Reversed-phase liquid chromatography in-line with negative ionization electrospray mass spectrometry for the characterization of the disulfide-linkages of an immunoglobulin gamma antibody

Chelius, Dirk; Wimer, Mary E. Huff; Bondarenko, Pavel V.

doi:10.1016/j.jasms.2006.07.008

Cited by 28 publications

(22 citation statements)

References 17 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Nevertheless, two IgG4s (Mylotarg/ gemtuzumab and Tysabri/ natalizumab), one IgG2 (Vectibix/ panitumumab) and one engineered mixed IgG2/4 (Soliris/ eculizumab) also reached the market recently [23] and dozens more are in clinical development [4,21,24]. Despite their higher heterogeneity and their different oligomeric status compared to IgG1 (half-antibodies and bispecific heterotetramers for IgG4s [13,25,26]; 2 vs 4 disulfide bridges linking the heavy chains and forming dimers for IgG2s [13,27], both isotypes are often selected and clinically developed to avoid cytotoxic effectors function, which are directly linked to Fc structure and to their glycosylation.…”

Section: Immunoglobulin Isotypes and Glycosylationmentioning

confidence: 99%

“…These micro-variants can be characterized by liquid chromatography (LC), electrophoresis and mass-spectrometry (MS) based methods [27,61,62]. Both "top-down" (direct mass measure of intact IgG and Light & Heavy Chains) and "bottom-up" (peptides, glycopeptides, glycans fingerprinting and structural analyses) approaches are necessary in a first-round antibody fine structural characterization (Fig.…”

Section: Analytical Characterization Of Glyco-formsmentioning

confidence: 99%

See 1 more Smart Citation

Trends in Glycosylation, Glycoanalysis and Glycoengineering of Therapeutic Antibodies and Fc-Fusion Proteins

Beck¹,

Wagner‐Rousset²,

Bussat³

et al. 2008

CPB

222

142

View full text Add to dashboard Cite

Monoclonal antibodies (MAbs) are the fastest growing class of human pharmaceuticals. More than 20 MAbs have been approved and several hundreds are in clinical trials in various therapeutic indications including oncology, inflammatory diseases, organ transplantation, cardiology, viral infection, allergy, and tissue growth and repair. Most of the current therapeutic antibodies are humanized or human Immunoglobulins (IgGs) and are produced as recombinant glycoproteins in eukaryotic cells. Many alternative production systems and improved constructs are also being actively investigated. IgGs glycans represent only an average of around 3% of the total mass of the molecule. Despite this low percentage, particular glycoforms are involved in essential immune effector functions. On the other hand, glycoforms that are not commonly biosynthesized in human may be allergenic, immunogenic and accelerate the plasmatic clearance of the linked antibody. These glyco-variants have to be identified, controlled and limited for therapeutic uses. Glycosylation depends on multiple factors like production system, selected clonal population, manufacturing process and may be genetically or chemically engineered. The present account reviews the glycosylation patterns observed for the current approved therapeutic antibodies produced in mammalian cell lines, details classical and state-of-the-art analytical methods used for the characterization of glycoforms and discusses the expected benefits of manipulating the carbohydrate components of antibodies by bio- or chemical engineering as well as the expected advantages of alternative biotechnological production systems developed for new generation of therapeutic antibodies and Fc-fusion proteins.

show abstract

Section: Immunoglobulin Isotypes and Glycosylationmentioning

confidence: 99%

Section: Analytical Characterization Of Glyco-formsmentioning

confidence: 99%

Trends in Glycosylation, Glycoanalysis and Glycoengineering of Therapeutic Antibodies and Fc-Fusion Proteins

Beck¹,

Wagner‐Rousset²,

Bussat³

et al. 2008

CPB

222

142

View full text Add to dashboard Cite

show abstract

“…However, modifications such as disulfide bonds are not typically fragmented by CID 19. In some cases, the fragmentation by CID in the negative ion mode can lead to the molecular weight of the disulfide-dissociated peptides, however, with limited or no peptide backbone sequence information, in addition to the low ionization efficiency in the negative ion mode 20–23…”

Section: Introductionmentioning

confidence: 99%

Mass Spectrometric Determination of Disulfide Linkages in Recombinant Therapeutic Proteins Using Online LC−MS with Electron-Transfer Dissociation

Jiang

et al. 2008

Anal. Chem.

