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ATTENTION!
DIRECTIONS HAVE CHANGED DUE TO ROADWORK
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ORAL PRESENTATIONS
Nanobodies® - macro challenges?
Hilde Revets, Ablynx
Nanobodies are antibody-derived therapeutic proteins that contain the unique structural and functional properties of naturally-occurring heavy-chain antibodies. Nanobodies combine the advantages of conventional antibodies with important features of small molecule drugs. Like conventional antibodies, Nanobodies show high target specificity, high affinity for their target and low inherent toxicity. However, like small molecule drugs they have the opportunity to target classes of targets such as GPCRs and ion channels, many of which are very difficult if not impossible to access with monoclonal antibodies. In addition, their inherent biophysical properties make them ideal candidates for alternative methods of drug delivery beyond the needle.
The Nanobody platform allows the ability to design modular drugs based on Nanobody building blocks combined with each other or with other protein domains or other molecules or drugs. These can combine more than one function in the final drug format. These formats are easy to construct and the modular proteins can often be expressed at high levels in bacteria or yeast.
Using case studies, data from initial discovery through the pre-clinical in vivo model validation and manufacturability will be presented to highlight the unique features of Nanobodies that make them well-suited for drug development, and discuss the differences and similarities in the discovery and development versus small molecules.
Fit-for-purpose development and validation of a challenging biomarker assay on Gyrolab for a novel Nanobody® drug candidate.
Ariëlla Van de Sompel, Maarten Van Roy, Kristi De Smet, Veronique Andriessen, Hans Ulrichts, Sofie Poelmans, Stefaan Rossenu, Laura Sargentini, Sandy Jacobs, Judith Baumeister and Josefin-Beate Holz.
The functions of the pleiotropic cytokine IL-6 are mediated through a receptor system consisting of gp130 and soluble or membrane IL-6 receptor (IL-6R). Deregulation of this system is implicated in a variety of diseases, including rheumatoid arthritis (RA). Blocking IL-6R results in clinical benefit as shown with tocilizumab, a marketed anti-IL-6R monoclonal antibody.
Nanobodies are therapeutic proteins based on the smallest functional fragments of heavy chain antibodies, naturally occurring in the Camelidae family. ALX-0061 is a highly specific anti-IL-6R Nanobody engineered to obtain half-life extension (HLE) in vivo, and high target affinity and potency. Monovalent interaction with IL-6R avoids unwanted cross-linking, while the HLE strategy employed excludes the need for a Fc-region, thus avoiding immune effector functions. These properties were considered important for a potentially improved safety profile compared to full IgG monoclonal antibodies targeting IL-6R.
A Gyrolab-based biomarker assay was developed for ALX-0061 in order to support preclinical toxicology studies in cynomolgus monkey. In plasma, free soluble IL-6R (sIL-6R) concentrations were quantified using the Gyrolab platform as this platform was considered optimal for not disturbing ALX-0061/IL-6R complexes during the analysis procedure. The method was successfully validated for its use to determine the proximal pharmacodynamic effect of ALX-0061.
During validation of the Gyrolab-based method to determine free sIL-6R levels in cynomolgus monkey plasma, whole CD uniformity was demonstrated and spiking and mix experiments showed no significant matrix effect. The validated range for quantification of free sIL-6R levels in cynomolgus monkey plasma was 0.2 to 20 ng/mL at CD level, which corresponded to 10 and 1000 ng/mL at plasma level. This dynamic range was acceptable as the natural baseline concentration of sIL-6R in cynomolgus monkey varies between 20-30 ng/mL. The limit of detection of the assay was calculated to be 1 ng/mL at plasma level. In-study validation showed that study samples can be stored at -80°C for at least 5 months and can undergo at least 2 F/T cycles without an effect on stability of free sIL-6R or Nanobody-sIL-6R complexes. Parallelism was also confirmed during in-study validation.
Results from the herein described assay were compared to an additional biomarker assay (ELISA) quantifying total sIL-6R. Both target biomarker assays were used to support the single dose dose-range finder study in cynomolgus monkey, in which ALX-0061 dose-dependently affected total and free sIL-6R levels. The measured total sIL-6R levels were inactive and complexed with ALX-0061 as a full suppression of the free sIL-6R concentrations was simultaneously obtained. Resulting circulating target levels in preclinical studies were used to build a PK/PD model in support of further non-clinical and clinical development.
