During development of adenovirus-based vaccines, samples have to be analyzed in order to either monitor the production process or control the quality and safety of the product. An important quality ...attribute is the total concentration of intact adenoviruses, which currently is determined by quantitative polymerase chain reaction (qPCR) or anion exchange-HPLC. Capillary Electrophoresis (CE) was evaluated as alternative to the current methods with the aim to have one single method that allows reliable and fast quantification of adenovirus particles throughout the full process. Intact adenoviruses samples from downstream processing and upstream processing were analyzed directly by CE with UV-detection at 214nm. Only the samples with high amounts of DNA required a simple sample pretreatment by benzonase. Adenovirus particles were separated from matrix components such as cell debris, residual cell DNA, and/or proteins on a PVA-coated capillary using a BGE consisting of 125mM Tris, 338mM tricine and 0.2% v/v polysorbate-20 at pH 7.7. Full factorial design of experiments was used for method optimization as part of the analytical quality by design (AQbD) method development approach. The method was validated for the quantification of adenoviruses on five representative samples from the manufacturing process in the range of 0.5×1011–1.5×1011 adenovirus particles per ml (~80 to 250pmol/l). The CE method showed intermediate precision of 7.8% RSD on concentration and an accuracy (spiked recovery) of 95–110%. CE proved highly useful for process development support and is being implemented for in-process control testing for adenovirus vaccine manufacturing.
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•A CE method was development and validated for adenovirus particle determination.•No peak interference observed from cell debris, protein, surfactant or DNA.•The method is accurate and precise for downstream and upstream processing samples.•The CE method was superior to the current method, quantitative PCR, in a method comparability study.•Analytical quality by design (AQbD) method development approach.
We measured picosecond time-resolved fluorescence of intact Photosystem I complexes from
Chlamydomonas reinhardtii and
Arabidopsis thaliana. The antenna system of
C. reinhardtii contains about 30–60 ...chlorophylls more than that of
A. thaliana, but lacks the so-called red chlorophylls, chlorophylls that absorb at longer wavelength than the primary electron donor. In
C. reinhardtii, the main lifetimes of excitation trapping are about 27 and 68 ps. The overall lifetime of
C. reinhardtii is considerably shorter than in
A. thaliana. We conclude that the amount and energies of the red chlorophylls have a larger effect on excitation trapping time in Photosystem I than the antenna size.
The cyanobacterium
Synechococcus PCC 7942 grown under iron starvation assembles a supercomplex consisting of a trimeric Photosystem I (PSI) complex encircled by a ring of 18 CP43′ or IsiA ...light-harvesting complexes Nature 412 (2001) 745. Here we present a spectroscopic characterization by temperature-dependent absorption and fluorescence spectroscopy, site-selective fluorescence spectroscopy at 5 K, and circular dichroism of isolated PSI–IsiA, PSI and IsiA complexes from this cyanobacterium grown under iron starvation. The results suggest that the IsiA ring increases the absorption cross-section of PSI by about 100%. Each IsiA subunit binds about 16–17 chlorophyll
a (Chl
a) molecules and serves as an efficient antenna for PSI. Each of the monomers of the trimeric PSI complex contains two red chlorophylls, which presumably give rise to one exciton-coupled dimer and at 5 K absorb and fluoresce at 703 and 713 nm, respectively. The spectral properties of these C-703 chlorophylls are not affected by the presence of the IsiA antenna ring. The spectroscopic properties of the purified IsiA complexes are similar to those of the related CP43 complex from plants, except that the characteristic narrow absorption band of CP43 at 682.5 nm is missing in IsiA.
We report a structural characterization by electron microscopy and image analysis of a supramolecular complex consisting of photosystem I and light-harvesting complex I from the unicellular green ...alga
Chlamydomonas reinhardtii. The complex is a monomer, has longest dimensions of 21.3 and 18.2 nm in projection, and is significantly larger than the corresponding complex in spinach. Comparison with photosystem I complexes from other organisms suggests that the complex contains about 14 light-harvesting proteins, two or three of which bind at the side of the PSI-H subunit. We suggest that special light-harvesting I proteins play a role in the binding of phosphorylated light-harvesting complex II in state 2.
Almost all photosystem I (PSI) complexes from oxygenic photosynthetic organisms contain chlorophylls that absorb at longer wavelength than that of the primary electron donor P700. We demonstrate here ...that the low-energy pool of chlorophylls in the PSI-LHCI complex from the green alga Chlamydomonas reinhardtii, containing five to six pigments, is significantly blue-shifted (A(max) at 700 nm at 4 K) compared to that in the PSI core preparations from several species of cyanobacteria and in PSI-LHCI particles from higher plants. This makes them almost isoenergetic with the primary donor. However, they keep the other characteristic features of "red" chlorophylls: clear spectral separation from the bulk chlorophylls, big Stokes shift revealing pronounced electron-phonon coupling, and large homogeneous and inhomogeneous broadening of approximately 170 and approximately 310 cm(-1), respectively.
We aimed to evaluate the relationship between the period of obesity onset in the life cycle of obese adults with their cardiometabolic profile and response to clinical treatment.
A cross-sectional ...study carried out in 124 individuals, ≥18 years old, who received clinical treatment for obesity based on the same protocol of care and were stratified according to the period of obesity onset in childhood/adolescence (early) or adulthood (adult). Cardiometabolic profiles (e.g., hypertension, dyslipidemia and diabetes) and weight loss during clinical treatment (mean follow-up of 2 years) were compared among the groups. A ≥10% weight loss of the baseline weight was considered a successful response to clinical treatment.
