Methods for Isolating and Quantifying Antigen from Vaccines

Inventors

Michael Graninger and Martin Kaliwoda

Assignees

1. BAXTER INTERNATIONAL INC, [US/US}; One Baxter Parway, Deerfield, IL 60015 (US).
2. BAXTER HEALTHCARE S.A. [CH/CH]; Thurgauerstrasse 130, CH-8152 Glattpark (Opfikon) (CH)

Abstract

The disclosure relates to the development of improved methods for quantifying antigen in a vaccine composition in the absence of available antigen standards. More specifically, the disclosure provides fast and robust methods of separating antigens from vaccine compositions, comprising the steps of solubilizing antigen without detergent and without alkylation, using acidification to prevent antigen subtypes from binding again, isolating antigen subtypes with chromatography, and quantifying the eluted antigen with amino acid analysis. The methods of the disclosure are applicable for use with a variety of antigens, thereby providing an improved method in the art of vaccine manufacturing to date.

CovalX Technology Used

HM1

Outcomes

In order to develop a new HPLC method that could be used to measure the total HA content of Influenza A/California/07/2009 (H1-N1) vaccines, the measurement of HA (HA1) without standard reagents was studied.

In order to quantify HA, samples (undiluted or diluted with deionized water) were mixed with reducing buffer (6 M guanidine hydrochloride, 266 mM Tris/HCl, 1 mM EDTA ( pH 8.3), 40 mM dithiothreitol) and incubated for one hour at 85 °C. 10% (v/v) 8.5% phosphoric acid was added to the samples to stop the reaction and the samples were centrifuged for 10 minutes at 18,407g. The proteins were separated by reversed phase-HPLC using a gradient made of the following:

• Eluent A: 0.1%  TFA in water
• Eluent B: 0.085% TFA in acetonitrile
• Eluent C: methanol

The control and samples were each injected separately with isocratic elution at 20% Eluent B with a flow rate of 0.4ml/min for 10 minutes and then eluted.

Using UV absorption at 214nm, the eluted proteins were detected and then HA1 was quantified and expressed as μg HA per ml of sample. Eluted HA1 was quantified by amino acid analysis for sample 2.

Inside glass ampoules, acid hydrolysis of protein was carried out by adding 2 nmol of 2-L-amino butyric acid as an internal standard and then collecting HPLC fractions directly into the ampoules where they were dried under vacuum. Each sample was dissolved in 200 μl 6N hydrochloric acid/0.2% Phenol with a nitrogen overlay before the ampoules were heat-sealed. The acid hydrolysis occurred over 18 hours at 115 °C before the ampoules were opened. 500 μL of deionized water was added to the ampoules before they were filtered (0.2μ) and transferred into Eppendorf vials to be dried under vacuum. Amino acid analysis was performed by dissolving the samples iin 0.1 N hydrochloric acid and labelling the amino acids with (a-amino butyric acid). The amino acids were separated by reversed phase-HPLCat 37 °C. To analyze the amino acid profile, 5 μL of sample were injected and a gradient was used containing the following:

• Eluent A: Deionized water
• Eluent B: Acetonitrile, gradient grade
• Eluent C: AccQ-Tag buffer concentrate, diluted 1:10 in deionized water

Using fluorescence detection (excitation 250 nm, emission 394 nm), the eluting fluorescent-labeled amino acids were detected. From this detection, the theoretical number of amino acids per HA1 and the molar amount of HA per injection were calculated based on information of the known sequence of HA for H1-N1.

The freeze-dried HPLC fraction was dissolved in a mixture of 5 μL 50% acetonitrile and 0.5% formic acid in water. Matrix (sinapic acid (10 mg/ml) in 50% acetonitrile, 0.5% formic acid in water) was added to this mixture before 1 μL of the final mixture was spotted on a MALDI target plate and dried at room temperature. The plate was analyzed using a mass spectrometer that had been modified with a CovalX HM1 detection system.

Patent Number

WO2012/145386 A1