In conclusion, the Gyrolab-based method was shown to be fit-for-purpose for the determination of free sIL-6R concentrations in cynomolgus monkey plasma samples. The assay was employed to confirm the proximal pharmacodynamic effect of ALX-0061 in cynomolgus monkey in which a good inverse correlation between total sIL-6R, free sIL-6R and ALX-0061 was demonstrated. A clinical trial application for the treatment of patients with active RA has been filed in Europe and a Phase I/II study is currently ongoing.
A new extrapolation method for the determination of kinetic rate- and affinity constants of a single ligand and analyte molecule with Surface Plasmon Resonance imaging.
Richard B.M. Schasfoort, PhD, IBIS Technologies B.V. Pantheon 5, 7483 AL Enschede, The Netherlands.
Label free biosensing instruments are applied to determine the kinetic rate- and affinity constants of e.g. antibody-antigen, drug-receptor interactions or in general any analyte-ligand interaction. For many applications the value of the parameter of the quality of binding or affinity equilibrium constant (KD) is of great importance because it determines the therapeutic relevant concentration range of the interactants. The better the affinity constant the lower the concentration of the therapeutic Ab-drug and the lower the induced immunogenicity effect. The determined values using biosensor instruments should be equal to the real affinity equilibrium constant in solution.
However various biosensor surfaces introduce several interfering effects as rebinding events, biphasic behavior and/or non 1:1 model interactions, mass transport limitation effects and parallel effects as non-specific binding, dimerization etc. In general all these interfering effects affect more or less the value of the affinity constant. As a rule the interference will decrease substantially when both ligand densities on the sensor chip and analyte concentrations are applied at very low but just detectable responses close to the zero-response of the biosensor instrument.
The IBIS MX96 array based SPR imaging instrument of IBIS Technologies, Enschede, The Netherlands allows to apply an array of ligand densities in 48- or even 96-plex format. By injecting analyte concentrations in serial dilution any combination of ligand versus analyte in a checkerboard is measured and the signals in the MX96 show more or less the interfering effects on the various spots. A new automated method will be revealed enabling to analyze the data in such a way that these defects are minimized by extrapolating a cloud of values from the spots for the rate constants ka , ka and equilibrium constants KD to response = zero. In this way, in principle, the affinity constant to the limit of a single analyte interactant with a single ligand molecule is calculated. Including applications for the IBIS MX96 as immunogenicity testing, epitiope mapping, Ab clustering, this new method will be highlighted in the presentation.
Use of chipLC-MS in biomarker discovery and validation – data processing and application aspects
P. Horvatovich, B. Hoekman, C. Christin, V. Mitra, N. Govorukhina,
R., Breitling [1], Frank Suits [2], R. Bischoff
University of Groningen, Center for Pharmacy, Department of Analytical Biochemistry, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands
[1] Faculty of Biomedical and Life Sciences, University of Glasgow, G12 8QQ Glasgow, UK and Groningen Bioinformatics Centre, University of Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands
[2] IBM TJ Watson Research Center, Yorktown Heights, New York, USA
Integration of liquid chromatography into microfluidic devices (chipLC) holds opportunities for biomarker research. This presentation will address the application of chipLC in biomarker discovery and validation, with special focus on practical aspects and data processing. Biomarker discovery by comparative sample analysis serves to establish testable hypotheses concerning molecular mechanisms of disease onset, progression and response the therapy. However, biomarker discovery relies on intricate methods that often do not allow to analyze large numbers of samples. Consequently the established hypotheses must be tested (validated) in larger sample sets focusing on a limited number of compounds.
My presentation will focus on assessing the quantitative performance of chip-LC-MS for biomarker discovery. I will notably focus on aspects of data processing methods for label-free LC-MS specially focusing on time alignment and quantification aspects. The msCompare framework, which allows to combine peak detection and peak alignment/matching modules from various open source data processing pipelines and includes a method to assess the relative performance of various homogenous or heterogeneous combination of quantitative data pipelines, will be presented. Applications to ongoing biomarker discovery projects for the early detection of cervical cancer or proteinurea will allow the audience to gain a better understanding of the possibilities and pitfalls.
