Contaminating agents in advanced therapy drugs: Bovine and porcine adventitious agents; Adventitious viruses; Retroviruses; Detection of retroviral agents: Amphotropic, xenotropic and ecotropic murine viruses.

Contaminating agents in advanced therapy drugs:

Bovine and porcine adventitious agents; Adventitious viruses; Retroviruses; Detection of retroviral agents: Amphotropic, xenotropic and ecotropic murine viruses.

(NFC-AEMPS [AEMPS Good Manufacturing Practices Standard]; European Community 2003/94/EC; Spain Decree 824/2010, June 25; FDA-PTC [Points-to-Consider] for Monoclonal Antibodies; FDA [ICH Guideline Q5A(R2)]-Viral Safety Evaluation of Biotechnology Products Derived from Cell Lines of Human or Animal Origin Guidance for Industry; EMEA/CPMP/BWP/268/95; EMEA/CHMP/BWP/398498/2005; EMA/CHMP/ICH/24235/2006-ICH Guideline Q9 on quality risk management)

Adventitious agents are biological agents (bacteria, fungi, parasites, mycoplasma, viruses or even prions) that are not introduced intentionally and that contaminate cell cultures. Although they may belong to any of the biological groups indicated, the main problem of interest is viruses, due to their specific requirements for detection.

Adventitious viruses can be difficult to detect, since conventional methods for their detection require observing the cytopathic effects generated in cell cultures, or demonstrating the hemagglutinating effect in cell culture supernatants for erythrocytes of some species/s (liquid culture medium that has been with cell cultures), or the hemadsorbent capacity (adherence of erythrocytes of some species/s to cell cultures developed in monolayers). These viruses can come from the cells themselves when they are obtained from infected animals, whwn using a virus to establish a cell line, or being accidentally introduced during the manipulation of cell cultures or by being present in some contaminated biological reagents (e.g. fetal bovine serum added to the culture medium solution as a source of proteins or porcine trypsin). Their presence must be analyzed to perform an adequate control that avoids contamination by them when the preparation of biological origin is administered as a medicine.

The main biological products in which the presence of adventitious viruses must be ruled out are the following: Monoclonal antibodies; Cell lines (master cell bank -MCB-); Working cells in use (WCB-); Cell culture supernatants; Unprocessed biological products obtained (Unprocessed bulk); or Finished biological medicines.

Several types of procedures can be used to detect adventitious viruses: 1) In vitro infectivity of cell cultures; 2) Amplification of nucleic acids (PCR, RT-PCR or massive sequencing -NGS-); 3) Generation of specific antibodies in rodents (mouse, rat, hamster); and 4) In vivo inoculation tests.

  1. In vitro infectivity methods in cell cultures allow the detection of many viruses, but they have the disadvantage that the appropriate cell line must be used according to the type of virus to be detected, and to demonstrate its presence the correct method must be followed (a.- visualization of cytopathic effect, if it occurs; b.- the generation of antigens expressed and released into the cell culture medium by means of erythrocyte agglutination -hemagglutination-, or on the cell surface of monolayer cultures during replication that can be detected by adherence to the surface of the cell monolayer -hemadsorption-). One of the problems is that cell cultures must be maintained for an appropriate time, up to 28 days, with intermediate passages at 7 or 14 days, with the consequent periodic changes of culture media, etc. The cells used should include at least one cell line from each species of origin, such as a human diploid cell line (e.g. MRC-5) and a monkey kidney cell line (e.g. Vero cells). Molecular methods may be used as an alternative.
  2. Methods based on the amplification of viral nucleic acids (RNA or DNA) require the use of primers specific to the virus to be detected, since families, and sometimes genera within a family, are phylogenetically different and do not share a common genomic sequence that allows amplification with a single pair of primers, as occurs with bacteria with the amplification of the 16S rRNA gene, or with fungi with the amplification of the 28S rRNA gene. This involves carrying out many different procedures, depending on the viruses whose presence is to be determined. 
  3. Methods based on the generation of specific antibodies inoculate the product into rodents and subsequently detect the specific antibodies generated. They can be performed in mice (Mouse Antibody Production Test -MAP-); when murine cell lines are used; antibody production in hamsters (Hamster Antibody Production -HAP-); or in rats (Rat Antibody Production -RAP-). These methods can now be replaced by molecular tests.
  4. In vivo inoculation methods can be performed with suckling mice, adult mice or embryonated chicken eggs. These methods require monitoring the condition of the animals or, in the case of embryonated chicken eggs, detecting the presence of hemagglutinating viruses. These methods can now be replaced by molecular tests. 

Adventitious viruses for which detection is considered recommended:

When non-human primate cell lines are used they should be screened for Simian herpesvirus, Simian Cytomegalovirus (sCMV), Simian Immunodeficiency virus, Simian T-cell lymphotropic virus (STLV), Human T-cell lymphotropic virus (HTLV), Simian Foamy virus, Human Immunodeficiency virus (HIV), Encephalomyocarditis virus, Simian Hemorrhagic Fever virus (SHF), Simian Varicella virus (sVZV), Adenovirus, SV40, Monkeypox, Rubella, Ebola virus, Simian Retrovirus (SRV), or any other zoonotic virus.

