Toward the goal of increasing the throughput of high-resolution mass characterization of intact antibodies, we developed a RapidFire-mass spectrometry (MS) assay using electrospray ionization. We achieved unprecedented screening throughput as fast as 15 s/sample, which is an order of magnitude improvement over conventional liquid chromatography (LC)-MS approaches. The screening enabled intact mass determination as accurate as 7 ppm with baseline resolution at the glycoform level for intact antibodies. We utilized this assay to characterize and perform relative quantitation of antibody species from 248 samples of 62 different cell line clones at four time points in 2 h using RapidFiretime-of-flight MS screening. The screening enabled selection of clones with the highest purity of bispecific antibody production and the results significantly correlated with conventional LC-MS results. In addition, analyzing antibodies from a complex plasma sample using affinity-RapidFire-MS was also demonstrated and qualified. In summary, the platform affords high-throughput analyses of antibodies, including bispecific antibodies and potential mispaired side products, in cell culture media, or other complex matrices.antibody screening | high throughput | intact protein mass spectrometry | electrospray U nambiguous characterization of analytes from complex matrices with high content information in a label-free format continues to expand the application of mass spectrometry (MS) in drug discovery (1-3). With the need for high accuracy, sensitivity, and selectivity, the rapidly improving MS instrumentations are emerging at the forefront of analyzing analytes with limited alternative assays for biologics developability (4-6), biotransformation, and high-throughput screening (HTS). For ultra-high-throughput screening, matrix-assisted laser desorption/ionization (MALDI) is amenable to speeds >100,000 samples per day (7-11). Through incorporating self-assembled monolayers for MALDI-time-of-flight (TOF) (SAMDI) technology (12-14), it is also possible to infer small molecule noncovalent hit identification from MALDI-MS detection under native conditions. While such MALDI-MS approaches have the capability for screening small molecule and peptide analytes, larger molecular weight proteins suffer from limited mass resolution and quantitative challenges. Alternatively, electrospray ionization MS (ESI-MS) can provide isotopic resolution for molecules as large as antibodies (15). Although modern mass spectrometers can scan as rapidly as tens of microseconds, highthroughput antibody analyses using ESI-MS is challenged by the relatively low ion sampling rate from chromatographic elution coupled to MS. For rapid sampling, Agilent RapidFire MS (RF-MS) utilizes a rapid trap and elute strategy to enable desalting and ion sampling coupled to a MS ion source. RF-MS has been shown to afford HTS triage for small molecules and proteins <30 kDa from biochemical buffers as fast as 15 s as opposed to minutes observed with conventional chromatography (16). Altern...

The use of biomaterials has been demonstrated as a viable strategy to promote cell survival and cardiac repair. However, limitations on combinational cell-biomaterial therapies exist, as cellular behavior is influenced by the microenvironment and physical characteristics of the material. Among the different scaffolds employed for cardiac tissue engineering, a myocardial matrix hydrogel has been shown to promote cardiogenesis in murine cardiac progenitor cells (mCPCs) in vitro. In this study, we investigated the influence of the hydrogel on Sca-1-like human fetal and adult CPCs (fCPCs and aCPCs) when encapsulated in three-dimensional (3D) material in vitro. fCPCs encapsulated in the myocardial matrix showed an increase in the gene expression level of cardiac markers GATA-4 and MLC2v and the vascular marker vascular endothelial growth factor receptor 2 (VEGFR2) after 4 days in culture, and a significant increase in GATA-4 up to 1 week. Increased gene expression levels of Nkx2.5, MEF2c, VEGFR2, and CD31 were also observed when aCPCs were cultured in the matrix compared to collagen. Cell survival was sustained in both hydrogels up to 1 week in culture with the myocardial matrix capable of enhancing the expression of the proliferation marker Ki-67 after 4 days in culture. When encapsulated CPCs were treated with H 2O2, an improved survival of the cells cultured in the myocardial matrix was observed. Finally, we evaluated the use of the myocardial matrix as hydrogel for in vivo cell transplantation and demonstrated that the gelation properties of the hydrogel are not influenced by the cells. In summary, we showed that the myocardial matrix hydrogel promotes human CPC cardiogenic potential, proliferation, and survival and is a favorable hydrogel for 3D in vitro culture. Furthermore, we demonstrated the in vivo applicability of the matrix as a potential vehicle for cell transplantation.

There are many pharmacokinetic challenges associated with administering protein therapeutics, including biotransformation via clipping, deamidation, isomerization, oxidation, etc. In the case of engineered multivalent tethered antibody formats, proteolysis or deconjugation at the fusion or conjugation site present further issues. Unlike degradations associated with antibody drug conjugates, such biotransformations of tethered antibody formats usually result in degraded products with large mass differences. These large differences can result in processing or mass spectrometry response bias among the resulting product species that can lead to inaccurate stability quantitation. Herein, we describe an assay strategy for characterizing and quantitating degradations accurately for multivalent antibodies by incorporating response bias corrections. For the multivalent tethered antibody molecules selected, an ∼30–80% difference in response, compared to the cleaved product, was observed. To correct for the response bias, selected tethered multivalent antibodies and an IgG antibody (representing the stable intact and the degraded product species, respectively) were spiked in serum at known ratios for analysis. Following affinity capture, we generated calibration curves (five-parameter logistic fit p < 0.05) by plotting the measured ratios of the MS ion responses against the known spiked-in ratios (CVs < 8% for calibration standards). The qualified calibration curve (accuracy within 8% and 2% for measuring degradations of 5% and 15% product, respectively) was then used, through interpolation, to determine stability profiles for the same multivalent tethered antibody formats from both in vitro serum and pharmacokinetic study samples.

Antibody-drug conjugates (ADCs) present unique challenges for ligand-binding assays primarily due to the dynamic changes of the drug-to-antibody ratio (DAR) distribution in vivo and in vitro. Here, an automated on-tip affinity capture platform with subsequent mass spectrometry analysis was developed to accurately characterize the DAR distribution of ADCs from biological matrices. A variety of elution buffers were tested to offer optimal recovery, with trastuzumab serving as a surrogate to the ADCs. High assay repeatability (CV 3%) was achieved for trastuzumab antibody when captured below the maximal binding capacity of 7.5 μg. Efficient on-tip deglycosylation was also demonstrated in 1 h followed by affinity capture. Moreover, this tip-based platform affords higher throughput for DAR characterization when compared with a well-characterized bead-based method. Graphical Abstract ᅟ.