (2000). In short, Epratuzumab samples were first dialyzed against deionized water followed by evaporation to dryness overnight.…”

Section: Tryptic Peptide Mappingmentioning

confidence: 99%

Fed‐batch bioreactor process scale‐up from 3‐L to 2,500‐L scale for monoclonal antibody production from cell culture

Yang

Stasny

et al. 2007

Biotech & Bioengineering

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This case study focuses on the scale-up of a Sp2/0 mouse myeloma cell line based fed-batch bioreactor process, from the initial 3-L bench scale to the 2,500-L scale. A stepwise scale-up strategy that involved several intermediate steps in increasing the bioreactor volume was adopted to minimize the risks associated with scale-up processes. Careful selection of several available mixing models from literature, and appropriately applying the calculated results to our settings, resulted in successful scale-up of agitation speed for the large bioreactors. Consideration was also given to scale-up of the nutrient feeding, inoculation, and the set-points of operational parameters such as temperature, pH, dissolved oxygen, dissolved carbon dioxide, and aeration in an integrated manner. It has been demonstrated through the qualitative and the quantitative side-by-side comparison of bioreactor performance as well as through a panel of biochemical characterization tests that the comparability of the process and the product was well controlled and maintained during the process scale-up. The 2,500-L process is currently in use for the routine clinical production of Epratuzumab in support of two global Phase III clinical trials in patients with lupus. Today, the 2,500 L, fed-batch production process for Epratuzumab has met all scheduled batch releases, and the quality of the antibody is consistent and reproducible, meeting all specifications, thus confirming the robustness of the process.

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“…Traditionally, structural characterization of mAbs has been performed by a "bottom-up" approach after first digesting them to peptides [4 -6]. Unfortunately, enzymatic digestion is a laborious, time-consuming process and it often introduces artificial modifications, such as cyclization of N-terminal glutamine and deamidation [5,7]. Alternatively, protein molecular mass analysis is relatively fast, does not require lengthy sample preparation, and induces fewer, if any, modifications [8] compared with the peptide mapping [5,7].…”

mentioning

confidence: 99%

“…Unfortunately, enzymatic digestion is a laborious, time-consuming process and it often introduces artificial modifications, such as cyclization of N-terminal glutamine and deamidation [5,7]. Alternatively, protein molecular mass analysis is relatively fast, does not require lengthy sample preparation, and induces fewer, if any, modifications [8] compared with the peptide mapping [5,7]. Analysis of intact monoclonal IgG antibodies and their large domains has been reported for matrix-assisted laser desorption/ ionization (MALDI) and electrospray ionization (ESI) sources and almost all mass analyzers including MALDI-time of flight (TOF) [9 -11], ESI quadrupole (Q) [4,[12][13][14], ion trap [15], orthogonal TOF [8,11,16,17], and the LTQ-Orbitrap during direct infusion [18].…”

mentioning

confidence: 99%

Mass measurement and top-down HPLC/MS analysis of intact monoclonal antibodies on a hybrid linear quadrupole ion trap-orbitrap mass spectrometer

Bondarenko

Second

Zabrouskov

et al. 2009

J. Am. Soc. Mass Spectrom.

140

129

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Mass and top-down analyses of 150-kDa monoclonal immunoglobulin gamma (IgG) antibodies were performed on an Orbitrap analyzer. Three different sample delivery methods were tested including (1) infusion of an off-line desalted IgG sample using nano-electrospray; (2) on-line desalting followed by a step elution with a high percentage of organic solvent; and (3) reversed-phase HPLC separation and on-line mass and top-down analyses of disulfide isoforms of an IgG2 antibody. The accuracy of mass measurements of intact antibody was within Ϯ2 Da (15 ppm). The glycoforms of intact IgG antibodies separated by 162 Da were baseline resolved. In-source fragmentation of the intact antibodies produced mainly 115 residue fragments including N-terminal variable domains of heavy and light chains. The sequence coverage (the number of cleavages) was greatly increased after reduction of disulfide bonds and HPLC/MS/MS analysis of light and heavy chains using collisioninduced dissociation in the ion trap of the LTQ-Orbitrap. This is an attractive alternative to peptide mapping for characterization and monitoring of post-translational modifications attributed to minimal sample preparation, high speed of the mass/top-down analysis, and relatively minor method-induced sample modifications. [1,2]. This has resulted in a strong need for a highthroughput methods for analysis of different antibody drug candidates. Mass spectrometry has become one of the most powerful techniques for the structural characterization of monoclonal antibodies (mAbs) [3]. Traditionally, structural characterization of mAbs has been performed by a "bottom-up" approach after first digesting them to peptides [4 -6]. Unfortunately, enzymatic digestion is a laborious, time-consuming process and it often introduces artificial modifications, such as cyclization of N-terminal glutamine and deamidation [5,7]. Alternatively, protein molecular mass analysis is relatively fast, does not require lengthy sample preparation, and induces fewer, if any, modifications [8] compared with the peptide mapping [5,7]. Analysis of intact monoclonal IgG antibodies and their large domains has been reported for matrix-assisted laser desorption/ ionization (MALDI) and electrospray ionization (ESI) sources and almost all mass analyzers including MALDI-time of flight (TOF) [9 -11], ESI quadrupole (Q) [4,[12][13][14], ion trap [15], orthogonal TOF [8,11,16,17], and the LTQ-Orbitrap during direct infusion [18]. Although a mass change in a population of mAbs can be used to monitor post-translational modifications [8], it cannot reveal the site of modification. For that purpose, fragmentation of intact proteins, also known as "topdown" mass spectrometry, has been developed during the past decade [19 -25]

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Validation of a peptide mapping method for a therapeutic monoclonal antibody: what could we possibly learn about a method we have run 100 times?

Cited by 95 publications

References 34 publications

Comparison of LC and LC/MS methods for quantifying N-glycosylation in recombinant IgGs

Comparison of LC and LC/MS methods for quantifying N-glycosylation in recombinant IgGs

Fed‐batch bioreactor process scale‐up from 3‐L to 2,500‐L scale for monoclonal antibody production from cell culture

Mass measurement and top-down HPLC/MS analysis of intact monoclonal antibodies on a hybrid linear quadrupole ion trap-orbitrap mass spectrometer

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