Post-acquisition correction of NMR spectra distorted by dynamic and static field inhomogeneity of cryogen-free magnets.

Post-acquisition correction of NMR spectra is an important part of NMR spectroscopy that enables refined NMR spectra to be obtained, clean from undesirable out-phasing, broadening and noising. We describe analytical and numerical mathematical methods for post-acquisition correction of NMR spectra distorted by static and dynamic magnetic field inhomogeneity caused by imperfections of main superconducting coils and the cold head operation, typical for cryogen-free magnets. For the dynamic inhomogeneity, we apply a variant of the general reference deconvolution method, complemented with our mathematical analysis of spectral parameters. For the static inhomogeneity, we apply the method of a delayed Fourier transform, also supported with our analytical calculations. We verify our approach by correction processing of high-field experimental liquid-state 1H NMR spectra of water and ethanol as well as solid-state 13C MAS NMR spectra of adamantane and obtain good results for both static and dynamic field distortions. This work complements our previous work on instrumental suppression of dynamic distortions caused by the cold head operation. The results presented contribute well to the general field of processing NMR spectra and serve towards a more extensive use of cryogen-free magnets in high-resolution NMR spectroscopy.

Cryogen-free 400 MHz (9.4 T) solid state MAS NMR system with liquid state NMR potential.

We show that the temporal magnetic field distortion generated by the Cold Head operation can be removed and high quality Solid-State Magic Angle Spinning NMR results can be obtained with a cryogen-free magnet. The compact design of the cryogen-free magnets allows for the probe to be inserted either from the bottom (as in most NMR systems) or, more conveniently, from the top. The magnetic field settling time can be made as short as an hour after a field ramp. Therefore, a single cryogen-free magnet can be used at different fixed fields. The magnetic field can be changed every day without compromising the measurement resolution.

A new method to measure the temporal magnetic field instabilities in cryogen-free magnets for magnetic resonance.

Despite the obvious advantages of cryogen-free magnets for NMR such as independence of liquid helium supply and the possibility to use the same magnet at different fields, the practical application of those magnets remains limited because of temporal magnetic field distortions associated with cryogen-free cold head operation. A new experimental method for the simple and reliable detection of the temporal field distortions is described in this paper. The accuracy of the magnetic field measurements by this method is two orders of magnitude higher than by conventional MetroLab Tesla meter. This has enabled us to make improvements in the design of cryogen-free magnets by reducing the amplitude of such field distortions down to sub ppb level. This then results in cryogen-free magnets that are suitable for MRI and MAS NMR applications.

A method for fast field settling in cryogen-free superconducting magnets for NMR.

We propose a fast algorithm to energise a cryogen free magnet to a highly persistent state. A decay rate as low as 0.021 â€‹ppm/h can be achieved in less than an hour after reaching the target field. The decay rate drops further to 0.0004 â€‹ppm/h in the following 48 â€‹h. This procedure can be applied at different values of target field, which makes it feasible to use a single magnet for study of various NMR lines at different fields. The mechanism of establishing a highly stable magnetic field can be understood on the basis of the magnetic properties of the superconducting wire, which were studied using a vibrating sample magnetometer. The results confirm the high quality of the superconducting wire and joints.

On the magnetic field stability of cryogen-free magnets for magnetic resonance applications.

The temporal magnetic field variation associated with Cold Head operation in cryogen-free magnets was studied. Three different mechanisms for such variations were tested separately and rated by their importance. It was found that mechanical displacement of the magnet inside the cryostat is the main issue of magnetic field perturbation. In a cryostat with the Gifford- McMahon type of cryocooler, motion of the displacer with magnetic material inside also produces significant field modulation. The temperature variation of the magnet, although noticeable, leads to smaller field distortions compared to the previous two factors. It was shown that the temporal magnetic field variation could be reduced down to below 20 â€‹ppb level that could be acceptable for MRI and MAS NMR applications. It was also shown that a single cryogen-free magnet could be easily used at different fields on a day-to-day basis without compromising the field stability unlike magnets housed in a liquid helium reservoir.