Early-onset obesity group (n = 40) was younger at the beginning of follow-up 35.8(10.3) vs. 47.9(10.5) years, p < 0.001 and presented a higher frequency of BMI≥40kg/m2 29(72.5) vs. 42(50.0) %, p = 0.018) than the adult-onset obesity group (n = 84). Duration of obesity and of follow-up was similar in both groups as well as mean weight loss during the treatment and frequencies of hypertension, diabetes, pre-diabetes or dyslipidemia.
Weight loss response to clinical treatment of obesity as well as prevalence of cardiometabolic risk factors were similar in early- and adult-onset obesity.
•Age of onset of obesity in life did not influence the weight loss response.•Cardiometabolic profile was similar according to age of onset of obesity.•Weight loss response in treatment was not associated with duration of obesity.
A CZE method was validated and implemented for fast and accurate in‐process determination of adenovirus concentrations of downstream process samples obtained during manufacturing of adenovirus ...vector‐based vaccines. An analytical‐quality‐by‐design approach was embraced for method development, method implementation, and method maintenance. CZE provided separation of adenovirus particles from sample matrix components, such as cell debris, residual DNA and proteins. The intermediate precision of the virus particle concentration was 6.9% RSD and the relative bias was 2.3%. In comparison, the CZE method is intended to replace a quantitative polymerase chain reaction method which requires three replicates in three analytical runs to achieve an intermediate precision of 8.1% RSD. Given that, in addition, the time from sampling till reporting results of the CZE method was less than 2 h, whereas quantitative polymerase chain reaction requires 3 days, it follows that the CZE method enables faster processing times in downstream processing.
•A validated method for quantitative protein profiling in adenovirus-based vaccines.•14 Adenovirus proteins baseline separated within 17 min by RP-UPLC.•Adenovirus-based samples directly injected, ...needing no sample pretreatment.•The method can detect and quantify protein degradants.
A method for the quantitative determination of the protein composition of adenovirus-vector based vaccines was developed. The final method used RP-UPLC with UV absorbance detection, a C4 column (300 Å, 1.7 μm, 2.1 × 150 mm), and a water- acetonitrile (ACN) gradient containing trifluoroacetic acid (TFA) as ion-pairing agent. The chromatographic resolution between the various adenovirus proteins was optimized by studying the effect of the TFA concentration and the column temperature, applying a full factorial design of experiments. A reproducible baseline separation of all relevant adenovirus proteins could be achieved within 17 min run time. Samples containing adenovirus particles could be directly injected into the UPLC system without sample pretreatment. The viruses reproducibly dissociate into proteins in the UPLC system upon contact with the mobile phase containing ACN. The new RP-UPLC method was successfully validated for protein profiling and relative quantification of proteins in vaccine products based on adenovirus vector types 26 and 35. The intermediate precision of the relative peak areas of all proteins was between 1% and 14% RSD, except for the peak assigned to protein V (26% RSD). The method proved to be stability indicating with respect to thermal and oxidation stress of the adenovirus-vector based vaccine and was successfully implemented for the characterization of adenovirus-based products.
Soret-excited resonance Raman spectra of two types of pheophytin-exchanged photosystem II RCs are reported. The cofactor composition of the reaction centers was modified by exchanging pheophytin a ...for 131-deoxo-131-hydroxypheophytin a, yielding one preparation with selective replacement of the photochemically inactive pheophytin (HB) and a second one exhibiting total replacement of HB and 40% replacement of HA, the primary electron acceptor. Resonance Raman spectra indicate that the other bound cofactors present are not significantly perturbed by Pheo substitution. The resonance Raman contributions from HA and HB in the carbonyl stretching region are identified at 1679 and 1675 cm-1, respectively, indicating that both pheophytin molecules in the photosystem II reaction center have hydrogen-bonded keto-carbonyl groups. This conclusion differs from what is observed in the functionally related RCs of purple non-sulfur bacteria, where the keto-carbonyl group of HB is not hydrogen bonded, but confirms predictions from models based on protein sequence alignments.
Current methods for the identification and/or quantification of viral proteins in influenza virus and virosome samples suffer from long analysis times, limited protein coverage and/or low accuracy ...and precision. We studied and optimized capillary gel electrophoresis (CGE) in order to achieve faster and enhanced characterization and quantification of viral proteins. Sample preparation as well the composition of the gel buffer was investigated in order to achieve adequate protein separation in relatively short times. The total sample preparation (reduction and deglycosylation) could be carried out efficiently within two hours. Hydrodynamic injection, separation voltage, and capillary temperature were optimized in full factorial design. The final method was validated and showed good performance for hemagglutinin fragment 1 (HA1), hemagglutinin fragment 2 (HA2), matrix protein (M) and nucleoprotein (NP). The CGE method allowed identification of different virus strains based on their specific protein profile. B/Brisbane inactivated virus and virosome samples could be analyzed within one day. The CGE results (titers) were comparable to single radial immune-diffusion (SRID), but the method has the advantage of a much faster time to results. CGE analysis of A/Christchurch from upstream process demonstrated the applicability of the method to samples of high complexity. The CGE method could be used in the same analyte concentration range as the RP-HPLC method, but showed better precision and accuracy. Overall, the total analysis time for the CGE method was much shorter, allowing analysis of 100 samples in 4 days instead of 10 days for SRID.
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•The total sample preparation was reduced from over 16 h to less than two hours.•Both the run time and the resolution were improved by a factor of two.•4 influenza proteins were quantified in influenza virus and virosome material.•The CGE method was validated and applied for influenza viruses and virosomes.
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