Quantification of emicizumab by mass spectrometry in plasma of people with hemophilia A: A method validation study.

Background: Emicizumab is a new treatment option for people with hemophilia A. Emicizumab was approved with a body-weight-based dosage regimen, without laboratory monitoring requirements. Guidelines, however, recommend measuring emicizumab concentrations when the presence of antidrug antibodies is suspected. Furthermore, drug monitoring can be useful in clinical decision making, in adherence checking, and for research purposes. Therefore, we developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for quantifying emicizumab. We performed a validation study on this LC-MS/MS method quantifying emicizumab in the plasma of people with hemophilia A. Methods: Sample preparation for LC-MS/MS analysis included ammonium sulfate protein precipitation and trypsin digestion. A signature peptide of emicizumab and a matching stable isotope-labeled internal standard were used to quantify emicizumab by LC-MS/MS analysis. Validation was performed in accordance with the "Guideline on Bioanalytical Method Validation" of the European Medicines Agency (EMA). The LC-MS/MS method was cross validated against a modified and calibrated (r 2 Diagnostics) one-stage clotting assay (OSA). Conclusions: The LC-MS/MS method demonstrated linearity over a wide range of emicizumab concentrations, far exceeding the concentrations observed in people with hemophilia A. Precision and accuracy were excellent, and all other validation parameters were also within the acceptance EMA criteria. Cross validation showed that the LC-MS/MS method and the OSA-based method can be used interchangeably for drug monitoring of emicizumab without the application of a correction factor.

A generic sample preparation method for the multiplex analysis of seven therapeutic monoclonal antibodies in human plasma or serum with liquid chromatography-tandem mass spectrometry.

Due to the increasing number of therapeutic monoclonal antibodies (mAbs) used in the clinic, there is an increasing need for robust analytical methods to quantify total mAb concentrations in human plasma for clinical studies and therapeutic drug monitoring. We developed an easy, rapid, and robust sample preparation method for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. The method was validated for infliximab (IFX), rituximab (RTX), cetuximab (CTX), dupilumab (DPL), dinutuximab (DNX), vedolizumab (VDZ), and emicizumab (EMZ). Saturated ammonium sulfate (AS) was used to precipitate immunoglobulins in human plasma. After centrifugation, supernatant containing albumin was decanted, and the precipitated immunoglobulin fraction was re-dissolved in buffer containing 6M guanidine. This fraction was then completely denatured, reduced, alkylated, and trypsin digested. Finally, signature peptides from the seven mAbs were simultaneously quantified on LC-MS/MS together with their internal standards stable isotopically labeled peptide counterparts. The linear dynamic ranges (1 - 512 mg/L) of IFX, CTX, RTX, and EMZ showed excellent (R2 > 0.999) linearity and those of DPL, DNX, and VDZ showed good (R2 > 0.995) linearity. The method was validated in accordance with the EMA guidelines. EDTA plasma, sodium citrate plasma, heparin plasma, and serum yielded similar results. Prepared samples were stable at room temperature (20°C) and at 5°C for 3 days, and showed no decline in concentration for all tested mAbs. This described method, which has the advantage of an easy, rapid, and robust pre-analytical sample preparation, can be used as a template to quantify other mAbs in human plasma or serum.

Stigmatellin probes the electrostatic potential in the QB site of the photosynthetic reaction center.

The electrostatic potential in the secondary quinone (QB) binding site of the reaction center (RC) of the photosynthetic bacterium Rhodobacter sphaeroides determines the rate and free energy change (driving force) of electron transfer to QB. It is controlled by the ionization states of residues in a strongly interacting cluster around the QB site. Reduction of the QB induces change of the ionization states of residues and binding of protons from the bulk. Stigmatellin, an inhibitor of the mitochondrial and photosynthetic respiratory chain, has been proven to be a unique voltage probe of the QB binding pocket. It binds to the QB site with high affinity, and the pK value of its phenolic group monitors the local electrostatic potential with high sensitivity. Investigations with different types of detergent as a model system of isolated RC revealed that the pK of stigmatellin was controlled overwhelmingly by electrostatic and slightly by hydrophobic interactions. Measurements showed a high pK value (>11) of stigmatellin in the QB pocket of the dark-state wild-type RC, indicating substantial negative potential. When the local electrostatics of the QB site was modulated by a single mutation, L213Asp → Ala, or double mutations, L213Asp-L212Glu → Ala-Ala (AA), the pK of stigmatellin dropped to 7.5 and 7.4, respectively, which corresponds to a >210 mV increase in the electrostatic potential relative to the wild-type RC. This significant pK drop (ΔpK > 3.5) decreased dramatically to (ΔpK > 0.75) in the RC of the compensatory mutant (AA+M44Asn → AA+M44Asp). Our results indicate that the L213Asp is the most important actor in the control of the electrostatic potential in the QB site of the dark-state wild-type RC, in good accordance with conclusions of former studies using theoretical calculations or light-induced charge recombination assay.

