NIST Interlaboratory Study on Glycosylation Analysis of Monoclonal Antibodies: Comparison of Results from Diverse Analytical Methods.

Glycosylation is a topic of intense current interest in the development of biopharmaceuticals because it is related to drug safety and efficacy. This work describes results of an interlaboratory study on the glycosylation of the Primary Sample (PS) of NISTmAb, a monoclonal antibody reference material. Seventy-six laboratories from industry, university, research, government, and hospital sectors in Europe, North America, Asia, and Australia submitted a total of 103 reports on glycan distributions. The principal objective of this study was to report and compare results for the full range of analytical methods presently used in the glycosylation analysis of mAbs. Therefore, participation was unrestricted, with laboratories choosing their own measurement techniques. Protein glycosylation was determined in various ways, including at the level of intact mAb, protein fragments, glycopeptides, or released glycans, using a wide variety of methods for derivatization, separation, identification, and quantification. Consequently, the diversity of results was enormous, with the number of glycan compositions identified by each laboratory ranging from 4 to 48. In total, one hundred sixteen glycan compositions were reported, of which 57 compositions could be assigned consensus abundance values. These consensus medians provide community-derived values for NISTmAb PS. Agreement with the consensus medians did not depend on the specific method or laboratory type. The study provides a view of the current state-of-the-art for biologic glycosylation measurement and suggests a clear need for harmonization of glycosylation analysis methods.

Improved LC/MS Methods for the Analysis of Metal-Sensitive Analytes Using Medronic Acid as a Mobile Phase Additive.

Phosphorylated compounds and organic acids with multiple carboxylate groups are commonly observed to have poor peak shapes and signal in LC/MS experiments. The poor peak shape is caused by the presence of trace metals, particularly iron, contributed from a variety of sources within the chromatographic system. To ameliorate this problem, different solvent additives were investigated to reduce the amount of metal in the flow path to achieve better analytical performance for these metal-sensitive compounds. Here, we introduce the use of a solvent additive that can significantly improve the peak shapes and signal of metal-sensitive metabolites for LC/MS analysis. Moreover, the additive is shown to be amenable for other metal-sensitive applications, such as the analysis of phosphopeptides and polar phosphorylated pesticides, where the instruments could be used in either positive or negative analysis mode.

Development of Comprehensive Online Two-Dimensional Liquid Chromatography/Mass Spectrometry Using Hydrophilic Interaction and Reversed-Phase Separations for Rapid and Deep Profiling of Therapeutic Antibodies.

Monoclonal antibodies (mAb) and related molecules are being developed at a remarkable pace as new therapeutics for the treatment of diseases ranging from cancer to inflammatory disorders. However, characterization of these molecules at all stages of development and manufacturing presents tremendous challenges to existing analytical technologies because of their large size (ca. 150 kDa) and inherent heterogeneity resulting from complex glycosylation patterns and other post-translational modifications. Multidimensional liquid chromatography is emerging as a powerful platform technology that can be used to both improve analysis speed for these molecules by combining existing one-dimensional separations into a single method (e.g., Protein A affinity separation and size-exclusion chromatography) and increasing the resolving power of separations by moving from one dimension of separation to two. In the current study, we have demonstrated the ability to combine hydrophilic interaction (HILIC) and RP separations in an online comprehensive 2D separation coupled with high resolution MS detection (HILIC × RP-HRMS). We find that active solvent modulation (ASM) is critical for coupling these two separation modes, because it mitigates the otherwise serious negative impact of the acetonitrile-rich HILIC mobile phase on the second dimension RP separation. The chromatograms obtained from these HILIC × RP-HRMS separations of mAbs at the subunit level reveal the extent of glycosylation on the Fc/2 and Fd subunits in analysis times on the order of 2 h. In comparison to previous CEX × RP separations of the same molecules, we find that chromatograms from the HILIC × RP separations are richer and reveal separation of some glycoforms that coelute in the CEX × RP separations.

Fc gamma receptor glycosylation modulates the binding of IgG glycoforms: a requirement for stable antibody interactions.

