Online multimethod platform for comprehensive characterization of monoclonal antibodies in cell culture fluid from a single sample injection - Intact protein workflow.

BACKGROUND: Therapeutic monoclonal antibodies (mAbs) comprise a large structural variability with respect to charge, size and post-translational modifications. These critical quality attributes (CQAs) need to be assessed during and after the production of mAbs. This normally requires off-line purification and sample preparation as well as several chromatographic selectivities, which makes the whole process time-consuming and error-prone. To improve on this, we developed an integrated and automated multi-dimensional analytical platform for the simultaneous assessment of multiple CQAs of mAbs in cell culture fluid (CCF) from upstream processes. RESULTS: The on-line system allows mAb characterization at the intact level, combining protein A affinity chromatography (ProtA) with size-exclusion, ion-exchange, and reversed-phase liquid chromatographic modes with UV and mass spectrometric detection. Multiple heart cuts of a single mAb elution band from ProtA are stored in 20-µL loops and successively sent to the multimethod options in the second dimension. ProtA loading and elution conditions and their compatibility with second-dimension LC modes were studied and optimized. Subsequently, heart-cutting and valve-switching schemes were investigated to achieve effective and reproducible analyses. The applicability of the developed workflow was demonstrated by the direct analysis (i.e. not requiring off-line sample preparation) of a therapeutic mAb in CCF, obtaining useful information on accurate molecular mass, glycosylation, and charge and size variants of the mAb product at the same time and in just over 1 h. SIGNIFICANCE: The developed multidimensional platform is the first system that allows for multiple fractions from a single ProtA band to be characterized using different chromatographic selectivities in a single run allowing direct correlation between CQAs. The performance of the system is comparable to established off-line methods, fully compatible with upstream process samples, and provides a significant time-reduction of the characterization procedure.

Supercharging reagents in LC-MS/MS hormone analyses: Enhancing ionization, not limit of quantification.

One of the critical steps during LC-MS/MS hormone analyses that affects the sensitivity of the assay is the ionization process. Enhancing ionization efficiencies by the addition of supercharging reagents might be one way to improve sensitivity and reduce the limit of quantification (LOQ). Therefore, we investigated whether the addition of the supercharging reagents m-nitrobenzyl alcohol (m-NBA), sulfolane, propylene carbonate, and o-nitroanisole (o-NA) increased ionization efficiency and improved assay LOQ of insulin, oxytocin, sex steroids, and corticosteroids in test solutions. Additionally, the influence of the supercharging reagents was tested in serum samples after sample pretreatment to determine whether ionization would be enhanced similarly in routine analyses and, subsequently, lead to improved sensitivity. The screening experiments showed that the impact of the supercharging reagents varied for each hormone; although the addition of m-NBA increased the signal of all hormones, the other reagents only enhanced ionization efficiencies for some hormones. While the addition of 0.05 v/v% m-NBA and 0.05 v/v% o-NA did result in an increase in peak area in both test solutions and serum samples, it did not significantly improve the signal-to-noise ratio, as a simultaneous increase in noise was observed. In conclusion, even though supercharging reagents can enhance ionization efficiencies of hormones significantly, the addition of these reagents does not result in an improved LOQ for hormone measurements with LC-MS/MS.

Hematocrit and standardization in DBS analysis: A practical approach for hormones mainly present in the plasma fraction.

BACKGROUND AND AIM: Dried blood spot (DBS) sampling has many advantages over conventionally used blood samples, but is thought to suffer from hematocrit related issues. The aim of our research was to investigate whether reliable results can be obtained without bothering about hematocrit effects in DBS analysis of analytes that are mainly present in the plasma compartment. MATERIALS AND METHODS: Venous blood samples with variation in hematocrit and spotted volume were prepared. Spot diameter and 25-OH Vitamin D3 and testosterone concentrations were measured. Moreover, DBS and plasma concentrations of 25-OH Vitamin D3, testosterone and hematocrit were determined in random patient samples. RESULTS: DBS spot size was linearly related to hematocrit. Measured DBS concentrations of 25-OH Vitamin D3 and testosterone were independent of hematocrit and spotted volume. Determining the relation between plasma and DBS concentration resulted in a factor that can be used to convert DBS concentrations to standardized plasma concentrations. CONCLUSION: Addressing the hematocrit issue is not necessary for hormones that are mainly present in the plasma compartment. The relation between plasma and DBS concentration can be used to convert DBS concentrations to standardized plasma concentrations which makes interpretation of DBS concentrations easier.