Continued interest in protein therapeutics has motivated the development of improved bioanalytical tools to support development programs. LC-MS offers specificity, sensitivity, and multiplexing ...capabilities without the need for target-specific reagents, making it a valuable alternative to ligand binding assays. Immunoaffinity purification (IP) and enzymatic digestion are critical, yet extensive and time-consuming components of the "gold standard" bottom-up approach to LC-MS-based protein quantitation. In the present work, commercially available technology, based on membrane-immobilized reagents in spin column and plate format, is applied to reduce IP and digestion times from hours to minutes. For a standard monoclonal antibody, the lower limit of quantitation was 0.1 ng μL
−1
compared to 0.05 ng μL
−1
for the standard method. A pharmacokinetics (PK) study dosing Herceptin in rat was analyzed by both the membrane and the standard method with a total sample processing time of 4 h and 20 h, respectively. The calculated concentrations at each time point agreed within 8% between both methods, and PK values including area under the curve (AUC), half-life (T1/2), mean residence time (MRT), clearance (CL), and volume of distribution (Vdss) agreed within 6% underscoring the utility of the membrane methodology for quantitative bioanalysis workflows.
Rapid spin membrane technology decreases the time for IP and digestion of therapeutic proteins.
The quantitation of therapeutic antibodies by mass spectrometry often utilizes a surrogate peptide approach following enzymatic digestion of the antibody. Although this approach has been widely ...adopted, it is labor intensive with limited throughput in most instances. In addition, this approach can pose challenges when attempting to infer details such as quantity and modification state of the intact analyte. Recent enhancements in instrumentation and sample preparation have enabled quantitation through mass spectrometry detection of the intact protein circumnavigating many limitations of the surrogate peptide approach. Presented here is a method for quantitative analysis of therapeutic monoclonal antibodies (mAb) at the fully intact level in a complex pharmacokinetic study. This methodology yielded sensitivity down to 0.1μg/mL from 30μL of a biological sample volume to be utilized across multiple preclinical species without the need for pooling.
The quantitation of therapeutic antibodies by MS often utilizes a surrogate peptide approach. Recent enhancements in instrumentation and sample preparation have enabled quantitation by detection of ...the intact molecule using MS.
A comparison of three methods for quantitative analysis of therapeutic monoclonal antibodies including analysis after deglycosylation, after hinge digestion and at the fully intact antibody level is reported. The optimized methodology provided sensitivity down to 0.1 μg/ml and a lower limit of quantitation of 0.5 ug/ml from a 30 μl sample volume.
Application of this approach to a pharmacokinetic study compared with a conventional surrogate peptide and a ligand-binding assays provided consistent data with direct detection of the dosed molecule.
The requirements for developing antibody biotherapeutics benefit from understanding the nature and relevant aspects of the entire molecule. The method presented herein employs on-line ...multidimensional LC-quadrupole time-of-flight (QTOF)-MS for the quantitative determination of an antibody isolated from biological samples while maintaining the intact native biologically active conformation of the antibody.
Following method optimization for a model antibody, an incurred biotherapeutic in cynomologus monkey was quantified in its intact top-down native conformation. A partial method validation demonstrated acceptable precision and accuracy although improved sensitivity requires further studies.
An on-line multidimensional LC-MS approach presents a proof-of-principle example for quantifying an intact, native antibody isolated from an incurred biological sample via immunoaffinity techniques coupled with top-down QTOF LC-MS bioanalysis.
Large molecule quantitation by LC-MS/MS commonly relies on bottom-up or so-called surrogate peptide measurements to infer the whole-molecule concentration. This can lead to questions about what is ...actually being measured in the assay (intact drug and/or other drug related material). An intact sequential affinity capture (ISAC) assay was developed utilizing two different immunoaffinity (IA) reagents. The reagents were selective for the heavy and light chain of a monoclonal antibody, which when used consecutively, ensures that only the intact form of the antibody is represented by the surrogate peptide. The approach provided comparable results to a traditional sandwich IA assay indicating similar capture populations. The use of an initial ISAC assessment of affinity capture purification, should add a degree of confidence in the use of a single IA-LC-MS/MS quantitation assay.
We have developed a new IR chromogenic cross-linker (IRCX) to aid in rapidly distinguishing cross-linked peptides from unmodified species in complex mixtures. By incorporating a phosphate functional ...group into the cross-linker, one can take advantage of its unique IR absorption properties, affording selective infrared multiphoton dissociation (IRMPD) of the cross-linked peptides. In a mock mixture of unmodified peptides and IRCX-cross-linked peptides (intramolecularly and intermolecularly cross-linked), only the peptides containing the IRCX modification were shown to dissociate upon exposure to 50 ms of 10.6-μm radiation. LC-IRMPD-MS proved to be an effective method to distinguish the cross-linked peptides in a tryptic digest of IRCX-cross-linked ubiquitin. A total of four intermolecular cross-links and two dead-end modifications were identified using IRCX and LC-IRMPD-MS. IRMPD of these cross-linked peptides resulted in secondary dissociation of all primary fragment ions containing the chromophore, producing a series of unmodified b- or y-type ions that allowed the cross-linked peptides to be sequenced without the need for collision-induced dissociation.