In the second part of my talk I will highlight aspects of the application or Selected Reaction Monitoring (SRM) for biomarker validation. Finally I will present our effort to build easy-to-use, web-based and high-throughput data processing services including protein quantification and identification for label-free (chip)LC-MS biomarker discovery and protein/peptide quantification by SRM for protein biomarker validation.
Microdialysis and LC-MS analysis
Robert Freije, Brains On-Line (Groningen, The Netherlands)
When profiling levels of neurotransmitters and other neurochemicals in the brain, microdialysis is an excellent method for quantitative determination of pharmaceutical effects over time in freely moving animals. The method does however put stringent demands on the bioanalytical system since concentrations of single neurotransmitters in specific brain areas may be low, and only small sample volumes are generally obtained.
SymDAQ (symmetrical dialdehyde quartenary ions) is a soft ionization label developed to improve analysis of neurotransmitters in microdialysate by HPLC coupled to tandem mass spectrometry. SymDAQ provides a rapid and sensitive bioanalytical method for the quantitation of dopamine, serotonin, norepinephrine, GABA, glutamate, glycine and histamine yielding detection limits as low as 20 pM for dopamine and 50 pM for serotonin and norepinephrine in microdialysate, making quantitative analysis in various brain areas possible. By automating reagent addition and derivatization, the total run-time can be as low as 14 minutes per sample.
Application of the SymDAQ label has been be extended to other areas such as metabolites of the urea cycle and neurotransmitters in other biofluids, for example human and rat plasma.
Bioanalytical support for peptide quantification in biological matrices
Lieve Dillen, Willy Cools, Liesbeth Vereyken and Filip Cuyckens
Janssen Research & Development, Drug Safety Science, Analytical Sciences, Non-regulated Bioanalysis
Over the last years, the focus of drug development is also shifted towards development of synthetic peptides as new medicines. Therefore, in many LCMS bioanalytical labs, peptides are introduced as new analytes and the bioanalytical scientist needs to address the quantitative challenges for peptide analysis.
We will discuss method development of a peptide and its biotinylated analogue. Strategy was to investigate whether enhanced extraction is possible for this biotinlyated peptide (making use of strepatvidine coated beads) and we compared to the protein precipitation method of the non-biotnylated analogue. Method development for 2 reference peptides will be presented as well. In the first example, the LC-MS/MS method showed disturbed chromatography due to the presence of a metabolite. For the third case performance of a typical peptide assay is demonstrated on the basis of accuracy and precision calculations. In the last part of the presentation we will show results on an evaluation of the use of high resolution mass spectrometry for peptide quantification and compare the results with performance of a LC-MS/MS method on a triple quadrupole instrument.
Application of LC-MS/MS for Microdosing studies
Ronald de Vries, Janssen R&D
Human micro dosing in phase 0 studies allows an early assessment of human pharmacokinetics and bioavailability of a drug candidate. Microdosing studies are designed to evaluate pharmacokinetics without inducing pharmacologic effects. According to the FDA guidance, a microdose is defined as less than 1/100th of the dose of a test substance calculated (based on animal data) to yield a pharmacologic effect of the test substance with a maximum dose of ? 100 micrograms. Another application of microdosing is determination of absolute bioavailibility by concomitant administration of a normal clinical p.o. dose together with an i.v. microdose of a labelled version of the drug. This approach eliminates the need for a cross-over design, makes the development of an i.v. formulation much easier and limits the number of supporting toxicology studies that are required.
At these low doses a highly sensitive method in the low pg/ml range is required.
Often, micro dose studies are analysed using AMS (accelerator mass spectrometry). This technique is extremely sensitive, but is expensive and has its method validation challenges.
Alternatively, micro dose studies can also be analysed by LC-MS/MS. In our lab we have run feasibility experiments to investigate in which cases it is possible to succesfully support micro dosing studies and in partcular microdosing Fabs studies (which are the most challenging) using LC-MS/MS and to define what the requirements, limitations, difficulties and points of attention are to take into account. In this presentation, a summary of the results will be given. The presentation will include a strategy to come to a highly sensitive method, suggestions for choice of the number, type and position of the (stable) labels and assessment of impact of the combination of high p.o. and (very) low i.v. dose on the assay (for instance how to cope with ion suppression and carry-over / contamination effects).
