Optimization of Flow Imaging Microscopy Setting Using Spherical Beads with Optical Properties Similar to Those of Biopharmaceuticals.

Flow imaging microscopy (FIM) is widely used to characterize biopharmaceutical subvisible particles (SVPs). The segmentation threshold, which defines the boundary between the particle and the background based on pixel intensity, should be properly set for accurate SVP quantification. However, segmentation thresholds are often subjectively and empirically set, potentially leading to variations in measurements across instruments and operators. In the present study, we developed an objective method to optimize the FIM segmentation threshold using poly(methyl methacrylate) (PMMA) beads with a refractive index similar to that of biomolecules. Among several candidate particles that were evaluated, 2.5-µm PMMA beads were the most reliable in size and number, suggesting that the PMMA bead size analyzed by FIM could objectively be used to determine the segmentation threshold for SVP measurements. The PMMA bead concentrations measured by FIM were highly consistent with the indicative concentrations, whereas the PMMA bead size analyzed by FIM decreased with increasing segmentation threshold. The optimal segmentation threshold where the analyzed size was closest to the indicative size differed between an instrument with a black-and-white camera and that with a color camera. Inter-instrument differences in SVP concentrations in acid-stressed recombinant adeno-associated virus (AAV) and protein aggregates were successfully minimized by setting an optimized segmentation threshold specific to the instrument. These results reveal that PMMA beads can aid in determining a more appropriate segmentation threshold to evaluate biopharmaceutical SVPs using FIM.

Critical Calibration of Mass Photometry for Higher-Mass Samples Such as Adeno-Associated Virus Vectors.

Mass photometry (MP) is a label-free, single-molecule technique that can determine molecular mass distribution with very low sample consumption in a short time. Because of the established experimental instrument and analytical software, MP measurements may be readily obtained; thus, the application of MP is expanding, especially in the fields of bioscience and biotechnology. However, because the MP data quality is strongly focus-dependent, optical settings must be intrinsically strict. In this study, we report the importance of the critical calibration of the mass photometer, which is required for the accurate estimation of high-molecular mass samples, such as adeno-associated virus vectors. Additionally, a method for optimizing the instrument settings, including the calibration of the stage, is presented.

Chemical-gas Sterilization of External Surface of Polymer-based Prefilled Syringes and Its Effect on Stability of Model Therapeutic Protein.

To reduce the risk of infection during intravitreal injections, the external surface of prefilled syringes (PFSs) must be sterilized. Usually, ethylene oxide (EO) gas or vaporized hydrogen peroxide (VHP) is used for sterilization. More recently, nitrogen dioxide (NO2) gas sterilization has been developed. It is known that gas permeability is approximately zero into glass-PFSs. However, polymer-PFSs (P-PFSs) have relatively high gas permeability. Therefore, there are concerns about the potential impact of external surface sterilization on drug solutions in P-PFSs. In this study, P-PFSs [filled with water for injection (WFI) or human serum albumin (HSA) solution] were externally sterilized using EO, VHP, and NO2 gases. For the WFI-filled syringes, the concentration of each gas that ingressed into the WFI was measured. For the HSA solution-filled syringes, the physical and chemical degradation of HSA molecules by each sterilant gas was quantified. For the EO- or VHP-sterilized syringes, the ingressed EO or hydrogen peroxide (H2O2) molecules were detected in the filled WFI. Additionally, EO-adducted or oxidized HSA molecules were observed in the HSA-filled syringes. In contrast, the NO2-sterilized WFI-filled syringes exhibited essentially immeasurable ingressed NO2, and protein degradation was not detected in HSA-filled syringes.

Dynamics of Conformational Changes in Full-Length Phytochrome from Cyanobacterium Synechocystis sp. PCC6803 (Cph1) Monitored by Time-Resolved Translational Diffusion Detection.

Phytochromes (Phys) are photoreceptor proteins that sense red/far-red light in plants, fungi, and bacteria. The proteins consist of a light-sensing photosensory module and a signaling output module, which is typically a histidine kinase (HK) domain in bacteriophytochromes. Although the time-resolved detection of the HK domain is essential for obtaining insights into the reaction mechanism of photoactivation, it has been very difficult to detect the change. Here, the reaction of Cph1, one of the Phys found in the cyanobacterium Synechocystis sp. PCC6803, was studied using time-resolved translational diffusion detection. It was found that the kinetics of the HK domain movement of the Cph1 dimer could be monitored successfully. The diffusion coefficient of the Cph1 dimer decreases significantly with a time constant similar to that of the final step of the reaction monitored by the transient absorption method (780 ms), whereas the monomer does not exhibit this change. We attribute this change to the closed-to-open type of conformational change in the HK domain of the Cph1 dimer without the secondary structure change. The fact that the rate is similar to that from the transient absorption method suggests that the proton uptake at His260 is the rate-determining step of the conformational change.

Photoinduced Orientation Change of the Dimer Structure of the Pr-I State of Cph1Δ2.

Phytochromes are red and far-red light sensor proteins found in many organisms. Photoisomerization of a chromophore triggers subsequent reactions leading to conformational changes that are essential for signaling. Conformational changes of the N-terminal sensor domain of cyanobacterium phytochrome 1 from Synechocystis sp. PCC6803 (Cph1Δ2) were studied in this report primarily by the transient grating (TG) method in the time domain. Although the reaction kinetics monitored by ultraviolet-visible absorption changes were insensitive to variations in pH, it was found for the first time that the diffusion coefficient and molecular volume monitored by the TG method are significantly dependent on pH. This pH sensitivity indicates that the conformational changes of Cph1Δ2 during the reaction are modulated by pH and was explained consistently by two contributions from two isomers (Pr-I and Pr-II) possessing different protonation states of His260. Thus, His260 is responsible for the conformational heterogeneity of Pr, as discussed previously, and this protonation difference leads to different conformational changes of the sensor domain of Cph1 during the reaction. The photoreaction dynamics of the Pr-I state was studied in detail, and a significant diffusion change was observed, which was attributed to a change in the orientation (quaternary structure) of the dimer with a time constant of 400 µs. Furthermore, it was revealed that this reorientation of the dimer was induced by a conformational change in the tongue region. New site-directed mutations to change the dimer-monomer equilibrium were reported.

Dynamics of the amino-terminal and carboxyl-terminal helices of Arabidopsis phototropin 1 LOV2 studied by the transient grating.

Recently, conformational changes of the amino-terminal helix (A'α helix), in addition to the reported conformational changes of the carboxyl-terminal helix (Jα helix), have been proposed to be important for the regulatory function of the light-oxygen-voltage 2 domain (LOV2) of phototropin 1 from Arabidopsis. However, the reaction dynamics of the A'α helix have not been examined. Here, the unfolding reactions of the A'α and Jα helices of the LOV2 domain of phototropin 1 from Arabidopsis thaliana were investigated by the time-resolved transient grating (TG) method. A mutant (T469I mutant) that renders the A'α helix unfolded in the dark state showed unfolding of the Jα helix with a time constant of 1 ms, which is very similar to the time constant reported for the wild-type LOV2-linker sample. Furthermore, a mutant (I608E mutant) that renders the Jα helix unfolded in the dark state exhibited an unfolding process of the A'α helix with a time constant of 12 ms. On the basis of these experimental results, it is suggested that the unfolding reactions of these helices occurs independently.