When cell lines of human origin are used, they should be screened for Epstein-Barr virus (EBV), Cytomegalovirus (CMV), Hepatitis B (HBV), Hepatitis C (HCV), Human Herpesvirus type 6 (HHV-6), Lymphocytic Choriomeningitis virus (LCM), Lymphotropic T-cell Virus (HTLV), Human Foamy virus, and any other possible virus based on the history of the donor from whom the cell line was derived. Cells from patients with Creutzfeld-Jakob disease (CJD) or TSE (Transmissible Spongiform Encephalopathies), or their relatives, should not be used.

For cell lines with exposure to raw materials of human or animal origin (e.g. bovine serum or porcine trypsin), testing for human, bovine, and porcine viruses must be performed. According to 9 CFR part 113.47, bovine viruses to be tested must be at least: Bovine viral diarrhea virus, Reovirus, Rabies virus, Bluetongue virus, Bovine Adenovirus, Bovine Parvovirus, and Bovine Respiratory Syncytial virus; while porcine viruses must be at least: Bovine Viral Diarrhea virus, Reovirus, Rabies virus, Porcine Adenovirus, Porcine Parvovirus, Transmissible Gastroenteritis virus (TGEV), Porcine Hemagglutinating Encephalitis virus (PHEV). Detection of additional viruses may be requested by the client.

When cells are used for the production of monoclonal antibodies, they should be analyzed for Sendai virus, Hantaan virus, Lymphocytic Corimeningitis virus, Minute virus of mice (MVM), among others.

Any cell line or the products generated must be tested to exclude the presence of Retroviruses. Many cell lines, such as murine cells used to produce monoclonal antibodies, can be infected with Murine Retroviruses. Retroviruses can be detected by electron microscopy, by co-culture with retrovirus-susceptible cells, or by the presence of reverse transcriptase with its PERT (product-enhanced reverse transcriptase) assay variant. At IVAMI, the detection of reverse transcriptase activity is performed by RT-PCR of the gene encoding the polyprotein of tobacco mosaic virus (BMV). Detection of additional retroviral agents, such as amphotropic, xenotropic, ecotropic murine retroviral agents, etc., can be requested.

Tests 

  • Detection of Bovine Adventitious agents (cell cultures) (MDBK and BT cell line cultures available, or other required). Tests with each of the cell lines for 28 days of culture. 
  • Detection of Bovine Adventitious agents (PCR for viruses with DNA genome:Bovine adenovirus; Bovine herpesvirus; Bovine parvovirus; Bovine papillomavirus; Bovine polyoma, or others required). 
  • Detection of Bovine Adventitious agents (PCR for viruses with RNA genome: Rabies virus, Bovine viral diarrhea virus, Bluetongue virus, Bovine coronavirus; Bovine enterovirus; Bovine parainfluenza; Bovine reovirus; Bovine respiratory syncytial virus; Bovine rhinovirus; Bovine rotavirus). 
  • Detection of Porcine Adventitious agents (cell cultures) (ST and PK-15 cell line cultures available or other required). Tests with each of the cell lines for 28 days of culture. 
  • Detection of Porcine Adventitious agents (PCR for viruses with DNA genome: Porcine Adenovirus; Porcine Herpesvirus types 1, 2 and 3; Porcine Circovirus types 1 and 2; Porcine cytomegalovirus » Porcine Herpesvirus type 2; Porcine Parvovirus; or others required). 
  • Detection of Porcine adventitious agents (PCR for viruses with RNA genome:Porcine coronaviruses (TGEV and PHEV); Porcine rotavirus A, B and C; Porcine Sapelovirus (PEV-8); or others required). 
  • Detection of human adventitious agents (PCR for viruses with DNA genome: Epstein-Barr virus (EBV); Cytomegalovirus (CMV); Human Herpes virus 6 (HHV-6); or others required). 
  • Detection of human adventitious agents (PCR for viruses with RNA genome: T-cell lymphotropic virus (HTLV); hepatitis B virus (HBV); hepatitis C virus (HCV); Lymphocytic Choriomeningitis virus; or others required). 
  • Detection of adventitious agents from non-human primates (apes) (PCR for viruses with DNA genome: Simian herpesvirus; Simian cytomegalovirus (sCMV); Simian varicella virus (sVZV); Adenovirus; Monkeypox; Polyomavirus Simian virus 40 (SV40); or others required). 
  • Detection of adventitious agents from non-human primates (apes) (PCR for viruses with RNA genome: Simian immunodeficiency virus; Simian T-cell lymphotropic virus (STLV); Human Foamy virus (HFV); Encephalomyocarditis virus; Simian hemorrhagic fever virus (SHF); Rubella; Ebola virus; Simian rotavirus; Simian Foamy virus (SFV); or others required). 
  • In vitro assay of adventitious viruses (by PTC: Points to Consider). 
  • In vitro assay of viral contaminants (FDA: Q5A(R2) Viral Safety Evaluation of Biotechnology Products Derived From Cell Lines of Human or Animal Origin Guidance for Industry). 
  • Detection of retroviral agents (detection of reverse transcriptase in cell culture samples with BMV [Brome Mosaic Virus] by RT-PCR). 
  • Detection of retroviral agents (amphotropic murine viruses-Murine Leukemia virus-MuLV-A) (RT-PCR). 
  • Detection of retroviral agents (Xenotropic murine viruses-MuLV-X) (RT-PCR). 
  • Detection of retroviral agents (Murine C-type retrovirus-MuLV-E) (RT-PCR). 
  • Mouse antibody production test (MAP: Mouse Antibodies Production). Not performed in our laboratory.