Analysis of therapeutic monoclonal antibodies by imaged capillary isoelectric focusing (icIEF).

Imaged capillary isoelectric focusing (icIEF) is a preferred analytical method for determining isoelectric points (pIs) and charge heterogeneity profiles in biotherapeutic proteins. In this study, we optimized the icIEF method for an in-house IgG1κ monoclonal antibody (mAb-1) and assessed its reproducibility, robustness, and autosampler stability. The optimized method was used to evaluate batch-to-batch consistency in pIs for multiple lots of mAb-1 and determine the relative percentages of charge variants. We also tested the method's performance using multiple lots of another IgG1 mAb, commercially available as Herceptin (trastuzumab). Additionally, we designed and assessed native and denaturing platform icIEF methods for 11 other marketed mAbs, with pIs ranging from 6.0 (eculizumab) to 9.22 (tocilizumab).

Dimethyl sulfoxide as a gas phase charge-reducing agent for the determination of PEGylated proteins' intact mass.

Determination of PEGylated proteins' intact mass by mass spectrometry is challenging due to the molecules' large size, excessive charges, and instrument limitations. Previous efforts have been reported. However, signal variability, ion coalescence, and a generally low degree of robustness have been observed. In this work, we have explored the capabilities of post-column infusion of dimethyl sulfoxide (DMSO) following reversed-phase liquid chromatography-mass spectrometry (RP-LCMS) to determine PEG-filgrastim' intact mass, and to characterize its PEG moiety. The method was optimized around reproducibility (six preparations, and three injection replicates) with an in-house prepared PEG-filgrastim standard. The method showed a mass accuracy of ≤1.2 Da. The average molecular weight (MWEO=483) was 40 147.9 Da. The number average molecular weight (Mn) and the weight average molecular weight (Mw) were observed to be 40 101.1 and 40 113.9 Da, respectively, both with an RSD of 0.03%. The molecular weight distribution of ethylene oxide (EO), the polydispersity index (PDI), was 1.0003 for all preparations with a minimum and maximum number of EO units of 448 ± 2 and 516 ± 2, respectively. The method was finally applied to commercially available Neulasta® lots where the Mn and Mw were 39 995.8 and 40 008.8 Da, respectively, both with an RSD of 0.1%. The minimum and maximum EO units across the lots were observed to be 444.5 ± 1.5 and 514 ± 3, respectively. The PDI for all Neulasta® lots was 1.0003. This study provides an insightful characterization of Neulasta® and describes a robust LC-MS methodology for the characterization of the PEGylated proteins.

Tick Ecdysteroid Hormone, Global Microbiota/Rickettsia Signaling in the Ovary versus Carcass during Vitellogenesis in Part-Fed (Virgin) American Dog Ticks, Dermacentor variabilis.

The transovarial transmission of tick-borne bacterial pathogens is an important mechanism for their maintenance in natural populations and transmission, causing disease in humans and animals. The mechanism for this transmission and the possible role of tick hormones facilitating this process have never been studied. Injections of physiological levels of the tick hormone, 20-hydroxyecdysone (20E), into part-fed (virgin) adult females of the American dog tick, Dermacentor variabilis, attached to the host caused a reduction in density of Rickettsia montanensis in the carcass and an increase in the ovaries compared to buffer-injected controls. This injection initiates yolk protein synthesis and uptake by the eggs but has no effect on blood feeding. Francisella sp. and R. montanensis were the predominant bacteria based on the proportionality in the carcass and ovary. The total bacteria load increased in the carcass and ovaries, and bacteria in the genus Pseudomonas increased in the carcass after the 20E injection. The mechanism of how the Rickettsia species respond to changes in tick hormonal regulation needs further investigation. Multiple possible mechanisms for the proliferation of R. montanensis in the ovaries are proposed.

Prevalence of Rickettsia Species (Rickettsiales: Rickettsiaceae) in Dermacentor variabilis Ticks (Acari: Ixodidae) in North Carolina.

The American dog tick, Dermacentor variabilis (Say), is a vector of spotted fever group (SFG) rickettsiae, including Rickettsia rickettsii the causative organism of Rocky Mountain spotted fever (RMSF). In North Carolina, SFG rickettsioses (including RMSF) are a leading cause of tick-borne illness. Knowledge of the infection rate and geographic distribution of D. variabilis ticks infected with Rickettsia spp. provides information on the spatial distribution of public health risk. Accordingly, we extracted genomic DNA from adult D. variabilis collected from field habitats in 32 North Carolina counties from 2009 to 2013. A nested PCR assay of the 23S-5S intergenic spacer (IGS) region of Rickettsia coupled with reverse line blot hybridization (RLBH) with species-specific probes was used to detect and identify rickettsiae to species. Approximately half of the 532 tick DNA samples exhibited a band of the expected size on agarose gels, indicating infection with Rickettsia spp. RLBH analyses showed R. amblyommatis (formerly 'Candidatus R. amblyommii'), R. parkeri, and R. montanensis were predominant, while other Rickettsia species detected included R. conorii-like, R. massiliae, R. rhipicephali, R. canadensis, R. bellii, and some unknown Rickettsia spp. Some ticks were infected with more than one Rickettsia species. Notably, several Rickettsia-positive ticks harbored R. rickettsii. DNA sequencing was performed on a portion of the 23S-5S IGS amplicons and the results were concordant with RLB assay results. We conclude that Rickettsia spp. are common in D. variabilis in North Carolina. Geographic patterns in the occurrence of Rickettsia-infected D. variabilis ticks across the counties sampled are discussed.

Development and Validation of an Improved PCR Method Using the 23S-5S Intergenic Spacer for Detection of Rickettsiae in Dermacentor variabilis Ticks and Tissue Samples from Humans and Laboratory Animals.

A novel nested PCR assay was developed to detectRickettsiaspp. in ticks and tissue samples from humans and laboratory animals. Primers were designed for the nested run to amplify a variable region of the 23S-5S intergenic spacer (IGS) ofRickettsiaspp. The newly designed primers were evaluated using genomic DNA from 11Rickettsiaspecies belonging to the spotted fever, typhus, and ancestral groups and, in parallel, compared to otherRickettsia-specific PCR targets (ompA,gltA, and the 17-kDa protein gene). The new 23S-5S IGS nested PCR assay amplified all 11Rickettsiaspp., but the assays employing other PCR targets did not. The novel nested assay was sensitive enough to detect one copy of a cloned 23S-5S IGS fragment from "CandidatusRickettsia amblyommii." Subsequently, the detection efficiency of the 23S-5S IGS nested assay was compared to those of the other three assays using genomic DNA extracted from 40 adultDermacentor variabilisticks. The nested 23S-5S IGS assay detectedRickettsiaDNA in 45% of the ticks, while the amplification rates of the other three assays ranged between 5 and 20%. The novel PCR assay was validated using clinical samples from humans and laboratory animals that were known to be infected with pathogenic species ofRickettsia The nested 23S-5S IGS PCR assay was coupled with reverse line blot hybridization with species-specific probes for high-throughput detection and simultaneous identification of the species ofRickettsiain the ticks. "CandidatusRickettsia amblyommii,"R. montanensis,R. felis, andR. belliiwere frequently identified species, along with some potentially novelRickettsiastrains that were closely related toR. belliiandR. conorii.