POSTER PRESENTATIONS
Addressing the Issues of Matrix Resolution and Measurement in Bioanalytical Assays
Presented by Diego Rodriguez Cabaleiro, Waters Europe, Guyancourt, France
Authors: Geneen Baynham [1], Paul D Rainville [1], Marian Twohig [1], Robert S Plumb [1] and Ian Wilson [2]
[1] Waters Corporation, Milford, MA 01757
[2] AstraZeneca, Macclesfield, UK.
In quantitative bioanalysis the analytical technique of choice is LC/MS/MS due to the high sensitivity and selectivity that it affords. Quantitative bioanalytical methods development is complicated by matrix interferences which can alter the response of the analyte.
Matrix effects, resulting from co-eluting matrix components that compete for charge in the ionisation process, manifest themselves as suppression or occasionally enhancement of the analyte signal. Matrix effects are caused by numerous factors all of which can cause significant errors in the accuracy and precision of a bioanalytical assay, Phospholipids, Subject differences, Impurities, Degradation Products, Co-eluting metabolites
Monitoring the presence and impact of matrix effects can complicate and prolong the development of a robust, sensitive assay.
In this poster we evaluate the use sub 2um particle LC on the matrix effects observed. We will also demonstrate how the background matrix interference can be monitored in a single run analytical using a mass spectrometer equipped with a novel fast switching quadrupole MS. We will also demonstrate how the matrix effects can be determined and quantified by an integrated matrix calculator tool. We will show how the use of these tools can significantly shorten the time taken to develop a sensitive, robust bioanalytical LC/MS/MS assay.
LC/MS/MS Quantification of Fluticasone Furoate in Human Plasma at the fg/mL Level
Presented by Diego Rodriguez Cabaleiro, Waters Europe, Guyancourt, France
Authors: Jennifer Keller [1]; Joanne Mather [2]; Nancy Zheng [1]; Zong-Ping Zhang [1]
[1} PPDI, Middleton,, WI;
[2] Waters Corporation, Milford, MA, USA
Novel Aspect:
New generation of LC/MS/MS methodology allowing previously unobtainable levels of sensitivity for the low exposure compound Fluticasone furoate.
Introduction
Fluticasone furoate is used in the management of asthma and chronic obstructive pulmonary disease; fluticasone is a trifluorinated corticosteroid which has potent anti-inflammatory activity. As this compound undergoes extensive first-pass metabolism, there is negligible systemic exposure. In order to accurately define the pharmacokinetics of this compound it is necessary to be able to detect the compounds down to the sub- pg/mL level. Historically LC/MS/MS instrumentation has not provided sufficient sensitivity to quantify the plasma concentrations of this compound at the fg/mL level. In this paper we demonstrate the quantification of fluticasone at the sub pg/mL level using LC/MS/MS.
Methods
The fluticasone furoate was extracted from 1 mL of human plasma using a LLE method. The final volume of the extract was 100µL. The chromatography was operated in reversed-phase UPLC with gradient and optimized to provide separation of the analytes of from the endogenous components in the sample over 5.5 minutes. Basic mobile phase (0.1% NH4OH) gave the greatest sensitivity for the analyte. The LC/MS analysis of the fluticasone was performed on an ACQUITY UPLC system employing sub 2µm particle technology coupled to a Xevo TQ-S tandem quadrupole mass spectrometer operated in MRM mode. A stable label isotope was used as the internal standard for the analyte in this study.
