2.2 Europe

1.Notes to applicants for marketing authorizations on the preclinical biological safety testing of medical products derived from biotechnology (and comparable products derived form chemical synthesis). CPMP: Ad Hoc Working Party on biotechnology/pharmacy and working party on safety of medicines, 1989.

2.Notes to applicants for marketing authorizations on the production and quality control of medical products derived by recombinant DNA technology. CPMP: Ad Hoc Working Party on biotechnology/pharmacy, 1994.

3.Safety studies for gene therapy products. CPMP: Ad Hoc Working Party on biotechnology/pharmacy and working party on safety of medicines, 1997.

2.3 International Conference on Harmonization (ICH)

1.Quality of biotechnological products: viral safety evaluation of biotechnology products derived form cell lines of human or animal origin (ICH Topic Q5A), 1997.

2.Quality of biotechnological products: Stability testing of biotechnological/biological products (ICH Topic Q5C), 1996.

3.Quality of biotechnological products: Derivation and characterization of cell substrates used for biotechnological/biological products (ICH Topic Q5D), 1998.

4.Preclinical safety evaluation of biotechnological-derived pharmaceuticals, 1997.

3.0 Human Viruses

3.1 Cytomegalovirus ( CMV )

CMV is an opportunistic pathogen that infects lung, kidney, gut, and other organs in situations where an individual is immunologically immature, such as in the fetus and neonate. Infection also occurs in situations of immunosuppression, e.g. transplant patients, patients undergoing chemotherapy and those with the HIV virus. CMV, like other herpesviruses, can remain as a latent infection with cells. Infection of patients can lead to serious disseminated diseases.

Detection of CMV was performed by PCR method. Primer pairs were designed to amplify the major immediately early gene, matrix protein (pp65), glycoprotein B, and pol regions of the CMV genome.

3.2 Hepatitis B Virus ( HBV )

HBV is an enveloped-DNA virus.  The HBV is a significant cause of posttransfusion hepatitis and also a major cause of chronic hepatitis and hepatoma worldwide. Detection of HBV was performed by PCR method. Primer pairs were designed to amplify the core antigen, pre-core, pre-S, and surface antigen regions of the HBV genome.

3.3 Hepatitis C Virus ( HCV )

HCV is an important human pathogen and accounts for the majority of transfusion-associated, non-A, non-B hepatitis (NANB) cases worldwide. HCV is a small, enveloped virus with a positive-strand RNA genome of approximately 10 Kb.

Detection of HCV was performed by RT-PCR method.  Primer pairs were designed to amplify the 5’ non-coding region of the HCV genome.

3.4 Herpes Simplex Virus (HSV-I/II)

Infection with HSV is extremely common and pathogenesis can vary depending on a variety of factors. These factors include age, immune status of the patient, the antigenic type of infecting virus (HSV type I or HSV type II), and the site of infection. HSV-I is responsible for acute necrosis encephalitis. HSV II is the predominant cause of genital neonatal herpes.

Detection of HSV-I/II was by using PCR method. Primer pairs were designed to amplify the DNA polymerase, thymidine kinase region of the HSV genome.

3.5 Human T-cell Leukemia Virus ( HTLV-I/II )                   

HTLV-I was the first human retrovirus described and is considered to be the causative agent of adult T-cell leukemia/lymphoma . HTLV-I has also been associated with a family of related neurological disorders. HTLV-I is transmitted through sexual contact , transfusion of infected blood, and via breast milk from infected mothers to their offspring . HTLV-II is a closely related retrovirus.

Detection of HTLV-I/II proviral DNA was performed by PCR. Primer pairs were designed to amplify the env, gag, and pol region of the HTLV genome.

3.6 Human Immunodeficiency Virus ( HIV-I/II )  

HIV-I is recognized as one of two etiologic agents of AIDS. Screening of HIV infection currently relies on the detection of specific antibodies to viral protein. However, HIV-specific antibodies are detectable on the average of 3 months after the infection of the virus has occurred. By using PCR-based assay techniques, it is useful for screening seropositive mothers of newborns for resolving the infection status of subjects with interdeterminant Western blot and with doubtful EIA reactivity, and detecting the HIV DNA in latent stage.

Primer pairs for PCR detection were designed to amplify the env, gag, and pol regions of HIV genome.

4.0  Porcine Virus

One potential problem for xenotransplantation is the possibility of a zoonotic infection resulting in the transfer of a known or unknown pathogen of non-human origin into the human population.  We will plan to develop a serious of tests for important zoonotic pathogen.

4.1 PERV (Porcine Endogenous Retrovirus)

A PERV capable of infecting human cells has been identified. The porcine xenografts might lead to PERV infection of recipients is critical for evaluating the safety of pig-to-human xenotransplantation.  PERV was widely distributed in the pig germline.  We have developed PERV-specific PCR and RT-PCR assays for detecting these viruses in pigs and patients.

Detection of PERV performs by PCR, RT-PCR, and Southern blot analysis. Primer pairs were designed to amplify the gag, pol, and env regions of PERV genome.

