Application Note

A Fast and High Precision Influenza Vaccine Potency Assay

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Introduction

David Wheatley, Principal Scientist, Debi Saunders, Staff Scientist, Kyle Jones, R&D Scientist and David Apiyo, Marketing Applications Manager, Pall Life Sciences

Vaccines are biological preparations that contain agents resem- bling disease causing microorganisms, and can improve immu- nity against a specific disease. They are typically prepared from inactivated or weakened forms of the microbe or its toxins, or surface proteins. Classical vaccines against the influenza virus are developed in embryonated hen eggs and may include whole virus, split virus or a purified subunit with every component other than hemagglutinin (HA) or neuraminidase (NA) removed. 1 The target molecule for the protective immune response triggered by vaccina- tion is generally accepted to be the HA molecule; a glycoprotein found on the surface of the influenza virus. Measuring the vaccine potency or the biologically active components is critical to the determination of the vaccine’s effective dose. In addition, the stability of the vaccine has major impact on its usage for immunization programs worldwide. Although real-time stability studies under different storage conditions is preferable, thermal stability testing using potency assays with samples subjected to heat or environ- mental stress conditions can be used as predicators of vaccine stability over time. 2Vaccines are biological preparations that contain agents resembling disease causing microorganisms, and can improve immu- nity against a specific disease. They are typically prepared from inactivated or weakened forms of the microbe or its toxins, or surface proteins. Classical vaccines against the influenza virus are developed in embryonated hen eggs and may include whole virus, split virus or a purified subunit with every component other than hemagglutinin (HA) or neuraminidase (NA) removed. 1 The target molecule for the protective immune response triggered by vaccina- tion is generally accepted to be the HA molecule; a glycoprotein found on the surface of the influenza virus. Measuring the vaccine potency or the biologically active components is critical to the determination of the vaccine’s effective dose. In addition, the stability of the vaccine has major impact on its usage for immunization programs worldwide. Although real-time stability studies under different storage conditions is preferable, thermal stability testing using potency assays with samples subjected to heat or environ- mental stress conditions can be used as predicators of vaccine stability over time.2

A fast and accurate determination of vaccine titer during manufacturing is important in understanding vaccine development process performance, and for correctly scaling each process step. The Single Radial Immunodiffusion (SRID) technique has been the most commonly used technique for vaccine titer determination. However, SRID is time consuming and generally exhibits poor precision. An alternative assay that can speed up the analysis process and provide accurate and precise potency data on different vaccine strains is therefore desirable. The Octet platform’s Bio-Layer Interferometry (BLI) technology combines the high-throughput characteristics of a 96-well or 384-well plate format with improvements in precision and reproducibility and is derived from a simpler and more directvaccine/antigen–antibody bind- ing measurement method. They provide process development groups with a robust and easy to use alternative to the SRID meth- od. BLI reduces the assay time from days to just a few hours for a 96-well plate of samples.

Octet system with sample plate loaded.

Figure 1: Octet system with sample plate loaded.

The relative standard deviation and dynamic range of a vaccine titer assay was tested for the influenza virus using the Octet platform and was found to be better than that encountered with SRID. Unlike in the SRID technique where detergents are used to expose the target HA molecule, with Octet systems, samples are analyzed in their natural state without the use of detergents. As a result, Octet systems are capable of analyzing whole virus, split virions and recombinant HA vaccine samples.

Materials And Reagents

  • Samples of inactivated virus and antibody standards were purchased from NIBSC, South Mimms, UK. Split virion vaccine samples were provided by Sanofi Pasteur, France, and recombinant hemagglutinin vaccine samples and antibodies were provided by Protein Sciences, USA.
  • Sample Diluent (Part No 18-1048), Protein A and G biosensors (Part No 18-5010 and 18-5082 respectively), were provided by Pall.
  • Black polypropylene 96-well sample plates from Greiner, Part No 655209 (Sigma Aldrich Part No M9685) were used.

Assay workflow for the influenza vaccine titer assay.

Figure 2: Assay workflow for the influenza vaccine titer assay. The assay is run using the Advanced quantitation setup in the Octet Data Acquisition software.

Method

The assay is based on the binding of the vaccine sample to polyclonal antibodies that recognize the influenza epitopes presented by the vaccine. Protein A or Protein G derivatized biosensors are first used to load the specific polyclonal antibody from the serum antibodies (Figure 2). The antibody-immobilized biosensors are then dipped into the vaccine samples and a response signal that depends on binding epitope recognition and vaccine concentration is registered.

To determine the optimal antibody concentration to capture onto the biosensor, a serial dilution of the serum antibody sample was performed using Pall ForteBio Sample Diluent.

