Using differential scanning calorimetry for the development of non-reduced capillary electrophoresis sodium dodecyl sulfate methods for monoclonal antibodies.

Analysis of non-reduced and reduced monoclonal antibodies (mAbs) by capillary electrophoresis-sodium dodecyl sulfate (CE-SDS) is routinely used to detect product size variants and process-related impurities. Levels of high molecular weight (HMW) forms obtained from this method usually trend comparably to those obtained by orthogonal methods such as size-exclusion ultra-high performance liquid chromatography (SE-UHPLC). However, in the presented case study, comparison of CE-SDS data for three IgG1 mAbs (trastuzumab, mAb1, and mAb2) showed a discrepancy between amounts of observed HMW forms in mAb2 compared with its native forms determined by SE-UHPLC (~17% vs. ~0.5%, respectively). SDS chemical denaturation, as measured by differential scanning calorimetry, demonstrated that the high thermal stability of mAb2 caused an unidentified HMW peak observed by non-reduced (NR)-CE-SDS, which was the result of improper denaturing, resulting in a partially folded species. More so, this strategy enabled the rapid identification of optimal SDS concentration and temperature conditions needed for suitable denaturation for mAb2. This case study presents an alternative option for quick optimization of NR-CE-SDS methods when characterizing mAbs or other thermally stable proteins. Also, this strategy can be used to understand basic biophysical mechanisms of protein unfolding and investigate the higher-order structure imparted by specific sequences and understand how these sequences might affect the results of an analytical method such as CE-SDS.

Multi-Site N-glycan mapping study 1: Capillary electrophoresis - laser induced fluorescence.

An international team that included 20 independent laboratories from biopharmaceutical companies, universities, analytical contract laboratories and national authorities in the United States, Europe and Asia was formed to evaluate the reproducibility of sample preparation and analysis of N-glycans using capillary electrophoresis of 8-aminopyrene-1,3,6-trisulfonic acid (APTS)-labeled glycans with laser induced fluorescence (CE-LIF) detection (16 sites) and ultra high-performance liquid chromatography (UHPLC, 12 sites; results to be reported in a subsequent publication). All participants used the same lot of chemicals, samples, reagents, and columns/capillaries to run their assays. Migration time, peak area and peak area percent values were determined for all peaks with >0.1% peak area. Our results demonstrated low variability and high reproducibility, both, within any given site as well across all sites, which indicates that a standard N-glycan analysis platform appropriate for general use (clone selection, process development, lot release, etc.) within the industry can be established.

A tunable silk-alginate hydrogel scaffold for stem cell culture and transplantation.

One of the major challenges in regenerative medicine is the ability to recreate the stem cell niche, which is defined by its signaling molecules, the creation of cytokine gradients, and the modulation of matrix stiffness. A wide range of scaffolds has been developed in order to recapitulate the stem cell niche, among them hydrogels. This paper reports the development of a new silk-alginate based hydrogel with a focus on stem cell culture. This biocomposite allows to fine tune its elasticity during cell culture, addressing the importance of mechanotransduction during stem cell differentiation. The silk-alginate scaffold promotes adherence of mouse embryonic stem cells and cell survival upon transplantation. In addition, it has tunable stiffness as function of the silk-alginate ratio and the concentration of crosslinker--a characteristic that is very hard to accomplish in current hydrogels. The hydrogel and the presented results represents key steps on the way of creating artificial stem cell niche, opening up new paths in regenerative medicine.

Divergent dispersion behavior of ssDNA fragments during microchip electrophoresis in pDMA and LPA entangled polymer networks.

Resolution of DNA fragments separated by electrophoresis in polymer solutions ("matrices") is determined by both the spacing between peaks and the width of the peaks. Prior research on the development of high-performance separation matrices has been focused primarily on optimizing DNA mobility and matrix selectivity, and gave less attention to peak broadening. Quantitative data are rare for peak broadening in systems in which high electric field strengths are used (>150 V/cm), which is surprising since capillary and microchip-based systems commonly run at these field strengths. Here, we report results for a study of band broadening behavior for ssDNA fragments on a glass microfluidic chip, for electric field strengths up to 320 V/cm. We compare dispersion coefficients obtained in a poly(N,N-dimethylacrylamide) (pDMA) separation matrix that was developed for chip-based DNA sequencing with a commercially available linear polyacrylamide (LPA) matrix commonly used in capillaries. Much larger DNA dispersion coefficients were measured in the LPA matrix as compared to the pDMA matrix, and the dependence of dispersion coefficient on DNA size and electric field strength were found to differ quite starkly in the two matrices. These observations lead us to propose that DNA migration mechanisms differ substantially in our custom pDMA matrix compared to the commercially available LPA matrix. We discuss the implications of these results in terms of developing optimal matrices for specific separation (microchip or capillary) platforms.

Free-solution electrophoretic separations of DNA-drag-tag conjugates on glass microchips with no polymer network and no loss of resolution at increased electric field strength.

Here, we demonstrate the potential for high-resolution electrophoretic separations of ssDNA-protein conjugates in borosilicate glass microfluidic chips, with no sieving media and excellent repeatability. Using polynucleotides of two different lengths conjugated to moderately cationic protein polymer drag-tags, we measured separation efficiency as a function of applied electric field. In excellent agreement with prior theoretical predictions of Slater et al., resolution is found to remain constant as applied field is increased up to 700 V/cm, the highest field we were able to apply. This remarkable result illustrates the fundamentally different physical limitations of free-solution conjugate electrophoresis (FSCE)-based DNA separations relative to matrix-based DNA electrophoresis. ssDNA separations in "gels" have always shown rapidly declining resolution as the field strength is increased; this is especially true for ssDNA > 400 bases in length. FSCE's ability to decouple DNA peak resolution from applied electric field suggests the future possibility of ultra-rapid FSCE sequencing on chips. We investigated sources of peak broadening for FSCE separations on borosilicate glass microchips, using six different protein polymer drag-tags. For drag-tags with four or more positive charges, electrostatic and adsorptive interactions with poly(N-hydroxyethylacrylamide)-coated microchannel walls led to appreciable band-broadening, while much sharper peaks were seen for bioconjugates with nearly charge-neutral protein drag-tags.