The document discusses validation studies for virus inactivation and removal in biotechnology products. It describes the aims of validation guidelines to provide guidance on designing validation studies, including selecting viruses and interpreting results to define effective process steps for inactivating and removing viruses. Validation is important for products derived from cell lines, blood, tissues or other biological sources to prevent viral transmission. Common virus inactivation methods discussed include solvent/detergent treatment, pasteurization, low pH treatment, and UV irradiation. Critical production parameters influencing effectiveness must be thoroughly investigated.

1. QUALITY OF BIOTECHNOLOGY PRODUCTS: VIRAL
SAFETY EVALUATION OF BIOTECHNOLOGICAL
PRODUCTS DERIVED FROM CELL LINES OF
HUMAN OR ANIMAL ORIGIN
DR. MUNIRA SHAHBUDDIN
LECTURE 6

2. 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

3. • 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 origin
3. Products derived from blood or urine or other biological fluid of human
or animal origin.

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

5. Extraction of heparin from porcine.
THE DIRTY PROCESS:

6. PREMARINE: Estrogen extracted from
pregnant female horse

8. HUMAN/ANIMAL BLOOD PLASMA:
Bloody business

9. • 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)

10. EXAMPLES OF VIRUS CONTAMINATION IN
VACCINES
1. 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.

11. UNDERSTANDING THE PROCEDURE AND
REASONS : HOW CONTAMINATION COULD HAPPEN
1. EXTRACTION PROCESS FROM SOURCES
2. CONTAMINATION FROM THE SURROUNDINGS AND HANDLING
PROCEDURES
3. TRANSPORTATION

12. 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.

13. • 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.

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

15. 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

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

19. The structure of HIV

20. 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.

21. 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.

22. • 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.