Native-MS analysis of monoclonal antibody conjugates by Fourier transform ion cyclotron resonance mass spectrometry

Antibody drug conjugates (ADCs) are an important class of therapeutic molecule currently being used to treat HER2-positive metastatic breast cancer, relapsed or refractory Hodgkin lymphoma, systemic anaplastic large cell lymphoma, relapsed or refractory B-cell precursor acute lymphoblastic leukemia and acute myeloid leukemia. An ADC typically consists of a small molecule or peptide-based cytotoxic moiety covalently linked, via lysine or cysteine residues, to amonoclonal antibody (mAb) scaffold. Mass spectrometric (MS) characterization of these molecules afford highly accurate molecular weight (MW) and drug-to-antibody ratio (DAR) determination, and is typically performed using orthogonal acceleration time-of-flight (oa-ToF) analysers and more recently Orbitrap instruments. Herein we describe for the first time the use of a 15 Tesla solariX Fourier transform ion cyclotron mass spectrometer to characterize an IgG1 mAb molecule conjugated with biotin via native lysine and cysteine residues, under native-MS and solution conditions. The cysteine biotin conjugates remained fully intact, demonstrating the ability of the FT-ICR to maintain the noncovalent interactions and efficiently transmit labile protein complexes. Native-MS was acquired and is displayed in magnitude mode using a symmetric Hann apodisation function. Baseline separation is achieved on all covalent biotin additions, for each charge state, for both the lysine and cysteine biotin-conjugates. Average DAR values obtained by native-MS for the lysine conjugate are compared to those derived by denaturing reversed phase liquid chromatography using an oa-ToF MS system (1.56 ±0.02 versus 2.24 ±0.02 for a 5-molar equivalent and 3.99 ±0.09 versus 4.43 ±0.01 for a 10-molar equivalent, respectively). Increased DAR value accuracy can be obtained for the higher biotin load, when using standard ESI conditions as opposed to nanoESI native-MS conditions. Both denatured LC-MS and native-MS spectral data were deconvoluted using a parsimonious based algorithm, without the need for parameter adjustment

Uncertainty Compensation in Human Attention: Evidence from Response Times and Fixation Durations

BACKGROUND: Uncertainty and predictability have remained at the center of the study of human attention. Yet, studies have only examined whether response times (RT) or fixations were longer or shorter under levels of stimulus uncertainty. To date, no study has examined patterns of stimuli and responses through a unifying framework of uncertainty. METHODOLOGY/PRINCIPAL FINDINGS: We asked 29 college students to generate repeated responses to a continuous series of visual stimuli presented on a computer monitor. Subjects produced these responses by pressing on a keypad as soon a target was detected (regardless of position) while the durations of their visual fixations were recorded. We manipulated the level of stimulus uncertainty in space and time by changing the number of potential stimulus locations and time intervals between stimulus presentations. To allow the analyses to be conducted using uncertainty as common description of stimulus and response we calculated the entropy of the RT and fixation durations. We tested the hypothesis of uncertainty compensation across space and time by fitting the RT and fixation duration entropy values to a quadratic surface. The quadratic surface accounted for 80% of the variance in the entropy values of both RT and fixation durations. RT entropy increased as a function of spatial and temporal uncertainty of the stimulus, alongside a symmetric, compensatory decrease in the entropy of fixation durations as the level of spatial and temporal uncertainty of the stimuli was increased. CONCLUSIONS/SIGNIFICANCE: Our results demonstrate that greater uncertainty in the stimulus leads to greater uncertainty in the response, and that the effects of spatial and temporal uncertainties are compensatory. We also observed compensatory relationship across the entropies of fixation duration and RT, suggesting that a more predictable visual search strategy leads to more uncertain response patterns and vice versa

The deuteron: structure and form factors

A brief review of the history of the discovery of the deuteron in provided. The current status of both experiment and theory for the elastic electron scattering is then presented.Comment: 80 pages, 33 figures, submited to Advances in Nuclear Physic

A922 Sequential measurement of 1 hour creatinine clearance (1-CRCL) in critically ill patients at risk of acute kidney injury (AKI)

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