Development of a High-Throughput Screening Platform to Study the Adsorption of Antigens onto Aluminum-Containing Adjuvants

Jully, Vanessa; Moniotte, Nicolas; Mathot, Frédéric; Lemoine, D.; Préat, Véronique

doi:10.1002/jps.24256

Cited by 5 publications

(8 citation statements)

References 48 publications

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“… 19 , 34 – 36 β-Casein was selected as the model protein to perform adsorption isotherms with AP. 16 Preliminary studies showed that adsorption and pH equilibration were achieved after 18 h of incubation with stirring for the mix AP/β-casein (data not shown). The adsorption of negatively charged β-casein (IEP 4.6–5.1) by negatively charged AP (PZC 4.5) at pH 7.0 ± 0.1 fitted the Langmuir model ( R 2 > 0.99).…”

Section: Resultsmentioning

confidence: 99%

“…Adsorption capacity of AP for β-casein was determined by performing adsorption isotherms and evaluated using a constant AP concentration (160 µg/mL) and varying β-casein concentrations (475–750 µg/mL). 16 A sodium chloride solution was used at a final volume of 500 µL in a 96-deep-well plate. The formulations were gently stirred using magnetic bars in a microplate for 18 h at room temperature, using a Vortex Lateral Tumble Stirrer VP 708-CON (V&P Scientific, San Diego, CA).…”

Section: Methodsmentioning

confidence: 99%

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Automated Gravimetric Calibration to Optimize the Accuracy and Precision of TECAN Freedom EVO Liquid Handler

et al. 2016

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High-throughput screening technologies are increasingly integrated into the formulation development process of biopharmaceuticals. The performance of liquid handling systems is dependent on the ability to deliver accurate and precise volumes of specific reagents to ensure process quality. We have developed an automated gravimetric calibration procedure to adjust the accuracy and evaluate the precision of the TECAN Freedom EVO liquid handling system. Volumes from 3 to 900 µL using calibrated syringes and fixed tips were evaluated with various solutions, including aluminum hydroxide and phosphate adjuvants, β-casein, sucrose, sodium chloride, and phosphate-buffered saline. The methodology to set up liquid class pipetting parameters for each solution was to split the process in three steps: (1) screening of predefined liquid class, including different pipetting parameters; (2) adjustment of accuracy parameters based on a calibration curve; and (3) confirmation of the adjustment. The run of appropriate pipetting scripts, data acquisition, and reports until the creation of a new liquid class in EVOware was fully automated. The calibration and confirmation of the robotic system was simple, efficient, and precise and could accelerate data acquisition for a wide range of biopharmaceutical applications.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Automated Gravimetric Calibration to Optimize the Accuracy and Precision of TECAN Freedom EVO Liquid Handler

et al. 2016

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“…The HTS platform can be incorporated during the vaccine formulation development processes to better understand the contributions of ligand exchange and electrostatic attractions governing the interactions between an antigen adsorbed onto aluminum-containing adjuvant, the assessment of the batch-tobatch consistency of aluminum-containing adjuvants, and the impact that formulation (e.g., salts) and process parameters could have on the adjuvant properties. The HTS platform brings together different applications already developed 41 into one unit (automated handling of excipients, antigens, and aluminum-based adjuvants followed by a characterization of the adsorption) to better understand the adsorption mechanisms of antigens onto aluminum-containing adjuvants.…”

Section: Discussionmentioning

confidence: 99%

“…Target pH was 7.0 ± 0.1 to allow electrostatic interactions as previously described. 41 Water for injection was used to reach the final volume. The ranges of aluminum and proteins concentrations are presented in Table 1 and were based on preliminary studies to cover the maximum adsorbed concentration of a protein to the aluminum salt (data not shown).…”

Section: Adsorption Isotherms Of Model Proteins and Antigens Onto Ahmentioning

confidence: 99%

“…For these evaluations, we have developed a highthroughput screening (HTS) platform that can produce adsorption isotherms and perform DiFMUP-based fluorescent spectroscopy on samples automatically titrated in microplates. 41 The effect of proteins/antigens on the "relative surface phosphophilicity" (RSP) of AH was calculated by comparing DiFMU production using AH with or without proteins/antigens. The RSP of AH was inversely proportional to affinity of adsorbing species tested.…”