The D1-D61N mutation in Synechocystis sp. PCC 6803 allows the observation of pH-sensitive intermediates in the formation and release of O2 from photosystem II.

The active site of photosynthetic water oxidation by Photosystem II (PSII) is a manganese-calcium cluster (Mn(4)CaO(5)). A postulated catalytic base is assumed to be crucial. CP43-Arg357, which is a candidate for the identity of this base, is a second-sphere ligand of the Mn(4)-Ca cluster and is located near a putative proton exit pathway, which begins with residue D1-D61. Transient absorption spectroscopy and time-resolved O(2) polarography reveal that in the D1-D61N mutant, the transfer of an electron from the Mn(4)CaO(5) cluster to Y(Z)(OX) and O(2) release during the final step of the catalytic cycle, the S(3)-S(0) transition, proceed simultaneously but are more dramatically decelerated than previously thought (t(1/2) of up to ~50 ms vs a t(1/2) of 1.5 ms in the wild type). Using a bare platinum electrode to record the flash-dependent yields of O(2) from mutant and wild-type PSII has allowed the observation of the kinetics of release of O(2) from extracted thylakoid membranes at various pH values and in the presence of deuterated water. In the mutant, it was possible to resolve a clear lag phase prior to the appearance of O(2), indicating formation of an intermediate before the onset of O(2) formation. The lag phase and the photochemical miss factor were more sensitive to isotope substitution in the mutant, indicating that proton efflux in the mutant proceeds via an alternative pathway. The results are discussed in comparison with earlier results obtained from the substitution of CP43-Arg357 with lysine and in regard to hypotheses concerning the nature of the final steps in photosynthetic water oxidation. These considerations led to the conclusion that proton expulsion during the initial phase of the S(3)-S(0) transition starts with the deprotonation of the primary catalytic base, probably CP43-Arg357, followed by efficient proton egress involving the carboxyl group of D1-D61 in a process that constitutes the lag phase immediately prior to O(2) formation chemistry.

Water oxidation by photosystem II: H(2)O-D(2)O exchange and the influence of pH support formation of an intermediate by removal of a proton before dioxygen creation.

Understanding the chemistry of photosynthetic water oxidation requires deeper insight into the interrelation between electron transfer (ET) and proton relocations. In photosystem II membrane particles, the redox transitions of the water-oxidizing Mn complex were initiated by nanosecond laser flashes and monitored by absorption spectroscopy at 360 nm (A(360)). In the oxygen evolution transition (S(3) + hν → S(0) + O(2)), an exponential decrease in A(360) (τ(O(2)) = 1.6 ms) can be assigned to Mn reduction and O(2) formation. The corresponding rate-determining step is the ET from the Mn complex to a tyrosine radical (Y(Z)(ox)). We find that this A(360) decrease is preceded by a lag phase with a duration of 170 ± 40 µs (τ(lag) at pH 6.2), indicating formation of an intermediate before ET and O-O bond formation and corroborating results obtained by time-resolved X-ray spectroscopy. Whereas τ(O(2)) exhibits a minor kinetic isotope effect and negligible pH dependence, formation of the intermediate is slowed significantly both in D(2)O (τ(lag) increase of ∼140% in D(2)O) and at low pH (τ(lag) of 30 ± 20 µs at pH 7.0 vs τ(lag) of 470 ± 80 µs at pH 5.5). These findings support the fact that in the oxygen evolution transition an intermediate is created by deprotonation and removal of a proton from the Mn complex, after Y(Z)(ox) formation but before the onset of electron transfer and O-O bond formation.

M234Glu is a component of the proton sponge in the reaction center from photosynthetic bacteria.

