Antibody-drug conjugates (ADCs) with biotin as a model cargo tethered to IgG1 mAbs via different linkers and conjugation methods were prepared and tested for thermostability and ability to bind target antigen and Fc receptor. Most conjugates demonstrated decreased thermostability relative to unconjugated antibody, based on DSC, with carbohydrate and amine coupled ADCs showing the least effect compared with thiol coupled conjugates. A strong correlation between biotin-load and loss of stability is observed with thiol conjugation to one IgG scaffold, but the stability of a second IgG scaffold is relatively insensitive to biotin load. The same correlation for amine coupling was less significant. Binding of antibody to antigen and Fc receptor was investigated using surface plasmon resonance. None of the conjugates exhibited altered antigen affinity. Fc receptor FcγIIb (CD32b) interactions were investigated using captured antibody conjugate. Protein G and Protein A, known inhibitors of Fc receptor (FcR) binding to IgG, were also used to extend the analysis of the impact of conjugation on Fc receptor binding. H10NPEG4 was the only conjugate to show significant negative impact to FcR binding, which is likely due to higher biotin-load compared with the other ADCs. The ADC aHISNLC and aHISTPEG8 demonstrated some loss in affinity for FcR, but to much lower extent. The general insensitivity of target binding and effector function of the IgG1 platform to conjugation highlight their utility. The observed changes in thermostability require consideration for the choice of conjugation chemistry, depending on the system being pursued and particular application of the conjugate.

Analytical ultracentrifugation (AUC) is a first principles based method to determine absolute sedimentation coefficients and buoyant molar masses of macromolecules and their complexes, reporting on their size and shape in free solution. The purpose of this multi-laboratory study was to establish the precision and accuracy of basic data dimensions in AUC and validate previously proposed calibration techniques. Three kits of AUC cell assemblies containing radial and temperature calibration tools and a bovine serum albumin (BSA) reference sample were shared among 67 laboratories, generating 129 comprehensive data sets. These allowed for an assessment of many parameters of instrument performance, including accuracy of the reported scan time after the start of centrifugation, the accuracy of the temperature calibration, and the accuracy of the radial magnification. The range of sedimentation coefficients obtained for BSA monomer in different instruments and using different optical systems was from 3.655 S to 4.949 S, with a mean and standard deviation of (4.304 ± 0.188) S (4.4%). After the combined application of correction factors derived from the external calibration references for elapsed time, scan velocity, temperature, and radial magnification, the range of s-values was reduced 7-fold with a mean of 4.325 S and a 6-fold reduced standard deviation of ± 0.030 S (0.7%). In addition, the large data set provided an opportunity to determine the instrument-to-instrument variation of the absolute radial positions reported in the scan files, the precision of photometric or refractometric signal magnitudes, and the precision of the calculated apparent molar mass of BSA monomer and the fraction of BSA dimers. These results highlight the necessity and effectiveness of independent calibration of basic AUC data dimensions for reliable quantitative studies.

Membrane-bound cytochrome P4503A4 (CYP3A4) is the major source of enzymatic drug metabolism. Although several structural models of CYP3A4 in various ligand complexes are available, none includes a lipid bilayer. Details of the effects of the membrane on protein dynamics and solvation, and access channels for ligands, remain uncertain. H/D exchange mass spectrometry (H/DXMS) with ligand free CYP3A4 containing a deletion of residues 3–12, compared to that of the full length wild type, in lipid nanodiscs afforded 91% sequence coverage. Deuterium exchange was fast in the F- and G-helices, HI loop, and C-terminal loop. In contrast, there is very low exchange in the F′-and G′-helices. The results are consistent with the overall membrane orientation of CYP3A4 suggested by published MD simulations and spectroscopic results, and the solvent accessibility of the F/G loop suggests that it is not deeply membrane-embedded. Addition of ketoconazole results in only modest, but global, changes in solvent accessibility. Interestingly, with ketoconazole bound some peptides become less solvent accessible or dynamic, including the F- and G-helices, but several peptides demonstrate modestly increased accessibility. Differential scanning calorimetry (DSC) of CYP3A4-nanodiscs suggests membrane-induced stabilization compared to that of aggregated CYP3A4 in buffer, and this stabilization is enhanced upon addition of the ligand ketoconazole. This ligand-induced stabilization is accompanied by a very large increase in ΔH for CYP3A4 denaturation in nanodiscs, possibly due to increased CYP3A4–membrane interactions. Together, the results suggest a distinct orientation of CYP3A4 on the lipid membrane, and they highlight likely solvent access channels, which are consistent with several MD simulations.

Background: P-glycoprotein plays a role in drug resistance and drug interactions. Results: Surface plasmon resonance with P-glycoprotein in lipid nanodiscs indicates that inhibitory antibodies uncouple the ATP hydrolysis of P-gp from its transport function. Conclusion: Inhibitory antibodies do not prevent P-gp flux through the catalytic cycle, but rather block drug efflux without inhibiting ATP hydrolysis. Significance: The results clarify the mechanism of inhibitory antibodies to P-gp.