Section: Introductionmentioning

confidence: 99%

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Mechanisms of Antigen Adsorption Onto an Aluminum-Hydroxide Adjuvant Evaluated by High-Throughput Screening

Jully

Mathot

Moniotte

et al. 2016

Journal of Pharmaceutical Sciences

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The adsorption mechanism of antigen on aluminum adjuvant can affect antigen elution at the injection site and hence the immune response. Our aim was to evaluate adsorption onto aluminum hydroxide (AH) by ligand exchange and electrostatic interactions of model proteins and antigens, bovine serum albumin (BSA), β-casein, ovalbumin (OVA), hepatitis B surface antigen, and tetanus toxin (TT). A high-throughput screening platform was developed to measure adsorption isotherms in the presence of electrolytes and ligand exchange by a fluorescence-spectroscopy method that detects the catalysis of 6,8-difluoro-4-methylumbelliferyl phosphate by free hydroxyl groups on AH. BSA adsorption depended on predominant electrostatic interactions. Ligand exchange contributes to the adsorption of β-casein, OVA, hepatitis B surface antigen, and TT onto AH. Based on relative surface phosphophilicity and adsorption isotherms in the presence of phosphate and fluoride, the capacities of the proteins to interact with AH by ligand exchange followed the trend: OVA < β-casein < BSA < TT. This could be explained by both the content of ligands available in the protein structure for ligand exchange and the antigen's molecular weight. The high-throughput screening platform can be used to better understand the contributions of ligand exchange and electrostatic attractions governing the interactions between an antigen adsorbed onto aluminum-containing adjuvant.

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The adsorption of allergoids and 3-O-desacyl-4′-monophosphoryl lipid A (MPL®) to microcrystalline tyrosine (MCT) in formulations for use in allergy immunotherapy

Bell

Heath

Hewings

et al. 2015

Journal of Inorganic Biochemistry

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Infectious disease vaccine potency is affected by antigen adjuvant adsorption. WHO and EMA guidelines recommend limits and experimental monitoring of adsorption in vaccines and allergy immunotherapies. Adsorbed allergoids and MPL® in MATA-MPL allergy immunotherapy formulations effectively treat IgE mitigated allergy. Understanding vaccine antigen adjuvant adsorption allows optimisation of potency and should be seen as good practice; however current understanding is seldom applied to allergy immunotherapies. The allergoid and MPL® adsorption to MCT in MATA-MPL allergy immunotherapy formulations was experimental determination using specific allergen IgE allerginicity and MPL® content methods. Binding forces between MPL® and MCT were investigated by competition binding experiments. MATA-MPL samples with different allergoids gave results within 100-104% of the theoretical 50μg/mL MPL® content. Unmodified drug substance samples showed significant desirable IgE antigenicity, 1040-170 QAU/mL. MATA-MPL supernatant samples with different allergoids gave results of ≤2 μg/mL MPL® and ≤0.1-1.4 QAU/mL IgE antigenicity, demonstrating approximately ≥96 & 99% adsorption respectively. Allergoid and MPL® adsorption in different MATA-MPL allergy immunotherapy formulations is consistent and meets guideline recommendations. MCT formulations treated to disrupt electrostatic, hydrophobic and ligand exchange interactions, gave an MPL® content of ≤2 μg/mL in supernatant samples. MCT formulations treated to disrupt aromatic interactions, gave an MPL® content of 73-92 μg/mL in supernatant samples. MPL® adsorption to l-tyrosine in MCT formulations is based on interactions between the 2-deoxy-2-aminoglucose backbone on MPL® and aromatic ring of l-tyrosine in MCT, such as C-H⋯π interaction. MCT could be an alternative adjuvant depot for some infectious disease antigens.

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Development of a High-Throughput Screening Platform to Study the Adsorption of Antigens onto Aluminum-Containing Adjuvants

Cited by 5 publications

References 48 publications

Automated Gravimetric Calibration to Optimize the Accuracy and Precision of TECAN Freedom EVO Liquid Handler

Automated Gravimetric Calibration to Optimize the Accuracy and Precision of TECAN Freedom EVO Liquid Handler

Mechanisms of Antigen Adsorption Onto an Aluminum-Hydroxide Adjuvant Evaluated by High-Throughput Screening

The adsorption of allergoids and 3-O-desacyl-4′-monophosphoryl lipid A (MPL®) to microcrystalline tyrosine (MCT) in formulations for use in allergy immunotherapy

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