Preliminary Data
As Fluticasone furoate is dosed by the inhaled route the majority of the drug is directed to the lungs. The remainder is eliminated in the liver to via hydrolysis of the S-fluoromethyl carbothioate function to form the inactive 17?-carboxylic acid metabolite. These factors result in circulating levels in the 0.5pg/mL – 1pg/mL level. The high protein binding, greater than 99%, also present a challenge when developing a high sensitivity assay. The various extraction procedures were evaluated such as liquid-liquid extraction (LLE), solid-phase extraction (SPE), solid-phase/liquid-liquid extraction (SLE+) and protein precipitation in tandem with SPE such as HLB by varying the following parameters: extraction buffers, extraction solvents, and PH. The combination of Zinc Sulphate dilution to LLE gave a reproducible high extraction efficiency sample prep approach with extraction recovery more than 95% and significantly lowered background noise. The sub 2um separation resolved the active compound from endogenous interferences and metabolites. The assay cycle time was 5.5 mins with a peak width of 2 secs at base. The novel ion guide optics in the MS resulted in sufficient sensitivity to detect and quantify the compound at very low levels. The limit of quantification fluticasone was determined to be 0.2 pg/mL. The peak width of the fluticasone analyte was calculated to be approximately 3 seconds at the base, this allows for a high resolution separation of the analyte from the endogenous material in the sample. The calibration was shown to be linear over the range of 0.2 - 100pg/mL. The signal to noise of the 0.2pg/mL standard was determined to be 7:1. This level of sensitivity allows for the accurate determination of the pharmacokinetics of the Fluticasone furoate in plasma.
Development of an ultra sensitive lc-ms/ms method for the simultaneous determination of ?9-tetrahydrocannabinol and one of its major metabolites ?9-(11-OH) tetrahydrocannabinol in human plasma
Mark Boterman [1], Tim Beumer [2], Freddy Oostebring [1], Andre Meijer [1] and Ep Oosting [1]
[1]Analytical Biochemical Laboratory B.V., W.A. Scholtenstraat 7, 9403 AJ Assen, The Netherland
[2]Echo Pharmaceuticals, Jonkerbosplein 52, 6534 AB Nijmegen, The Netherlands
Since the discovery of an endogenous cannabinoid system, research into the pharmacology and therapeutic potential of cannabinoids has steadily increased. To date ?9-tetrahydrocannabinol (THC) has been employed in the treatment of numerous diseases. However, more therapeutic indications for THC are currently extensively investigated. Consequently, more sensitive and specific analytical methods are needed for pharmacokinetic studies. Therefore, the objective of the present study was to develop and validate an ultra sensitive and robust LC-MS/MS method for the combined determination of THC and ?9-(11-OH)-tetrahydrocannabinol (11-OH-THC, a major metabolite of THC) in human EDTA plasma. The analytical range for both THC and 11-OH-THC was 10.0-5000 pg/mL.
Methods
Human EDTA plasma samples, using deuterated analogs as internal standards, were subjected to a solid phase extraction step using C18 end capped SPE columns (3cc 100 mg), followed by a derivatization step with dansylchloride in an alkaline environment. Subsequently, the derivatized samples were subjected to a liquid/liquid extraction. Finally the samples were chromatographed on a Kinetex 2.6µ PFP 100A (100 x 3.00 mm) column. A full validation of the method was performed according to the current guidelines for bioanalytical method validation and in accordance with Good Laboratory Practice guidelines.
Results
The assay for THC and 11-OH-THC was validated in the concentration range of 10.0 - 5000 pg/mL and a Lower Limit Of Quantification (LLOQ) of 10.0 pg/mL was achieved in human EDTA plasma for both compounds. The method showed excellent accuracies and precisions for all QC levels which were determined in three validation runs and was found valid with respect to recovery, specificity, 10-fold dilution, matrix effect and stability (bench-top (24h), on machine (111h at 15°C) and freeze/thaw (three additional cycles) stability).
Conclusions
Currently, bioanalytical methods are available for the determination of THC and 11-OH-THC in human plasma with LLOQs down to 100 pg/mL. ABL has successfully developed and validated an extremely sensitive and robust bioanalytical LC-MS/MS method for the determination of THC and 11-OH-THC in human EDTA plasma with a 10-fold increased sensitivity for both compounds. The method will be applied to analyse samples from several clinical studies, including a clinical study with Namisol® (an oral tablet with THC) in patients with dementia. In this clinical phase II study the pharmacokinetic profile of THC and its metabolite 11-OH-THC and the relationship between plasma concentrations of THC and 11-OH-THC and clinical effects (behavioral disturbances) in these subjects will be determined. In conclusion, unlike other alternative methods available, this method offers an unique opportunity to investigate the pharmacokinetics of THC and 11-OH-THC in the lower plasma concentration ranges.
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