Column chromatography for the development of pandemic and seasonal influenza vaccines

Olga ChervyakovaResearch Institute for Biological Safety Problems, RK ME&S5th Meeting with International Partners on Prospects for Influenza Vaccine Technology Transfer to Developing Country Vaccine Manufacturers

Belgrade, Serbia, 27-28 March 2012

Influenza virus purification process

• Size-exclusion chromatography (Sepharose CL-6B)

Load: not exceeding 25% of column volume

Virus is eluted in the void volume

2

Clarification by Filtration (2.0/1.2 µm)

Ultrafiltration & Diafiltration (cut-off 100-300 kD)

Virus concentration (10-50 x), >90% protein removal

3

Influenza Vaccine

Pandemic

H1N1

H5N1

Seasonal

H1N1, H3N2

B

Pandemic influenza vaccines: Development and Research 4

Formulation (adjuvant addition)

Filling

Packaging

Purification& Filtration

Clarification by Filtration (2.0/1.2 µm)

Ultrafiltration & Diafiltration (cut-off 100 kD)

Size-exclusion chromatography (Sepharose CL-6B)

Microfiltration (0.22 µm)

Production of antigens

Inoculation into the 10-11-day old fertilized eggs

Harvest and inactivation (formalin, 3 days)

Development processKazfluvac

recombinant strainA/AstanaRG/6:2/2009 (H5N1),

produced by reverse genetics at RIBSP

Refluvacrecombinant strain

NIBRG-121 xp (H1N1), produced by reverse genetics at NIBSC

Pandemic influenza vaccines: Development and Research 5

Bulk Specifications: H1N1Single harvest

Bulk

ItemsQualifying standards

Batch Results

2009/1 2009/2 2009/3

Identity wt-like Confirm Confirm Confirm

HA Content(µg/ml)

≥ 30 44.9 69.1 85.0

Sterilityto meet

requirementConfirm Confirm Confirm

Ovalbumin(µg/ml)

≤ 2 4.5 2.0 2.0

Total Protein(µg/ml)

≤ 6-fold HA content

220 340 390

Endotoxin(EU/ml)

≤ 100 <50 <50 <50

Pandemic influenza vaccines: Development and Research6

Bulk Specifications : H5N1Single harvest

Bulk

ItemsQualifying standards

Batch Results

2009/1 2009/2 2009/3

Identity wt-like Confirm Confirm Confirm

HA Content(µg/ml)

≥ 30 30 31 31

Sterilityto meet

requirementConfirm Confirm Confirm

Ovalbumin(µg/ml)

≤ 2 0.5 0.5 0.5

Total Protein(µg/ml)

≤ 6-foldHA content

116 120 121

Endotoxin(EU/ml)

≤ 100 <50 <50 <50

7Pandemic influenza vaccines: Development and Research

Production of pilot vaccine batches

VaccineHA content

(µg/dose)Quantity(dose)

Kazfluvac (H5N1)15.0 1320

7.5 1320

Refluvac (H1N1)

15.0 1320

7.5 1320

3.75 1320

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Items Qualifying standards

Kazfluvac (H5N1) Refluvac (H1N1)

2010/1 2010/2 2010/3 2010/1 2010/2 2010/3

Identity wt-like Confirm Confirm Confirm Confirm Confirm Confirm

AppearanceSuspension with loose sediment

Confirm Confirm Confirm Confirm Confirm Confirm

Sterility To meet requirement Confirm Confirm Confirm Confirm Confirm Confirm

pH 6.6 – 7.3 7.1 7.1 7.1 7.1 7.2 7.1

Pyrogenicity apyrogenic Confirm Confirm Confirm Confirm Confirm Confirm

Toxicity To meet requirement Confirm Confirm Confirm Confirm Confirm Confirm

Thiomersal (µg/ml) 100±±±±15 103 103 100 102 100 101

Al+3 ion (mg/ml) 1.0±0.2 1.1 1.1 1.0 1.0 1.0 1.0

Formaldehyde (%) ≤0.008 0.006 0.006 0.006 0.006 0.006 0.006

Immunogenicity To meet requirement Confirm Confirm Confirm Confirm Confirm Confirm

Pandemic influenza vaccines: Development and Research

Quality control of pilot vaccine batches

9Pandemic influenza vaccines: Development and Research

Phase I/II clinical trials: Kazfluvac H5N1

PhaseInvestigational

vaccine

Volunteers*Number of persons with protective

antibody titers (>1:40)

Vaccine

groupControl

After 1st

vaccination (%)

After 2nd

vaccination (%)

