The ELISA Detectability and Potency of Pegfilgrastim Decrease in Physiological Conditions: Key Roles for Aggregation and Individual Variability.

PEGylated recombinant human granulocyte colony stimulating factor (pegfilgrastim) is used clinically to accelerate immune reconstitution following chemotherapy and is being pursued for biosimilar development. One challenge to overcome in pegfilgrastim biosimilar development is establishing pharmacokinetic (PK) similarity, which is partly due to the degree of PK variability. We herein report that commercially available G-CSF and PEG ELISA detection kits have different capacities to detect pegfilgrastim aggregates that rapidly form in vitro in physiological conditions. These aggregates can be observed using SDS-PAGE, size-exclusion chromatography, dynamic light scattering, and real-time NMR analysis and are associated with decreased bioactivity as reflected by reduced drug-induced cellular proliferation and STAT3 phosphorylation. Furthermore, individual variability in the stability and detectability of pegfilgrastim in human sera is also observed. Pegfilgrastim levels display marked subject variability in sera from healthy donors incubated at 37 °C. The stability patterns of pegfilgrastim closely match the stability patterns of filgrastim, consistent with a key role for pegfilgrastim's G-CSF moiety in driving formation of inactive aggregates. Taken together, our results indicate that individual variability and ELISA specificity for inactive aggregates are key factors to consider when designing and interpreting studies involving the measurement of serum pegfilgrastim concentrations.

Microsatellite instability in saliva from patients with hereditary non-polyposis colon cancer and siblings carrying germline mismatch repair gene mutations.

Microsatellites are short tandem repeats of deoxyribonucleic acid (DNA) sequences which are distributed throughout the genome. Tumors in patients with Lynch syndrome tend to accumulate mutations in microsatellites at a much higher rate than other sequences in the genome resulting in microsatellite instability (MSI). This is due to germline mutations in mismatch repair (MMR) genes. Using small pool-polymerase chain reaction (SP-PCR), previous studies have shown that mutant alleles can be detected in microsatellites of DNA from peripheral blood lymphocytes (PBLs) of Lynch syndrome patients at frequencies that were low, but significantly higher than frequencies in PBLs of age-matched non-Lynch syndrome controls. In the present study, SP-PCR detection of frequency of mutant MSI alleles (FMMA) was performed on PBLs and saliva samples from four sets of families. Each family set consisted of a mutation carrying affected proband (initial tumor bearer), a germline mutation-carrier sibling without tumors, and an age-matched normal control, either related (for 3 family sets) without mutation carrier status or unrelated (for 1 family set) without mutation carrier status. FMMAs of saliva and PBL DNA were compared between each proband, sibling and control for each family set, and between family sets. In all five statistically significant saliva comparisons identified between germline mutation carriers (FMMA: 0.080-0.261) and normal controls (FMMA: 0.003-0.087), the measured FMMAs were always higher in the carriers (p < 0.05). A logistic regression model of the data showed a significant increase in FMMAs in saliva DNA from siblings with MMR mutation compared to the normal controls (p < 0.001). These results indicated that the increased FMMAs observed in the saliva DNA as well as PBL DNA of MMR gene mutation carriers compared to normal controls are real and repeatable. Furthermore, the logistic regression also indicated that the FMMAs seen in saliva were nearly double those seen in PBLs (p < 0.001). Saliva testing, a less-invasive procedure than PBL testing, is more sensitive and appears to be a viable alternative for identifying MSI in carriers with MMR mutations.

Spectroscopic characterization of novel polycyclic aromatic polymers.

A series of novel polyphenylenevinylene (PPV) derivative polymers were studied by absorption and photoluminescence spectroscopies. The effect of the sequential introduction of polycyclic aromatic ring substituents into the delocalized backbone was examined with relation to hypsochromatic and bathochromatic shifting. While the replacement of the phenyl units by naphthyl units results in a substantial hypsochromic shift of both the absorption and emission spectra, their subsequent substitution by anthryl units results in a bathochromic shift. The system is modeled according to, and is found to be consistent with, a previous study of donor-acceptor polyenes of varying length. The electronic structure of the backbone is found to be a balance between that of the high electron affinity polycyclic ring system and the contribution to conjugation across the linking vinyl unit. The model is adapted based on electron affinities of the constituent units, and a clear structure-property relationship for the absorption and emission properties of the system is elucidated. The Stokes shift is examined and is seen to be well-correlated with the vinyl contribution to the electron affinity total (EAtotal). The trends observed in the optical properties of the polymeric system are supported by Raman spectroscopy, whereby the spectral signature of the connecting vinyl bond is seen to soften in a fashion which is correlated with the modeled electron affinity parameters.