Figure 3 shows the biosensor binding profiles of a range of anti- body dilutions starting from 1/10 to 1 /2000 of the neat antibody samples obtained from NIBSC as California/7/2009 (H1N1); an A strain inactivated virus standard and antibody pair. Typical Octet assay antibody loading time is 300 seconds with a shake speed ranging from 400 –1000 RPM. The antibody loading time can vary significantly from virus strain to strain and should be evaluated.

While high precision Streptavidin (SAX) biosensors are recommend- ed for multi-step quantitation assays, Protein A or G biosensors can be used especially when dealing with non-purified IgG samples. The biosensors were first tested in five replicates for each lot in a biosen- sor lot to lot robustness study for antibody loading (Figure 4, Table 1). The biosensors were found to be highly robust to loading variations. However, it is critical to include a referencing biosensor (zero analyte) in each assay to subtract off potential post antibody baseline drift. The second stage of the assay optimization involves dipping the antibody-loaded biosensors into a fixed concentration of the vac- cine sample to monitor response (Figure 5). Both antibody loading and antigen binding during assay optimization are performed in sequence on the Octet instrument using the Advanced Quantitation experimental setup in the Octet Data Acquisition software.

Biosensors Average response % CV
Lot 1 0.489 2.41
Lot 2 0.463 2.63
Lot 3 0.455 1.53
Lot to Lot % CV   3.78

Table 1: Protein A biosensors lot to lot loading response monitored at T = 500 seconds.

Antibody Load Scouting using Protein A biosensors

Figure 3: Antibody Load Scouting using Protein A biosensors – A/California/7/2009(H1N1)

Antibody loading at 1/250 dilution using different lots of Protein A biosensors

Figure 4: Antibody loading at 1/250 dilution using different lots of Protein A biosensors

The lowest coating antibody concentration (1/250 dilution in this example) that gives maximal vaccine binding should be selected for the studies. For influenza virus, the sample binding step is typi- cally 300 seconds except for split virion analysis, where the step should take less than 30 seconds as virus heterogeneity can occur with time. To determine titer, a standard curve is required and is generated using a titration of standard samples whose concentra- tion is known. The response signals obtained from the standard samples are analyzed by calculating the binding rate for the initial slope of the binding curve. The measured binding rate is then plot- ted as a function of the standard samples concentration and the data fit to a dose response equation resulting in a standard curve from which unknown sample titre can be determined.

The antigen binding response for the three most concentrated anti- body solutions overlap (Figure 5). The lack of distinction in response from these three antibody coating concentrations suggest biosensor binding saturation, which may imply that steric hindrance could play a role in antigen binding. As a result, the next lower antibody concentration should be selected for the studies.

Fixed concentration of antigen

Figure 5: Fixed concentration of antigen bound to the different antibody concentrations from Figure 3 – A/California/7/2009 (H1N1).

  Antibody Loading Vaccine Binding
Whole Virus Recombinant Hemagglutinin Split Virion Whole Virus Recombinant Hemagglutinin Split Virion
Assay Time (s) 300 300 300 300 300 30
Hold Time (s) 600 600 600 N/A N/A N/A
Shake Speed (rpm) 400

Table 2: Typical conditions for vaccine titer assays. Hold time refers to temperature equilibration time with samples in the instrument prior to the start of the assay.

Tips For Optimizing Vaccine Titer Assays

  • Select the appropriate biosensors for the study. For immobilization of non-purified samples such as serum antibodies, Protein A or Protein G biosensors (ProA or ProG) are recommended. When purified samples are available for capture, it is preferable to use High-Precision Streptavidin biosensors (SAX). In this case, the purified capture molecule should be biotinylated prior to use.
  • Use the same batch/lot number of biosensors for both the stan- dard curve and samples.
  • Ensure that biosensors are hydrated in assay buffer for at least 10 minutes.
  • Determine the shake speed for both the antibody loading and the antigen binding steps to obtain optimal conditions. Two shake speeds, 400 RPM and 1000 RPM, are recommended for evaluation prior to the start of the assay. The same shaking speed should be used for both the standard curve and unknown con- centration samples.
  • Standard curves should have a minimum of eight data points, in- cluding a reference or zero concentration point for data subtrac- tion, and should be run in replicates. The reference data should be acquired using media similar to the vaccine analyte.
  • Check raw data for overlapping binding curves in both the antibody loading and antigen binding scouting steps. This will highlight data that cannot be differentiated at these levels. For antibody loading, choose the highest concentration of antibody that does not overlap (typically around 2 nm binding). For antigen binding, discount all standard levels that overlap. Figure 5 shows examples of these overlaps in binding curves.
  • In some cases, depending on size, particles such as viruses will generate a negative signal, so the acquired data will need to be ‘flipped’ in the data analysis window prior to fitting.
  • Read time should be set at 300 seconds for inactivated and recombinant hemagglutinin vaccine samples. Split Virion sample read times should be ascertained from the raw data (typically < 30 seconds), and the appropriate window used to capture the linear region in the positive binding response.
  • Antibody and vaccine samples give different results for each strain, and so conditions such as shake speed, analysis time, antibody loading and linear range should be evaluated for each strain. Typical conditions for the assay are shown in Table 2.