128

View full text Add to dashboard Cite

In the biotechnology industry, the generation of incorrectly folded recombinant proteins, either from an E.coli expression system or from an over-expressed CHO cell line (disulfide scrambling), is often a great concern as such incorrectly folded forms may not be completely removed in the final product. Thus, significant efforts have been devoted to map disulfide bonds to assure drug quality. Similar to ECD, disulfide bond cleavages are preferred over peptide backbone fragmentation in ETD. Thus, an on-line LC-MS strategy combining collision induced dissociation (CID-MS 2 ), electron transfer dissociation (ETD-MS 2 ), and CID of an isolated product ion derived from ETD (MS 3 ) has been used to characterize disulfide-linked peptides. Disulfide-linked peptide ions were identified by CID and ETD fragmentation, and the disulfide-dissociated (or partially dissociated) peptide ions were characterized in the subsequent MS 3 step. The on-line LC-MS approach is successfully demonstrated in the characterization of disulfide linkages of recombinant human growth hormone (Nutropin), a monoclonal antibody (Herceptin) and tissue plasminogen activator (Activase). The characterization of disulfide-dissociated or partially dissociated peptide ions in the MS 3 step is important to assign the disulfide linkages, particularly, for intertwined disulfide bridges and the unexpected disulfide scrambling of tissue plasminogen activator. The disulfide-dissociated peptide ions are shown to be obtained either directly from the ETD fragmentation of the precursors (disulfide-linked peptide ions) or indirectly from the charge-reduced species in the ETD fragmentation of the precursors. The simultaneous observation of disulfide-linked and disulfide-dissociated peptide ions with high abundance provided not only facile interpretation with high confidence but also simplified the conventional approach for determination of disulfide linkages, which often requires two separate experiments (with and without chemical reduction). The on-line LC-MS with ETD methodology represents a powerful approach to aid in the characterization of the correct folding of therapeutic proteins.

show abstract

“…isulfide bonds are one of the most important post-translational modification (PTM) processes because of their unique role in determining the three-dimensional structures and stabilities of proteins [1][2][3]. Although various PTM sites in peptides have been identified using tandem mass spectrometry (MS n ) [4 -11], disulfide bonds are not readily characterized by MS n studies of protonated peptides [2].…”

mentioning

confidence: 99%

Mapping disulfide bonds in insulin with the route 66 method: Selective cleavage of S-C bonds using alkali and alkaline earth metal enolate complexes

Kim

Beauchamp

2009

J. Am. Soc. Mass Spectrom.

View full text Add to dashboard Cite

Simple and fast identification of disulfide linkages in insulin is demonstrated with a peptic digest using the Route 66 method. This is accomplished by collisional activation of singly and doubly charged cationic Na ϩ and Ca 2ϩ complexes generated using electrospray ionization mass spectrometry (ESI-MS). Collisional activation of doubly charged metal complexes of peptides with intermolecular disulfide linkages yields two sets of singly charged paired products separated by 66 mass units resulting from selective SOC bond cleavages. Highly selective elimination of 66 mass units, which corresponds to the molecular weight of hydrogen disulfide (H 2 S 2 ), is observed from singly charged metal complexes of peptides with disulfide linkages. The mechanism proposed for these processes is initiated by formation of a metal-stabilized enolate at Cys, followed by cleavage of the SOC bond. Further activation of the products yields sequence information that facilitates locating the position of the disulfide linkages in the peptic digest fragments. For example, the doubly charged Ca 2ϩ complex of the peptic digest product GIVEQCCASVCSL/FVNQHLCGSHL yields paired products separated by 66 mass units resulting from selective SOC bond cleavages at an intermolecular disulfide linkage under low-energy collision-induced dissociation. , Co 2ϩ , and Zn 2ϩ ) complexes of oxytocin exhibit dissociation pathways related to SOS and SOC bond cleavages under electroncapture dissociation (ECD) conditions [14] and under sustained off-resonance irradiation collision-induced dissociation (SORI-CID) conditions [15]. Selective SOS bond cleavages in cationic gold(I) complexes of peptides under low-energy CID conditions were also reported [13,16]. We have recently reported what we call the Route 66 method for locating disulfide bonds in peptides. This methodology is based on the highly selective elimination of H 2 S 2 (66 mass units) from singly charged sodiated and alkaline earth metal (Mg 2ϩ and Ca 2ϩ ) bound peptide cations with disulfide linkages under CID conditions [12]. The process is initiated starting with a metal-stabilized enolate anion at Cys, followed by cleavage of the SOC bond. Further MS n spectra reveal additional details of the peptide structure in the region between the newly formed dehydroalanine (dA) residues.In this study, we report application of the Route 66 method for locating the position of disulfide linkages in insulin. This dipeptide hormone, widely used as a model system for the identification of disulfide bonds along with sequence information, offers the challenge of three closely spaced disulfide bonds, including both intramolecular and intermolecular linkages [17][18][19]. A number of studies have reported sequence analysis of insulin using mass spectrometry [19 -23]. Complete sequence analysis of insulin was demonstrated using the reduced protein with low-energy CID [22]. ECD of the oxidized B-chain of insulin yields almost complete

show abstract

Reversed-phase liquid chromatography in-line with negative ionization electrospray mass spectrometry for the characterization of the disulfide-linkages of an immunoglobulin gamma antibody

Cited by 28 publications

References 17 publications

Trends in Glycosylation, Glycoanalysis and Glycoengineering of Therapeutic Antibodies and Fc-Fusion Proteins

Trends in Glycosylation, Glycoanalysis and Glycoengineering of Therapeutic Antibodies and Fc-Fusion Proteins

Mass Spectrometric Determination of Disulfide Linkages in Recombinant Therapeutic Proteins Using Online LC−MS with Electron-Transfer Dissociation

Mapping disulfide bonds in insulin with the route 66 method: Selective cleavage of S-C bonds using alkali and alkaline earth metal enolate complexes

Contact Info

Product

Resources

About