5.0 Mycoplasma test

Mycoplasma contamination of cell cultures is a common occurrence, and is capable of altering virtually property and parameter measured in cell cultures, including virus production. These eight organisms (including M. arginini, M. fermentans, M. hominis, M. hyorhinis, M. orale, M. pirum, M. salivarium, and Acholeplasma laidlawii) account for more than 95% of mycoplasma contaminants found in cell cultures. There is a virtually universal requirement that all biological products intended for use in man or animals are free from mycoplasma, spiroplasma and acholeplasma contamination.

5.1 Polymerase Chain Reaction (PCR) Mycoplasma Detection

PCR is the fastest and most sensitive method of mycoplasma detection available. The method can detect as few as 20~180 colony-forming units (CFUs) per 1 ml sample. It detects over 20 different mycoplasma species, including the eight most commonly encountered mycoplasma contaminants in cell culture.

5.2  Culture Methods

The Direct Culture consists of inoculation of mycoplasma broth and agar media with suspensions of the cell culture. Test samples are incubated at 37℃ under aerobic and anaerobic conditions and observed for growth and/or pH changes. Agar plates are examined microscopically for at least three weeks.

5.3  Hoechst DNA Staining

The Indirect Hoechst DNA Staining consists of incubating the suspected cell culture for five days with an indicator cell line (Vero cell), which supports the growth of fastidious mycoplasma. The culture is then stained with bisbenzamide DNA-fluorochrome stain to detect mycoplasma DNA.

6.0 Sterility

The presence of bacteria or fungi contaminants in cell cultures seriously compromises virtually all research or production work involving culture technology.  Although many contamination events are overt and readily apparent to the responsible investigator, others are insidious and more difficult to detect.

6.1 Culture method

Tests for bacterial and fungal contaminants use Trypticas soy broth, Brain heart infusion broth, Sabouraud dextrose broth, Blood agar plate and MacConkey agar plate inoculated with the test sample and incubated at 26±2℃ and 37±2℃ respectively for 14 days.

Sterility testing for cell lines, primary cells or ingredients of animal origin (9 CFR) (except live vaccines) by the direct incubation method.  This assay meets the FDA recommendations (9 CFR 113.26) (21 CFR 610.12), CVMP and EP2.6.1 requirements for testing for bacteria and fungi in biological products from animals or animal products.  The presence of microorganisms is detected by concentrating the microorganisms by incubating in an appropriate medium.  Trypticas soy broth, Brain heart infusion broth, Sabouraud dextrose broth are used for the growth of aerobic bacteria and fungi.  Blood agar plate and MacConkey agar plates are used for the growth of both anaerobic and aerobic bacteria.

6.2 Endotoxin assay

        Bacterial endotoxin test for biopharmaceutical or biotechnology product was conducted by LAL testing.

6.3 Bioburden assay

Bioburden assay estimates a total viable aerobic count of microbial contamination and can be conducted on bulk products prior to sterilization. Validation studies to ensure potential contamination of reagents are within acceptable standards for the shelf life period can also be designed.

7.0 Characterization of cell

7.1 Isoenzyme Analysis

Different cell enzyme patterns may be compared qualitatively between different cell since enzyme activities are expressed by different, though related species of molecules. These isoenzyme, such as lactate dehydrogenase, purine nucleoside phosphorylase, glucose-6-phosphate dehydrogenase, malate dehydrogenase, peptidase B, aspartate aminotransferase, and mannose phosphate isomerase, may be separated electrophoretically and the distribution patterns obtained are found to be characteristic of different species.  The isoenzyme analysis technique can be used to detect interspecies cross-contamination of cell cultures and to authenticate cell species of origin.

7.2 DNA Fingerprinting (STR fingerprinting)

All eukaryotic genomes contain regions of simple repeatitive DNA, called short tandem repeats ( STR ) or microsatellites, which consist of tandem repeats of a  small number of bases. The number of repeats at STR locus can be highly variable among individuals, resulting in length polymorphisms that can be detected by relatively simple PCR-based assays.  The STR fingerprinting system can be used to detect intraspecies cross-contamination of cell cultures, to authenticate cell of human, to establish DNA databanks, to process paternity samples, and to monitor bone marrow transplants.

8.0  Final products testing

8.1 DNA Contamination Test

The DNA contamination test includes a series of extracted test samples and a known amount of DNA. PCR and Southern blot assay can be done with species-specific DNA primers and probes for samples derived from porcine.

8.2 Cytotoxicity

The methods for detecting carcinogens and mutagens with the Salmonella mutagenicity test were described previously (Ames et al., 1975). The test is highly efficient in detecting carcinogens as mutagens. Many carcinogens and non- carcinogens have been tested and we are currently compiling results obtained using the test in this and many laboratories throughout the world. The test has been adapted for use in detecting chemicals which are potential human carcinogens or mutagens by adding homogenates of rat (or human) liver directly (S9 mix) to the petri plates thus incorporating an important aspect of mammalian metabolism into the in vitro test. In this way, a wide variety of carcinogens requiring metabolic activation can be detected easily as mutagens.  Bacteria for gene mutation were used including S. typhimurium TA97, S. typhimurium TA98, S. typhimurium TA100, S. typhimurium TA102.