I7.5 µg/dose 12 6 16.7 66.7

15 µg/dose 12 6 72.7 90.9

II7.5 µg/dose 40 - ** 52.5 -

15.0 µg/dose 40 - 57.5 -

*Ages of Volunteers: 18-60 yrs** not examined

I.I.Mechnikov Research Institute of Vaccines and Sera, RF RAMS (Moscow) Research Institute of Influenza, NWB, RF RAMS (St.Petersburg)

10Pandemic influenza vaccines: Development and Research

Phase I/II clinical trials: Refluvac H1N1

Research Institute of Influenza, NWB of RAMS, Russian Federation, St.Petersburg

PhaseInvestigational

vaccine

Volunteers*Number of persons

with protective

antibody titers (>1:40)

(%)Vaccine group Control

I

3.75 µg/dose 12 6 75.00

7.5 µg/dose 12 6 75.00

15.0 µg/dose 12 6 83.33

II3.75 µg/dose 40 - 85.00

7.5 µg/dose 39 - 92.30

*Ages of Volunteers: 18-60 yrs

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� Results of clinical trials show that Kazfluvac(H5N1) and Refluvac(H1N1) are well tolerated,

low reactogenic, safe and meet European СРМР ЕМЕА requirements on immunogenicity of

inactivated influenza vaccines.

� On the basis of the results of the clinical trials applications for governmental

registration of vaccines Refluvac (3.75 µg/dose ) for influenza A/H1N1v protection of

the population aged between 18-60 years and Kazfluvac (7.5 µg/dose ) as an influenza

A/H5N1 prophylactic means were filed.

Pandemic influenza vaccines: Development and Research

Phase I/II clinical trials: conclusion

12

Influenza Vaccine

Pandemic

H1N1

H5N1

Seasonal

H1N1, H3N2

B

13Seasonal influenza vaccines: Development and Research

Optimization of purification conditions:NIBRG-121 xp (inactivated virus)

StepBatch

20111201/1 20111208/2 20111227/3

Harvest/pooling

Inactivation

Clarification (2.0/1.2 µm)

Ultrafiltration/diafiltration (cut-off 100 kD)

Protein removal (%) 98.30 97.24 98.48

HA total (SRID) (%) 100 100 100

Gel-filtration (Sepharose CL-6B)

Protein removal (%) 99.81 99.71 99.61

HA total (SRID) (%) 28.1 20.7 38.2

Detergent treatment/removal

0.22 µm filtration

HA total (SRID) (%) 23.9 15.3 33.5

Ovalbumin (µg/mL) 1.4 1.1 5.0

14Seasonal influenza vaccines: Development and Research

Optimization of purification conditions:NIBRG-121xp (native virus)

StepBatch

20120122/4 20120201/5 20120206/6 20120213/7 20120217/8 20120224/9

Harvest/pooling

Clarification (2.0/1.2 µm)

Ultrafiltration/diafiltration

(cut-off 300 kD)

Protein removal (%) 90.20 96.62 97.28 99.19 97.55 94.88

HA total (SRID) (%) 100 100 100 100 100 100

Gel-filtration (Sepharose CL-6B)

Protein removal (%) 96.27 99.35 98.58 99.50 98.07 96.22

HA total (SRID) (%) 59.0 46.0 51.4 52.1 100.0 85.7

Inactivation

Detergent treatment/removal

0.22 µm filtration

HA total (SRID) (%) 14.8 17.0 33.0 37.4 60.4 32.9

Ovalbumin (µg/mL) 0.75 1.5 0.65 0.25 1.2 0.5

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Bulk Specifications : H1N1

Seasonal influenza vaccines: Development and Research

Items Qualifying standardsBatch Results

2012/1 2012/2 2012/3 2012/4

Identity wt-like Confirm Confirm Confirm Confirm

Total Protein(µg/ml)

≤ 6-foldHA content

0.475 0.325 0.490 0.247

HA Content(µg/ml)

≥ 90 113.82 76.12 124.46 47.09

Endotoxin(EU/ml)

≤ 100 5.0 1.25 1.25 1.25

Ovalbumin(µg/ml)

≤ 2 1.3 0.25 1.2 0.5

Sterility To meet requirement Confirm Confirm Confirm Confirm

16Seasonal influenza vaccines: Development and Research

Conclusion

•Tangential diafiltration in combination with column chromatography may be successfully used for influenza virus purification.

•The method is suitable for different influenza virus subtypes. In dialysis and chromatography the same buffers are used regardless of the virus subtype.

•Chromatographic profiles of the different influenza virus subtypes are identical. Virus is eluted in the void volume.

•≥99.0% removal of protein; ovalbumin content <2 µg/mL; HA yields > 30%

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Thank you for your attention!