Estimating real-time infiltration for use in residential ventilation control.

Minimum outdoor air ventilation rates specified in standards such as ASHRAE Standard 62.2 are generally based on envelope airtightness, building floor area, geographical location, and number of occupants. ASHRAE Standard 62.2 allows for a constant infiltration credit, which reduces the required mechanical ventilation. However, infiltration rates vary based on the weather and system operation. Thus, mechanical systems could potentially operate less if the real-time (RT) infiltration rate were known and used to adjust the mechanical ventilation rate. CONTAM models of two test houses on the campus of the National Institute of Standards and Technology were verified with measurements and used to simulate hourly infiltration rates in three cities. The infiltration rates were passed to a theoretical controller that changed the hourly mechanical ventilation rate to meet the ventilation requirement. Simulated energy use and relative annual occupant exposure for this RT control strategy was compared with ventilating at a constant rate. Implementation of the RT control strategy resulted in annual average energy savings of $66USD across both houses and three cities without increasing the annual occupant exposure compared with ventilating continuously at a constant rate. The authors discuss the advantages and limitations of the proposed real-time ventilation control strategy.

In Vivo Stability Profiles of Anti-factor D Molecules Support Long-Acting Delivery Approaches.

The collection of aqueous humor (phase 1 b/2 Mahalo study) from patients dosed intravitreally with anti-factor D (AFD; FCFD4514S, lampalizumab), a humanized antibody fragment previously under investigation to treat geographic atrophy (GA) secondary to age-related macular degeneration, presented a unique opportunity to examine AFD properties in clinical samples. We investigated AFD stability and target-binding characteristics to set up strategies for engineering and evaluating optimized molecules that enable less frequent dosing. Two variants, AFD.v8 and AFD.v14, were evaluated as alternatives to AFD for longer-acting treatments. Mass spectrometry, surface plasmon resonance, and immunoassay were used to assess AFD stability and binding activity in aqueous humor samples from Mahalo patients. In vitro stability and binding activity of AFD, AFD.v8, and AFD.v14 were assessed in human vitreous humor versus buffer at 37 °C over 16 weeks and in vivo in rabbits over 28 days along with pharmacokinetic determinations. In human aqueous humor, AFD specific binding was >85% through 30 days, and deamidation was <3% through 60 days, consistent with the AFD stability and binding activity in vitreous humor from humans in vitro and rabbits in vivo. Target binding, stability, and rabbit pharmacokinetic parameters of AFD.v8 and AFD.v14 were similar to those of AFD. Physiological stability and activity of AFD translated across in vitro and in vivo studies in humans and rabbits. The two variants AFD.v8 and AFD.v14 demonstrated comparable potency and pharmacokinetics. These findings, along with previously demonstrated improved solubility of AFD.v8 and AFD.v14, provide proof-of-concept for developing other similar long-acting therapeutic variants.

Optimization and qualification of the single molecule array digital immunoassay for IL-12p70 in plasma of cancer patients.

AIM: Cytokine/chemokine levels can reflect the pharmacodynamics of checkpoint inhibitors. The single molecule array (Simoa) HD-1 is a sensitive next-generation immunoassay platform for quantification of low abundance proteins, with potential for cancer immunotherapy mechanism of action studies. RESULTS: The Simoa IL-12p70 reagents, standard curve and test conditions were optimized for improved precision and linearity of dilution in plasma of cancer patients. The assay achieved a lower limit of quantification of 0.08 pg/ml, with 27/29 samples recording above lower limit of quantification, precision ≤20% CV and accuracy within 80-120%. CONCLUSION: Simoa enabled quantification of IL-12p70 at sub-pg/ml levels in cancer patients and was superior to Simple Plex™ and Aushon® in overall performance. This study qualifies the user-modified IL-12p70 immunoassay to measure pharmacodynamic changes in plasma during cancer immunotherapy.

Bioanalytical qualification of clinical biomarker assays in plasma using a novel multi-analyte Simple Plex™ platform.

