QUALITY OF BIOTECHNOLOGY PRODUCTS: VIRAL SAFETY

EVALUATION OF BIOTECHNOLOGICAL PRODUCTS DERIVED FROM CELL

LINES OF HUMAN OR ANIMAL ORIGIN

DR. MUNIRA SHAHBUDDINLECTURE 6

VIRUS VALIDATION STUDY: THE DESIGN, CONTRIBUTION AND INTERPRETATION OF STUDIES. VALIDATING THE INACTIVATION AND REMOVAL OF VIRUSES• Introduction

The principal aims of the guidelines are to provide guidance on the design of a validation study including the choice of viruses to be used and on interpretation of the ensuing data especially with respect to defining a process step which can be considered very effective in the inactivation and/or removal of viruses

• The guideline concern the validation of virus inactivation and/or removal procedures in all categories of medicinal and biotechnological products for human use with the exceptional of live viral vaccines including genetically engineered live vectors.

• Types of products 1. Products derived in vitro culture of cell lines of human or animal origin.2. Products derived in vivo culture of cell lines or from organ or tissue of human

or animal origin3. Products derived from blood or urine or other biological fluid of human or

animal origin.

WHY THE PROCEDURE IS VERY IMPORTANT?• Possible risk of viral and protein transmission• Hygiene and sanitation – how clean is clean?

Extraction of heparin from porcine.THE DIRTY PROCESS:

PREMARINE: Estrogen extracted from pregnant female horse

HUMAN/ANIMAL BLOOD PLASMA:Bloody business

• Viral contamination of a biological may arise from the source materials e.g. cell banks, blood, tissues and protein of animal/human origin.• Adventitious agents introduced during the production process e.g. the

use of animal serum in cell culture medium. • Human error – require effective standard operating procedure (SOP)

EXAMPLES OF VIRUS CONTAMINATION IN VACCINES1. Yellow fever vaccines : contaminated by Avian Leukocyte Virus due

to naturally infected eggs.2. Poliovirus and adenovirus vaccines prepared in 1950s – the primary

cultures of kidney cells from Rhesus monkey originally harbouring infection of SV40.

3. Human blood plasma – that can be contaminated with HIV, HBV or HCV.

4. Growth hormone extracted from human/animals’ cadavers – in the implicated transmission of prion diseases.

UNDERSTANDING THE PROCEDURE AND REASONS : HOW CONTAMINATION COULD HAPPEN

1. EXTRACTION PROCESS FROM SOURCES2. CONTAMINATION FROM THE SURROUNDINGS AND HANDLING

PROCEDURES3. TRANSPORTATION

SOURCES OF VIRAL CONTAMINATION• Sources material may be contaminated with a virus, indigenous to the

species of origin. Blood may contain HIV, HBV, parcovirus 19 and HAV.• Murine viruses some of which are pathogenic to man may contain

murine hybridomas• Cell lines which are intended to be used for genetic manipulation can

also be compromised• Cells may have a latent or persistent infection e.g. herpes virus or

retrovirus – maybe transmitted vertically from one cell generation to the next as viral genome and may be expressed of a production of cell line.

• The process construction of a production of cell lines.• Adventitious virus may be introduced by the use of animal product in

the production process e.g cell culture may be contaminated with bovine viruses through the use of serum from bovine.

VIRUS REMOVAL PROCEDURE• FILTRATION• - micro and nanofiltration• CHROMATOGRAPHY• - affinity chromatography

VIRUS INACTIVATION• The aim of validation proves will effectively inactivate viruses which

are known to contaminate the starting materials or which conceivably do so,• And to provide indirect evidence that the production process might

inactivate/remove novel or unpredicted virus contamination

UNDERSTANDING THE STRUCTURE OF VIRUSES• Enable scientists to figure mechanisms of virus inactivation

The structure of HIV

PRODUCTION PARAMETERS WHICH INFLUENCE THE EFFECTIVENESS OF A PROCESS STEP TO INACTIVATE/REMOVE VIRUS SHOULD BE EXPLORED AND INVESTIGATED THROUGHLY.

• Critical parameters include: flow rates, mixing rates, column dimension• Physicochemical parameters such as protein content, pH,

moisture content etc.

VIRUS INACTIVATION

• 2.1 Solvent/detergent (S/D) inactivation• This process does not denature proteins, because the detergents only affect lipids and lipid

derivatives. There is a 100% viral death achieved by this process and the equipment is relatively simple and easy to use. Equipment designed to purify post-virus inactivated material would be necessary to guard against contamination of subsequent process streams.

• 2.2 Pasteurization• Because pasteurization involves increasing the temperature of solution to a value that will

sufficiently denature the virus, it does not matter whether the virus has an envelope or not because the envelope alone cannot protect the virus from such high temperatures. However, there are some proteins which have been found to act as thermal stabilizers for viruses. Of course, if the target protein is not heat-resistant, using this technique could denature that target protein as well as the viral impurity. Typical incubation lasts for 10 hours and is performed at 60°C.

• 2.3 Acidic pH inactivation• Some viruses, when exposed to a low pH, will denature spontaneously. Similar to

pasteurization, this technique for viral inactivation is useful if the target protein is more resistant to low pHs than the viral impurity. This technique is effective against enveloped viruses, and the equipment typically used is simple and easy to operate. This type of inactivation method is not as effective for non-enveloped viruses however, and also requires elevated temperatures.

• 2.4 Ultraviolet (UV) inactivation• UV rays can damage the DNA of living organisms by creating nucleic acid dimers.

However, the damages are usually not important due to low penetration of UVs through living tissues. UV rays can be used, however, to inactivate viruses since virus particules are small and the UV rays can reach the genetic material, inducing the dimerisation of nucleic acids.