Quantitative Analysis of Vaccine Antigen Adsorption to Aluminum Adjuvant Using an Automated High-Throughput Method

Patrick L. Ahl; Sheng-Ching Wang; Ramesh Chintala; Christopher Mensch; William J. Smith; Marc Wenger; Jeffrey Blue

doi:10.5731/pdajpst.2017.008250

Abstract

Aluminum-containing adjuvants have been widely used in vaccine formulations to safely and effectively potentiate the immune response. The examination of the extent of antigen adsorption to aluminum adjuvant is always evaluated during the development of aluminum adjuvant containing vaccines. A rapid, automated, high-throughput assay was developed to measure antigen adsorption in a 96-well plate format using a TECAN Freedom EVO^® (TECAN). The antigen adsorption levels at a constant adjuvant concentration for each sample were accurately measured at 12 antigen/adjuvant (w/w) formulation ratios. These measurements were done at aluminum adjuvant concentrations similar to normal vaccine formulations, unlike previous non-automated and automated adjuvant adsorption studies. Two high-sensitivity analytical methods were used to detect the non-absorbed antigens. The antigen-to-adjuvant adsorption curves were fit to a simple Langmuir adsorption model for quantitatively analyzing the antigen to the adjuvant adsorption level and strength. The interaction of two model antigens, bovine serum albumin and lysozyme, with three types of aluminum adjuvant, were quantitatively analyzed in this report. Automated, high-throughput methodologies combined with sensitive analytical methods are useful for accelerating practical vaccine formulation development.

LAY ABSTRACT: Vaccines are probably the most effective public health method to prevent epidemics of many infectious diseases. Many of the most effective vaccines contain aluminum adjuvant. This report describes novel technology that can be used to better optimize the efficacy and stability of aluminum adjuvant–containing vaccines.

Abbreviations:

AAHS: amorphous aluminum hydroxyphosphate sulfate adjuvant
Al: elemental aluminum
AlOH: aluminum hydroxide adjuvant
AP: aluminum phosphate adjuvant
BPP: Biologics Pilot Plant
BSA: bovine serum albumin
BSA-Alexa₄₈₈: bovine serum albumin labeled with Alexa₄₈₈ fluorophore
Dtap-IVP/Hib: diphtheria, tetanus, cellular pertussis, inactivated polio virus, haemophilus influenza b vaccine
CV%: coefficient of variation %
FDA: Food and Drug Administration
Hep A: hepatitis A virus
Hep B: hepatitis B virus
HPV: human papilloma virus
HT: high-throughput
LINEST: Microsoft Excel^® function that calculates the statistics of values fitting a line using the least squares method
Lyso: lysozyme
Lyso-FITC: lysozyme labeled with FITC fluorophore
MMD: Merck Manufacturing Division (West Point, PA)
MW: molecular weight
P: elemental phosphorous
PCV13: pneumo conjugate vaccine 13 serotype antigens
pI: protein isoelectric point
PZC: point of zero charge
TECAN: TECAN Freedom EVO^® laboratory automation instrument
VLP: virus-like particle

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