Results – Inactivated Virus

The virus standard was tested from a range of 1 to 140 μg/mL based on the HA concentration provided by NIBSC. The linear range was established to be between 5 and 75 μg/mL with a linear regression value of 0.9951, as shown in Figure 6. Once the linear range and antibody loading concentration are determined, the assay is ready to be used for sample titre determination.

 Linear range of A/H1N1 California virus standard layout.

Figure 6: Linear range of A/H1N1 California virus standard layout.

Heat denatured H3/TX recombinant hemaglutinin sample

Figure 7: Heat denatured H3/TX recombinant hemaglutinin sample vs. control. Both samples are shown in red against the standard curve points shown in blue.

Stability Indicating Assay - Heat Denatured Samples

A robust assay capable of distinguishing between native and deactivated antigen would also be suitable as a stability assessment assay. Such an assay can then be used to determine the stability of the sample under accelerated degradation conditions such as temperature, pH and oxidative conditions.

The recombinant hemagglutinin H3/TX sample was diluted to 50 μg/mL using Pall ForteBio Sample Diluent and was divided into two aliquots. One aliquot was treated at 95 °C for five minutes while the other was left at room temperature as a control. The samples were then analyzed using the using the method described above with the H3/TX antibody bound to the biosensors. Figure 7 shows that the native sample gave an average recovery concentration of 53.6 μg/mL, while the heat-treated sample showed a loss of response to an average recovery concentration of 7.2 μg/mL. This proves the assay can successfully test heat- treated, stability-indicating samples and can distinguish between native and deactivated antigen.

A Comparison Between Srid and octet Analysis For Split Virion Samples

SRID, the most often used technique for influenza virus titre, is a gel-based assay that is easy to use and relatively inexpensive to setup. It can, however, take as long as three days to run, and can produce subjective data. 3 Antigen diffusion into the agarose gel alone can take as long as 16 hours.

Platform Assay time for 96-samples
SRID Several Days
OctetRed96 < 180 min
OctetRed384 < 90 min
Octet HTX < 20 min

Table 3: SRID technique and Octet platforms assay time comparison.

Due to differences in sample processing, the two techniques measure different molecular aspects of the sample. SRID measures the HA content after lysing with zwittergent 314, while the Octet system measures the diluted sample as is. The Octet platform is a much quicker technique with easier sample processing, involv- ing only a simple dilution in Sample Diluent. It also analyzes the vaccine sample itself and not a secondary sample that has been changed by the addition of a reagent. Precision and dynamic range produced by the Octet assay is also significantly better than what can be obtained from SRID.

B/Massachusetts
  Octet Assay (μg/mL) SRID (μg/mL)
Replicate Group Conc. Avg High Low Conc. %CV Conc. Avg High Low Conc. %CV
A 723.65 821 658 8.84 711 792 620 8.04
B 664.18 753 589 7.97 697 850 554 13.20
C 737.58 796 686 5.21 697 802 538 10.04

Table 3: Comparison of SRID vs Octet assay data for split virion vaccine samples.

Conclusion

Octet platform assays offer a number of advantages over the SRID assay for vaccine titre analysis:

  • Higher quality data: The Octet assay has a wider dynamic range (5–75 μg/mL), with greater precision and accuracy.
  • Faster results with plate-based format: It uses a 96 or 384- well plate format and can analyze a full plate in under three hours, including sample preparation time.
  • Simple sample prep: Samples only need to be diluted with no complex sample preparation. In-process and purified samples can be analyzed without encountering matrix effects.
  • Direct vaccine measurement: the data collected is for the vaccine itself and not a derived analyte produced from denaturing the sample with reagents such as Zwittergent 314.
  • Use the same assay for all strains: A change in vaccine strains does not require a change in equipment or method. A limited requalification with the appropriate biosensor and strain specific
    antibody is sufficient.
  • Determine vaccine stability: The assay can analyze heat inactivated samples, hence can also be used as a vaccine stability indicating technique.

References

  1. Phillip D Minor, Assaying the potency of influenza vaccines, Vaccines 2015, 3, 90–104.
  2. WHO Department of Immunization, Vaccines and Biologicals. Temperature sensitivity of vaccines. World Health Organization, 2006 (http://www.who.int/vaccines-documents/DocsPDF06/847. pdf ).
  3. Aziza P. Manceur, Amine A. Kamen, Critical review of current and emerging quantification methods for the development of influenza candidates, Vaccines, 2015, 33, 5913–5919.
  4. Yingxia Wen et al, Conformationally selective biophysical assay for influenza vaccine potency determination, Vaccines, 2015, 33, 5342-5349.

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