Bacterial reaction centers use light energy to couple the uptake of protons to the successive semi-reduction of two quinones, namely Q(A) and Q(B). These molecules are situated symmetrically in regard to a non-heme iron atom. Four histidines and one glutamic acid, M234Glu, constitute the five ligands of this atom. By flash-induced absorption spectroscopy and delayed fluorescence we have studied in the M234EH and M234EL variants the role played by this acidic residue on the energetic balance between the two quinones as well as in proton uptake. Delayed fluorescence from the P(+)Q(A)(-) state (P is the primary electron donor) and temperature dependence of the rate of P(+)Q(A)(-) charge recombination that are in good agreement show that in the two RC variants, both Q(A)(-) and Q(B)(-) are destabilized by about the same free energy amount: respectively approximately 100 +/- 5 meV and 90 +/- 5 meV for the M234EH and M234EL variants, as compared to the WT. Importantly, in the M234EH and M234EL variants we observe a collapse of the high pH band (present in the wild-type reaction center) of the proton uptake amplitudes associated with formation of Q(A)(-) and Q(B)(-). This band has recently been shown to be a signature of a collective behaviour of an extended, multi-entry, proton uptake network. M234Glu seems to play a central role in the proton sponge-like system formed by the RC protein.

Turnover of ubiquinone-0 at the acceptor side of photosynthetic reaction center.

The steady-state operation of photosynthetic reaction center from Rhodobacter sphaeroides was investigated by measuring the rate of cytochrome photo-oxidation under intensive continuous illumination (808 nm, 5 W cm(-2)). The native quinone UQ10 in Q(B) binding site of the reaction center was substituted by tailless UQ0 and the binding parameters and the turnover rate of the UQ0 was studied to test the recently discovered light-intensity dependent acceptor side effect (Gerencsér and Maróti 2006). The binding parameters of UQ0 (k(on) = 2.1 x 10(5) M(-1) s(-1) and k(off) = 100 s(-1)) were characteristic to the RC exposed to high light-intensity. The dissociation constant (K (D) = 480 microM) determined under high light intensity is 2-3 times larger than that determined from flash-experiments. The light-intensity dependent acceleration of cytochrome turnover measured on reaction center of inhibited proton binding was independent of the type of the quinone and was sensitive only to the size ("pressure") of the quinone pool. The dissociation constants of different types of semiquinones show similarly high (several orders of magnitude) increase in the modified conformation of the Q(B) binding pocket due to high intensity of illumination. This result indicates the exclusive role of the quinone headgroup in the binding of semiquinone to different conformations of the protein.

Mechanism of quinol oxidation by ferricenium produced by light excitation in reaction centers of photosynthetic bacteria.

The kinetics and thermodynamics of cyclic electron transfer through the isolated reaction center protein of photosynthetic bacterium Rhodobacter sphaeroides were determined in detergent (Triton X-100) solution. The redox reactions between the reducing (ubiquinol-0 or ubiquinol-10) and oxidizing species (ferricenium, ferricytochrome, or ferricyanide) produced chemically or by light excitation of the protein were monitored by absorption changes of the reactants and by acidification of the solution accompanied with the disappearance of the quinol. The bimolecular rate constants of reactions of anionic ubiquinol-0 with different oxidizing agents showed large variation: 5 x 10(8) M(-1) s(-1) for ferricenium, 3.5 x 10(5) M(-1) s(-1) for ferricyanide, and 1.5 x 10(5) M(-1) s(-1) for ferricytochrome. Although the redox partners were created in pairs by the same protein promptly after light excitation, their bimolecular redox reaction was not observed even in the case of the fastest reacting partners of ferricenium and ubiquinol-0. Instead, they equilibrate with the corresponding (donor and acceptor) pools before the electron is transferred. The (logarithms of the) observed rate constants of quinol oxidation showed steep pH-dependence for water soluble ubiquinol-0 (slope +1) and mild pH-dependence for hydrophobic ubiquinol-10 (slope approximately 0.25). Combined with studies of the ionic strength dependence of the rate, it was concluded that the electron-transfer pathways of ubiquinol-0 and ubiquinol-10 oxidation started from their anionic and neutral forms, respectively. The mild pH-dependence of the rate of ubiquinol-10 oxidation came from the electrostatic interactions between ferricenium and the pH-dependent surface charges of the reaction center. The results help to understand, monitor, and design (cyclic) electron flow in bioenergetic proteins.

Uncoupling of electron and proton transfers in the photocycle of bacterial reaction centers under high light intensity.