AIM: Immune-checkpoint inhibitors are presumed to break down the tolerogenic state of immune cells by activating T-lymphocytes that release cytokines and enhance effector cell function for elimination of tumors. Measurement of cytokines is being pursued for better understanding of the mechanism of action of immune-checkpoint inhibitors, as well as to identify potential predictive biomarkers. RESULTS: In this study, we show bioanalytical qualification of cytokine assays in plasma on a novel multi-analyte immunoassay platform, Simple Plex™. The qualified assays exhibited excellent sensitivity as evidenced by measurement of all samples within the quantifiable range. The accuracy and precision were 80-120% and 10%, respectively. CONCLUSION: The qualified assays will be useful in assessing mechanism of action cancer immunotherapies.

Commercial biomarker assays: friend and foe.

AIM: Commercial kits can provide a convenient solution for measuring circulating biomarkers to support drug development. However, their suitability should be assessed in the disease matrix of interest. METHODOLOGY: Twelve biomarkers were evaluated in samples from patients with rheumatoid arthritis. We used immunoassay kits from five vendors on three multiplexed platforms. Kit suitability was evaluated on the basis of detectability and prespecified performance acceptance criteria. RESULTS: Assays had varying levels of sensitivity and susceptibility to interference by matrix components. Only a few assays in the multiplexed kits were found to be suitable. In general, kits for analytes that passed our assay criteria showed good correlation between vendors. CONCLUSION: The data from this study demonstrate that the majority of assays on multiplexed kits evaluated either lacked sensitivity and/or had poor performance, which diminishes the utility of the multiplexing approach.

A mechanistic pharmacokinetic/pharmacodynamic model of factor D inhibition in cynomolgus monkeys by lampalizumab for the treatment of geographic atrophy.

Lampalizumab is an antigen-binding fragment of a humanized monoclonal antibody against complement factor D (CFD), a rate-limiting enzyme in the activation and amplification of the alternative complement pathway (ACP), which is in phase III clinical trials for the treatment of geographic atrophy. Understanding of the pharmacokinetics, pharmacodynamics, and biodistribution of lampalizumab following intravitreal administration in the ocular compartments and systemic circulation is limited but crucial for selecting doses that provide optimal efficacy and safety. Here, we sought to construct a semimechanistic and integrated ocular-systemic pharmacokinetic-pharmacodynamic model of lampalizumab in the cynomolgus monkey to provide a quantitative understanding of the ocular and systemic disposition of lampalizumab and CFD inhibition. The model takes into account target-mediated drug disposition, target turnover, and drug distribution across ocular tissues and systemic circulation. Following intravitreal administration, lampalizumab achieves rapid equilibration across ocular tissues. Lampalizumab ocular elimination is relatively slow, with a τ1/2 of approximately 3 days, whereas systemic elimination is rapid, with a τ1/2 of 0.8 hours. Target-independent linear clearance is predominant in the eye, whereas target-mediated clearance is predominant in the systemic circulation. Systemic CFD synthesis was estimated to be high (7.8 mg/day); however, the amount of CFD entering the eye due to influx from the systemic circulation was small (<10%) compared with the lampalizumab dose and is thus expected to have an insignificant impact on the clinical dose-regimen decision. Our findings support the clinical use of intravitreal lampalizumab to achieve significant ocular ACP inhibition while maintaining low systemic exposure and minimal systemic ACP inhibition.

Complement inhibition in cynomolgus monkeys by anti-factor d antigen-binding fragment for the treatment of an advanced form of dry age-related macular degeneration.