A majority of high-grade (HG) serous ovarian cancer (SOC) patients develop resistant disease despite high initial response rates to platinum/paclitaxel-based chemotherapy. We identified shed/secreted ...proteins in preclinical models of paclitaxel-resistant human HGSOC models and correlated these candidate proteins with patient outcomes using public data from HGSOC patients. Proteomic analyses of a HGSOC cell line secretome was compared to those from a syngeneic paclitaxel-resistant variant and from a line established from an intrinsically chemorefractory HGSOC patient. Associations between the identified candidate proteins and patient outcome were assessed in a discovery cohort of 545 patients and two validation cohorts totaling 795 independent SOC patients. Among the 81 differentially abundant proteins identified (q < 0.05) from paclitaxel-sensitive vs -resistant HGSOC cell secretomes, AKAP12 was verified to be elevated in all models of paclitaxel-resistant HGSOC. Furthermore, elevated AKAP12 transcript expression was associated with worse progression-free and overall survival. Associations with outcome were observed in three independent cohorts and remained significant after adjusted multivariate modeling. We further provide evidence to support that differential gene methylation status is associated with elevated expression of AKAP12 in taxol-resistant ovarian cancer cells and ovarian cancer patient subsets. Elevated expression and shedding/secretion of AKAP12 is characteristic of paclitaxel-resistant HGSOC cells, and elevated AKAP12 transcript expression is a poor prognostic and predictive marker for progression-free and overall survival in SOC patients.
Skin cancer and photodamage are problems commonly addressed by dermatologists. Despite the opportunities for chemoprevention with broad-spectrum sunscreen, there is little research investigating ...patient knowledge of proper sunscreen guidelines, or patient perception of physician counseling.
The author sought to determine patient knowledge of the American Academy of Dermatology guidelines for proper sunscreen use and to obtain patient-reported rates of physician counseling regarding sunscreen.
We used a 12-question, multiple choice, anonymous survey to collect data.
The study setting was a private dermatology clinic near Detroit, Michigan.
Two hundred ninety- four adult patients presenting for routine office visits were included in the study.
About 59 percent of the subjects selected the recommended frequency of proper sunscreen use and 60 percent selected the recommended minimum sun protection factor. The minimum amount of sunscreen to cover the body, time of application before sun exposure, and time between reapplications of sunscreen did not receive a majority response. Differences in knowledge were seen between the sexes and skin types. Forty-four percent of patients previously received sunscreen counseling. Patients older than 40 years of age (39.3% vs. 18.4%,
=0.04), those who were fair skinned (62.5% vs. 23.8%), established patients (40.7% vs. 8.3%,
<0.0001), and those with a skin cancer (58.3% vs. 28%,
<0.0001) were more likely to report previous counseling.
The majority of the study subjects never received counseling and lacked adequate knowledge of sunscreen guidelines. In order to obtain adequate primary prevention of skin cancer, it is essential to provide patients with further counseling and education on proper sunscreen use.
We modified a dual pressure linear ion trap Orbitrap to permit infrared multiphoton dissociation (IRMPD) in the higher energy collisional dissociation (HCD) cell for high resolution analysis. A ...number of parameters, including the pressures of the C-trap and HCD cell, the radio frequency (rf) amplitude applied to the C-trap, and the HCD DC offset, were evaluated to optimize IRMPD efficiency and maintain a high signal-to-noise ratio. IRMPD was utilized for characterization of phosphopeptides, supercharged peptides, and N-terminal modified peptides, as well as for top-down protein analysis. The high resolution and high mass accuracy capabilities of the Orbitrap analyzer facilitated confident assignment of product ions arising from IRMPD.
Reduce animal usage for discovery-stage PK studies for biologics programs using microsampling-based approaches and microscale LC-MS.
We report the development of an automated DBS-based serial ...microsampling approach for studying the PK of therapeutic proteins in mice. Automated sample preparation and microflow LC-MS were used to enable assay miniaturization and improve overall assay throughput. Serial sampling of mice was possible over the full 21-day study period with the first six time points over 24 h being collected using automated DBS sample collection. Overall, this approach demonstrated comparable data to a previous study using single mice per time point liquid samples while reducing animal and compound requirements by 14-fold.
Reduction in animals and drug material is enabled by the use of automated serial DBS microsampling for mice studies in discovery-stage studies of protein therapeutics.
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