Photosynthetic reaction centers produce and export oxidizing and reducing equivalents in expense of absorbed light energy. The formation of fully reduced quinone (quinol) requires a strict (1:1) stoichiometric ratio between the electrons and H(+) ions entering the protein. The steady-state rates of both transports were measured separately under continuous illumination in the reaction center from the photosynthetic bacterium Rhodobacter sphaeroides. The uptake of the first proton was retarded by different methods and made the rate-limiting reaction in the photocycle. As expected, the rate constant of the observed proton binding remained constant (7 s(-)(1)), but that of the cytochrome photooxidation did show a remarkably large increase from 14 to 136 s(-)(1) upon increase of the exciting light intensity up to 5 W/cm(2) (808 nm) at pH 8.4 in the presence of NiCl(2). This corresponds to about 20:1 (e(-):H(+)) stoichiometric ratio. The observed enhancement is linearly proportional to the light intensity and the rate constant of the proton uptake by the acceptor complex and shows saturation character with quinone availability. For interpretation of the acceleration of cytochrome turnover, an extended model of the photocycle is proposed. A fraction of photochemically trapped RC can undergo fast (>10(3) s(-)(1)) conformational change where the semiquinone loses its high binding affinity (the dissociation constant increases by more than 5 orders of magnitude) and dissociates from the Q(B) binding site of the protein with a high rate of 4000 s(-)(1). Concomitantly, superoxide is being produced. No H(+) ion is taken up, and no quinol is created by the photocycle which is operating in about 25% of the reaction centers at the highest light intensity (5500 s(-)(1)) and slowest proton uptake (3.5 s(-)(1)) used in our experiments. The possible physical background of the light-induced conformational change and the relationship between the energies of dissociation and redox changes of the quinone in the Q(B) binding sites are discussed.

The use of the SPSA method in ECG analysis.

The classification, monitoring, and compression of electrocardiogram (ECG) signals recorded of a single patient over a relatively long period of time is considered. The particular application we have in mind is high-resolution ECG analysis, such as late potential analysis, morphology changes in QRS during arrythmias, T-wave alternants, or the study of drug effects on ventricular activation. We propose to apply a modification of a classical method of cluster analysis or vector quantization. The novelty of our approach is that we use a new distortion measure to quantify the distance of two ECG cycles, and the class-distortion measure is defined using a min-max criterion. The new class-distortion-measure is much more sensitive to outliers than the usual distortion measures using average-distance. The price of this practical advantage is that computational complexity is significantly increased. The resulting nonsmooth optimization problem is solved by an adapted version of the simultaneous perturbation stochastic approximation (SPSA) method of. The main idea is to generate a smooth approximation by a randomization procedure. The viability of the method is demonstrated on both simulated and real data. An experimental comparison with the widely used correlation method is given on real data.

Effect of binding of Cd2+ on bacterial reaction center mutants: proton-transfer uses interdependent pathways.

In bacterial reaction center of Rhodobacter sphaeroides, Cd2+ binds in stoichiometric amount to the protein. In the wild type, this results into a notable decrease of the rates of electron-transfer between the two quinone acceptors after the first (kAB(1)) and second flash (kAB(2)). We have studied these effects in two single mutants, L209PY and L209PF. L209Pro is situated in a protein region rich in hydrogen-bond networks involving water molecules. We show that (1) the combined effects of Cd2+ binding and point mutations have a cumulative consequence in the two mutants, decreasing very substantially the observed rates of electron-transfer. Interestingly, the [Cd2+] titration curves of kAB(2) in the L209PY and L209PF mutants are nearly superimposable to those previously reported for the M17DN and L210DN mutants (Paddock, M. L., Feher, G., and Okamura, M. Y. (2000) Proc. Natl. Acad. Sci U.S.A. 97, 1548-1553). These observations suggest a common effect of all of these mutations (L209, M17, L210) on the protonation state of the histidine cluster to which Cd2+ binds; (2) in the L209PY mutant, the pH titration curves of kAB(1), kAB(2), and k(H)(+), the proton-transfer rate at the second flash, are systematically downshifted by 1.5-2 pH units in the presence of 300 microM Cd2+, similarly to the wild type RCs (Gerencser, L., and Maroti, P. (2001) Biochemistry 40, 1850-1860). We propose that Cd2+ binding influences the electrostatics of interdependent ways of proton penetration within the protein, involving at least, directly or indirectly, L209P, L210D, and M17D, probably in conjunction with hydrogen-bonded connected water molecules.