Anti-factor D (AFD; FCFD4514S, lampalizumab) is a humanized IgG Fab fragment directed against factor D (fD), a rate-limiting serine protease in the alternative complement pathway (AP). Evaluation of AFD as a potential intravitreal (IVT) therapeutic for dry age-related macular degeneration patients with geographic atrophy (GA) is ongoing. However, it is unclear whether IVT administration of AFD can affect systemic AP activation and potentially compromise host-immune responses. We characterized the pharmacologic properties of AFD and assessed the effects of AFD administered IVT (2 or 20 mg) or intravenous (0.2, 2, or 20 mg) on systemic complement activity in cynomolgus monkeys. For the IVT groups, serum AP activity was reduced for the 20 mg dose group between 2 and 6 hours postinjection. For the intravenous groups, AFD inhibited systemic AP activity for periods of time ranging from 5 minutes (0.2 mg group) to 3 hours (20 mg group). Interestingly, the concentrations of total serum fD increased up to 10-fold relative to predose levels following administration of AFD. Furthermore, AFD was found to inhibit systemic AP activity only when the molar concentration of AFD exceeded that of fD. This occurred in cynomolgus monkeys at serum AFD levels ≥2 µg/ml, a concentration 8-fold greater than the maximum serum concentration observed following a single 10 mg IVT dose in a clinical investigation in patients with GA. Based on these findings, the low levels of serum AFD resulting from IVT administration of a clinically relevant dose are not expected to appreciably affect systemic AP activity.

A novel homogeneous Biotin-digoxigenin based assay for the detection of human anti-therapeutic antibodies in autoimmune serum.

Electrochemiluminescence (ECL) assays have been widely used for the detection of anti-therapeutic antibodies (ATAs) against biotherapeutics. With the discontinuation of BioVeris (BV) ECL platform, an alternative technology was needed to replace BV assays to ensure continuous support of multi-year clinical studies. After evaluation of several immunoassay platforms, a novel homogeneous Biotin-digoxigenin (DIG) based bridging ELISA format was selected to develop an anti-rhuMAbX antibody screening assay to test serum samples from rheumatoid arthritis (RA) patients. With a homogeneous overnight sample incubation, the Biotin-DIG ELISA achieved comparable relative sensitivity and free drug tolerance to the previous BV ATA assay for rhuMAbX. To abrogate potential auto-antibody interference in RA sera, various assay conditions were thoroughly evaluated and a horseradish peroxidase (HRP)-conjugated chicken anti-DIG antibody was selected as the detection conjugate. Other potential interferences from serum Biotin, naturally occurring anti-avidin antibodies, and concomitant medications such as digoxin and hydrocortisone, which have similar structures to digoxigenin, were also investigated. Under optimized final assay conditions, the Biotin-DIG assay showed a relative sensitivity of approximately 11 ng/mL using a polyclonal anti-complementarity determining region (CDR) enriched positive control; the assay could detect 500 ng/mL of the positive control in the presence of approximately 27 µg/mL of rhuMAbX in RA serum. In addition, a confirmatory step was optimized for the assay based upon pre-incubating serum samples with an excess of free drug. Overall, the Biotin-DIG assay met the performance requirements for an ATA screening assay and had comparable sensitivity and drug tolerance to the BV assay; therefore this assay was a suitable replacement for the BV assay used for previous clinical studies of rhuMAbX. The Biotin-DIG based assay format can be broadly used as an effective screening platform for the detection of anti-therapeutic antibodies.

The utility of ISCOMATRIX adjuvant for dose reduction of antigen for vaccines requiring antibody responses.

The capacity of an adjuvant to reduce the amount of antigen required in vaccines would be beneficial in a variety of settings, including situations where antigen is difficult or expensive to manufacture, or in situations where demand exceeds production capacity, such as pandemic influenza. The ability to reduce antigen dose would also be a significant advantage in combination vaccines, and vaccines that by necessity must contain multiple antigens to accommodate variability between strains or genotypes. ISCOMATRIX adjuvant was compared to aluminium hydroxide adjuvant (Al(OH3)) for induction of antibody responses and dose sparing of a recombinant HIV gp120 vaccine. Neutralising antibody responses were significantly greater, at the same protein dose, when the gp120 protein was formulated with ISCOMATRIX adjuvant compared to Al(OH3). Moreover, strong responses were achieved with up to 100-fold lower doses of gp120 using ISCOMATRIX adjuvant. Therefore, ISCOMATRIX adjuvant has the potential to substantially reduce the dose of antigen required in human vaccines, without compromising the immune response.