institute of virology, vaccines and sera belgrade, serbia · influenza vaccine production from 1960...

Institute of Virology, Vaccines and Sera

Belgrade, Serbia

INACTIVATION PROCESSES

Aleksandar Radojević, MSc Biochem

Influenza Vaccine Production

Department

Influenza vaccine production From 1960 to 2005, the Institute Torlak was producing whole virus, inactivated,

trivalent influenza vaccine in embryonated hen's eggs.

From 2006 to 2010, Influenza vaccine production department had been under

reconstruction to meet GMP requirements.

Since January 2011, influenza vaccine production has been started, and all

production steps have been optimized.

Influenza vaccine

• Whole virus

• Inactivated

• Trivalent

• According to cGMP and Ph.Eur.

I n f l u e n z a v a c c i n e p r o d u c t i o n - u p s t r e a m p r o c e s s -

inoculation

Incubation

clarificationharvesting

Influenza vaccine production- inactivation-

- Inactivation of virus

suspension with

formaldehyde in the

concentration less than

0.2 g/l

Influenza vaccine production - downstream process -

Ultracentrifugation in sucrose

gradient

Tangential flow filtration

Sterile filtrationStorage

Inactivation

Inactivation is a critical step in the

inactivated influenza vaccine

production

Inactivation

•When is it done?

•Which substance is used as an inactivating agent?

•What are inactivation conditions?

Inactivation

When is inactivation done?

According to Ph.Eur.:

-Time: inactivation is initiated as soon as possible after harvesting,

-Production steps before and after: before or after the inactivation process, the monovalent pooled harvest is concentrated and purified by high-speed centrifugation or some other suitable method

Inactivation

Which substances are used as inactivating agents?

According to Ph.Eur.:

-if formaldehyde solution is used, the

concentration does not exceed 0.2 g/l of CH2O at

any time during inactivation,

-if beta-Propiolactone is used, the concentration

does not exceed 0.1 per cent V/V at any time

during inactivation

Inactivation

Inactivation conditions?

- Mixing duration,

- Inactivation temperature,

- Storage conditions

Inactivation

Inactivation process is to be validated for each

virus type before start of the production.

The virus is inactivated by a method that has been

demonstrated on three consecutive batches to be

consistently effective for the manufacturer.

Inactivation

The inactivation process shall have been shown to be

capable of inactivating the influenza virus without

destroying its antigenicity.

The process should cause minimum alteration of the

haemagglutinin and neuraminidase antigens.

The inactivation also has to inactivate bioburden.

Our Inactivation process

Unchangeable conditions:

- After clarification, followed by ultracentrifugation

- Inactivating agent: formaldehyde,

- Mixing duration: one hour

- Mixing temperature: T=RT

- Storage temperature: T= 5±3°C (in accordance with

Ph.Eur)

Changeable parameters are determined for every type

and strain of influenza virus by inactivation validation

-Formaldehyde concentration

-Inactivation duration


Inactivation validation

+ live virus present

- live virus absent

/ test not done

Inactivation with 0.005% CH2O

Sample

NoSAMPLING TIME DATE RESULT

1 after 60 min of mixing and

homogenization13.02.12. +

2 after 24 h of inactivation 14.02.12. +

3 after 48 h of inactivation 15.02.12. -






B strain



- live virus absent

/ test not done


Sample



homogenization14.02.12. -







8 after 168 h of inactivation 21.02.12. /

B strain



- live virus absent

/ test not done


Sample


1 at the moment of adding

HCHO01.02.11. +





4 after 24 h of inactivation 02. 02.11. -





H1N1 strain



- live virus absent

/ test not done


Sample



HCHO01.02.11. -





4 after 24 h of inactivation 02. 02.11. -





H1N1 strain



- live virus absent

/ test not done


Sample



HCHO13.10.11. +


homogenization13.10.11. /








H3N2 strain

During validation, a few control tests are executed to

determine critical parameters:

-Residual infectious virus (RIV)

-ALV and mycoplasmas

-NA and HA identity

-Bioburden


• Tests have to confirm:

− no residual infectious virus

− ALV and mycoplasmas are inactivated

− HA and NA are present and active

− bioburden is inactivated and product is sterile



• Having in mind the results of inactivation validation,

we chose: • For B strain – 0.005% CH2O, 7 days in refrigerator

• For H1N1 strain – 0.01% CH2O, 5days in refrigerator

• For H3N2 strain – 0.02% CH2O, 5 days in refrigerator

• Our product is fully in compliance with the latest

standards and requirements

Aleksandar Radojević, MSc Biochem

Associate in Influenza vaccine production department

Tel: +381 11 398 90 48; +381 64 84 66 320

Fax: +381 11 246 88 83

e-mail: [email protected]

Address:

Institute of Virology, Vaccines and Sera – Torlak

458 Vojvode Stepe St., 11152 Belgrade

P.O. Box 1, R Serbia

Tel: +381 11 397 66 74

Fax: +381 11 247 18 38

e-mail: [email protected]

www.torlakinstitut.com

Contact

Institute of Virology, Vaccines and Sera

Belgrade, Serbia

institute of virology, vaccines and sera belgrade, serbia · influenza vaccine production from 1960...

Documents