FcγRs play a critical role in the immune response following recognition of invading particles and tumor associated antigens by circulating antibodies. In the present study we investigated the role of FcγR glycosylation in the IgG interaction and observed a stabilizing role for receptor N-glycans. We performed a complete glycan analysis of the recombinant FcγRs (FcγRIa, FcγRIIa, FcγRIIb, FcγRIIIa(Phe158/Val158), and FcγRIIIb) expressed in human cells and demonstrate that receptor glycosylation is complex and varied between receptors. We used surface plasmon resonance to establish binding patterns between rituximab and all receptors. Complex binding was observed for FcγRIa and FcγRIIIa. The IgG-FcγR interaction was further investigated using a combination of kinetic experiments and enzymatically deglycosylated FcγRIa and FcγRIIIa(Phe158/Val158) receptors in an attempt to determine the underlying binding mechanism. We observed that antibody binding levels decreased for deglycosylated receptors, and at the same time, binding kinetics were altered and showed a more rapid approach to steady state, followed by an increase in the antibody dissociation rate. Binding of rituximab to deglycosylated FcγRIIIa(Phe158) was now consistent with a 1:1 binding mechanism, while binding of rituximab to FcγRIIIa(Val158) remained heterogeneous. Kinetic data support a complex binding mechanism, involving heterogeneity in both antibody and receptor, where fucosylated and afucosylated antibody forms compete in receptor binding and in receptor molecules where heterogeneity in receptor glycosylation plays an important role. The exact nature of receptor glycans involved in IgG binding remains unclear and determination of rate and affinity constants are challenging. Here, the use of more extended competition experiments appear promising and suggest that it may be possible to determine dissociation rate constants for high affinity afucosylated antibodies without the need to purify or express such variants. The data described provide further insight into the complexity of the IgG-FcγR interaction and the influence of FcγR glycosylation.

LED controlled flow photolysis for concentration gradients in microfluidic systems.

Many of the channels and reservoirs in microfluidic systems are used simply to allow liquids with different compositions to be delivered to where they are needed. An alternative approach is to use dissolved photochemicals and variable intensity LEDs to generate composition changes in situ. We applied this approach to generate concentration gradients of HCl for gradient ion chromatography.

Porous polymer monoliths for extraction: diverse applications and platforms.

Polymer monoliths are becoming increasingly popular as sorbent materials, and, along with silica monoliths, they are sometimes touted as replacements for the particulate stationary phases used in HPLC. This critical and prospective review shows how polymer monoliths are in fact finding numerous extraction roles that do not resemble HPLC. They are showing great promise as extractors in a remarkable range of platforms, formats and hyphenated systems with functions ranging from chromatographic preconcentration to large-scale preparative extraction. Monolith surface chemistry, morphology and the approaches to monolith synthesis are discussed with regards to these emerging roles.

Identification of homemade inorganic explosives by ion chromatographic analysis of post-blast residues.

Anions and cations of interest for the post-blast identification of homemade inorganic explosives were separated and detected by ion chromatographic (IC) methods. The ionic analytes used for identification of explosives in this study comprised 18 anions (acetate, benzoate, bromate, carbonate, chlorate, chloride, chlorite, chromate, cyanate, fluoride, formate, nitrate, nitrite, perchlorate, phosphate, sulfate, thiocyanate and thiosulfate) and 12 cations (ammonium, barium(II), calcium(II), chromium(III), ethylammonium, magnesium(II), manganese(II), methylammonium, potassium(I), sodium(I), strontium(II), and zinc(II)). Two IC separations are presented, using suppressed IC on a Dionex AS20 column with potassium hydroxide as eluent for anions, and non-suppressed IC for cations using a Dionex SCS 1 column with oxalic acid/acetonitrile as eluent. Conductivity detection was used in both cases. Detection limits for anions were in the range 2-27.4ppb, and for cations were in the range 13-115ppb. These methods allowed the explosive residue ions to be identified and separated from background ions likely to be present in the environment. Linearity (over a calibration range of 0.05-50ppm) was evaluated for both methods, with r(2) values ranging from 0.9889 to 1.000. Reproducibility over 10 consecutive injections of a 5ppm standard ranged from 0.01 to 0.22% relative standard deviation (RSD) for retention time and 0.29 to 2.16%RSD for peak area. The anion and cation separations were performed simultaneously by using two Dionex ICS-2000 chromatographs served by a single autoinjector. The efficacy of the developed methods was demonstrated by analysis of residue samples taken from witness plates and soils collected following the controlled detonation of a series of different inorganic homemade explosives. The results obtained were also confirmed by parallel analysis of the same samples by capillary electrophoresis (CE) with excellent agreement being obtained.

Boronate functionalised polymer monoliths for microscale affinity chromatography.

Novel macroporous monolithic stationary phase materials suitable for microscale boronate affinity chromatography were developed.