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036_sushma_rampal_article.pdf

036_sushma_rampal_article.pdf

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38
Introduction
Immunoglobulins or antibodies are solubleserum glycoproteins involved in passiveimmunity against foreign antigens. Mono-specific or monoclonal antibodies (MAb) havebeen developed as therapeutic reagentsbecause of their specificity towards particularmolecular targets associated with diseasemanifestation. There exists a high degree of structural and functional heterogeneity amongantibodies, due in large part to the diversity of associated glycan populations. Glycosylationon therapeutic monoclonal antibodies is acritical post-translational modification that hasbeen associated with bioactivity, structure,and pharmacokinetics. A number of differentcarbohydrate moieties can potentiallyassociate with MAbs, but it is generallythought that a core group of bi-antennary andhigh-mannose structures make up the mostcommon species.MAb carbohydrate heterogeneity analysis andquantitation is essential as oligosaccharideslinked to their Fc region play an importantrole in regulation of cell-dependentcytotoxicity (CDC) and antibody-dependentcellular cytotoxicity (ADCC)
1
. Increase interminal galactose (Gal) on MAb N-linkedoligosaccharides has been implicated in up-regulation of CDC
2
. Therefore, separation andquantification of Gal-containingoligosaccharides is beneficial to betterunderstand MAb function. Glycan speciesvarying in terminal Gal content can be readilyseparated and analyzed using existing CEtechnology. Glycan sample preparationincludes addition of both charge andfluorescence properties, allowingoligosaccharides to be electrophoreticallyseparated and then detected using laser-induced fluorescence (LIF) technology. First,oligosaccharides are removed from theAsn297 residue of the MAb backbone usingthe N-glycosidase PNGase F. This is followedby derivatization of the fluorophoreaminopyrene tri-sulfonic acid (APTS) viareductive amination at the reducing end othe oligosaccharide (Figure 1A).Electrophoretic separation can be performedutilizing a polymeric separation matrixconsisting of 0.4% polyethylene oxide (PEO).
Separation of Fucosylated andnon-Fucosylated CarbohydratesAssociated with Monoclonal Antibodiesusing Capillary Electrophoresis
Sushma Rampal, Oscar Salas, and Mark Lies
March 2011
In order to gain a comprehensive understanding of therapeutic Monoclonal Antibody (MAb) function, it is necessary to critically characterizeglycosylation associated with them. Carbohydrates, and therefore glycosylation, are known to play an important role in the structure, function,and clearance of MAbs and have been shown to be responsible for invoking immune responses in humans. Changes in carbohydratecomposition or concentration can significantly impact the overall efficacy of therapeutic MAbs and can also lead to side effects. Presence of fucose on monoclonal antibody associated N-linked oligosaccharides is a notable glycan modification and has been linked to a decrease inantibody dependent cellular cytotoxicity (ADCC). Accurate analysis of fucosylated and afucosylated oligosaccharides is therefore critical for acomplete understanding of MAb microheterogeneity. Capillary Electrophoresis (CE) technology has been successfully used to separate majorIgG N-linked oligosaccharides G0, G1, and G2 structures from one another. The basis for this separation relies on electrophoresis of oligosaccharides labeled with amino pyrene tri-sulfonic acid (APTS). The complexity of glycans associated with many molecules calls for highresolution separation in order to assess heterogeneity among carbohydrate isomers and co-migrating carbohydrate species. Since CE isalready an established technology for automated and quantitative analysis of N-linked oligosaccharides, we set out to develop a methodologyby which fucosylated oligosaccharides can be differentiated from afucosylated species. Optimization of chemistry and CE methods enabledseparation of fucosylated and non-fucosylated carbohydrates from each another.
Figure 1. Schematic of glycan analysis sample preparation.A. Glycan cleavage and APTS derivatization strategy for analysis of N-linked oligosaccharides. B. Fucosylated N-linked oligosaccharide.
 
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Beckman Coulter has developed andcommercialized technology (Beckman Coulterp/n 477600) to help automate and simplify thisprocess. It has been shown that the principlefor this CE separation of oligosaccharides isbased on both their mobility andhydrodynamic volume
3
. This is illustrated inpart by the fact that positional isomers,although identical in mass, can be resolvedfrom one another (Figure 2). The presence of acore fucose moiety on MAb N-linkedoligosaccharides has been associated with adecrease in ADCC activity and thus reducedefficacy
4
(Figure 1B). It is suggested thattherapeutic MAbs produced in various celllines such as the commonly used ChineseHamster Ovary (CHO) cells contain glycansthat are >90% fucosylated
5
. Because of this,separation of these species is necessary foraccurate analysis. Due to a size difference assmall as 16 daltons between fucosylated andafucosylated glycans, as well as the presenceof numerous positional isomers, separationhas proven to be difficult. Current methodshave been incapable of resolving all of themajor co-migrating glycan species from oneanother. The success for CE technology toresolve terminal Gal differences onoligosaccharide species suggests that itshould also be capable of separatingfucosylated from afucosylated species
Methods and Materials
All separations were performed using the PA800 plus Pharmaceutical Analysis Systemconfigured with a 488 nm solid state laser andLIF detection using an emission band-passfilter of 520 nm ± 10 nm (Beckman CoulterInc). N-CHO capillaries were used forseparation of oligosaccharides. All other assayconditions were as described in the standardoperating procedure for the CarbohydrateLabeling and Analysis Assay Kit, (BeckmanCoulter p/n 477600) with the exception thatthe carbohydrate separation buffer wassubstituted with a new separation bufferformulation where indicated. Finalconcentration for oligosaccharide sampleswas 1.25 mM. Glycan standards forfucosylated and afucosylated species of G0,G1, G1’, and G2 were purchased from GlykoProZyme, Inc. (Hayward, CA). The therapeuticMAb was obtained from Genentech, Inc. (SSan Francisco, CA).Experimental details for this work were asfollows:Carbohydrate separation gels usedCarbohydrate assay gel (containspolyethylene oxide (PEO)) buffer or,New separation gel buffer was 1:1 mixture of:Carbohydrate separation gel buffer (PEO) –BEC p/n 477623dsDNA1000 separation gel buffer (LPA) –BEC p/n 477628Capillary length: total length = 60.2 cm,length to detector = 50 cmCapillary diameter: 50 mm I.D.Injection conditions: 0.5 psi for 10 secSeparation Voltage: 30 kvField Strength: 500 volts/cmCapillary cartridge temperature: 20° CSample storage temperature: 10° C
Results and Discussion
 The goal of this study was to achieveseparation of the major glycan speciesassociated with monoclonal antibodies. Thisglycan population includes positional isomersas well as fucosylated and afucosylatedoligosaccharides. We set out to characterize
Figure 2. Separation of G0, G1, and G2 glycan species. Representative data (top trace) shows separation of N-linked oligosaccharides G0, G1, G1’, and G2 using the Carbohydrate Labeling & Analysis Assay Kit (Beckman Coulter p/n 477600). G1positional isomers are resolved from one another illustrating the mobility- and hydrodynamic volume- based separation. The bottom trace shows separation of a glucose ladder standard. The ‘G’ designation for the glucose ladder standards refers to the number of glucose subunits making up that standard.Figure 3. G0, G1, and G2 oligosaccharides. G0, G1, and G2 oligosaccharides are commonly associated with monoclonal antibodies. The ‘G’ designation refers to the number of galactose subunits occupying the bi-antennary termini of the oligosaccharides. Note G1 can exist as either one of two positional isomers differing in only the location of the terminal galactose.

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