The fraction of IgG antibodies with anti-oligomeric Aβ affinity and surface sialic acid was compared between Octagam and Gammagard intravenous immunoglobulin (IVIG) using two complementary surface plasmon resonance methods. These comparisons were performed to identify if an elevated fraction existed in Gammagard, which reported small putative benefits in a recent Phase III clinical trial for Alzheimer’s Disease. The fraction of anti-oligomeric Aβ IgG was found to be higher in Octagam, for which no cognitive benefits were reported. The fraction and location of surface-accessible sialic acid in the Fab domain was found to be similar between Gammagard and Octagam. These findings indicate that anti-oligomeric Aβ IgG and total surface sialic acid alone cannot account for reported clinical differences in the two IVIG products. A combined analysis of sialic acid in anti-oligomeric Aβ IgG did reveal a notable finding that this subgroup exhibited a high degree of surface sialic acid lacking the conventional α2,6 linkage. These results demonstrate that the IVIG antibodies used to engage oligomeric Aβ in both Gammagard and Octagam clinical trials did not possess α2,6-linked surface sialic acid at the time of administration. Anti-oligomeric Aβ IgG with α2,6 linkages remains untested as an AD treatment.

Methods for the initial steps of surface plasmon resonance analysis of membrane proteins incorporated in lipid nanodiscs are described. Several types of BiacoreTM sensor chips are available and require distinct strategies to immobilize proteonanodiscs on the chip surface. The procedures for immobilization on three of these chips (NTA, antibody coupled CM5, and L1) are described and results are demonstrated for a model system with cytochrome P4503A4 (CYP3A4) in nanodiscs binding to a polyclonal anti-CYP3A4 antibody. Advantages and disadvantages of each chip type are considered.

Hepatic problems related to a Fontan circulation have been highlighted and elastography using ultrasound is a non-invasive tool that can measure the severity of hepatic stiffness. We investigated the hepatic stiffness using shear wave elastography (SWE) and related factors in patients with a Fontan circulation. This study enrolled 64 patients with a Fontan circulation who underwent cardiac catheterization and abdominal ultrasound from 2011 to 2015. The correlation between the laboratory tests, hemodynamic factors by cardiac catheterization, and SWE was evaluated. The patients were classified into non-cirrhotic level (≥ 2.0 m/s) and cirrhotic level (< 2.0 m/s) groups by the SWE value. The mean age was 17.6 years and the mean duration after the Fontan operation was 12.1 years. The mean value of SWE in patients (1.95 m/s) was higher than the normal (< 1.3 m/s). The SWE was higher in patients without than those with a fenestration (2.03 vs. 1.75 m/s, P = 0.003). In a multiple regression analysis between SWE and other factors, the CVP, fenestration, and lipoprotein Apo B had a significant correlation. In a multivariate analysis of cirrhotic level group, the CVP was the only significant factor. The hepatic stiffness had significantly progressed in most patients with a Fontan circulation. A low CVP and Fontan circulation with a fenestration might reduce the progression of the hepatic stiffness.

BackgroundThe rate of the prenatal diagnosis of congenital heart disease is increasing along with advances in fetal echocardiography techniques. Here, we aimed to investigate the trend of the use of fetal echocardiography over time and to compare the medical costs of congenital heart disease treatment according to whether fetal echocardiography was performed.MethodsWe reviewed our hospital’s database, and patients who underwent the first surgery for congenital heart disease within 30 days of birth during 2005–2007, 2011–2013, and 2017–2019 were included. The severity of congenital heart disease diagnosed in each case was evaluated according to The Society of Thoracic Surgeons-European Association for Cardio-Thoracic Surgery Congenital Heart Surgery Mortality Scores (STS-EACTS Mortality Scores) and Mortality Categories (STAT Mortality Categories).ResultsIn total, 375 patients were analyzed, and fetal echocardiography use increased significantly after the 2010s compared with in 2005–2007 (19.1% vs. 39%, p = 0.032 in Mortality Category 1–3; 15.5% vs. 69.5%, p = 0.000 in Mortality Category 4–5). Additionally, the mean STS-EACTS Mortality Score was higher in prenatally diagnosed patients than in postnatally diagnosed patients (2.287 vs. 1.787, p = 0.001). In the recent period, there was no significant difference in hospitalization durations and medical costs according to whether or not fetal echocardiography was performed.ConclusionsThis single center study showed the use of fetal echocardiography is increasing. Further, prenatal diagnosis with fetal echocardiography causing no differences in medical costs in recent years. Therefore, we suggest that fetal echocardiography can be applied more widely without increasing the economic burden.