Transmission Electron Microscopy (TEM) plays a major role in biological sciences and in the assessment of biological safety, particularly in virology. The technique allows visualization of virus particles, particle size, morphology, and intra- and extra-cellular location, both in biological fluids and in vitro.  TEM methods are used extensively for the identification and enumeration of virus particles in both diagnostic virology and biological safety.1-11 TEM is recommended in several guidelines, specifying which materials require testing including: cell lines, culture supernatants and fermenter bulk harvests.  Although relatively insensitive, TEM is a ‘catch-all’ test for viral detection which other techniques, such as in vitro and in vivo assays, or ‘specific’ molecular assays, such as qPCR, may miss.

TEM is included in a group of complimentary tests designed to provide characterization information, viral clearance data, absence of adventitious agents and product information for regulatory submissions, required or recommended by the Food and Drug Administration (FDA), Center for Biologics Evaluation and Research (CBER), International Committee of Harmonisation (ICH), European Medicines Agency (EMEA), European Pharmacopoeia (EP) and World Health Organisation (WHO).12-18

In a biologics manufacturing environment, various sample types are examined which include: master cell banks  (MCB), working cell banks (WCB), cells at limit (CAL), end of production cell banks (EoPCB), post-production cell banks (PPCB)), fermenter bulk harvests, virus seeds (MVSS/WVSS), and vaccine final product. 

Examination of Cell Cultures

TEM has a role in the characterization of cells and viruses, and also in contaminant identification.  It is routinely used as an end-point study in tandem with F-PERT in co-cultivation studies and in induction studies on mammalian and insect cell lines.  During the production of virus from inoculated or permissive cells, TEM can be used to identify the optimal timeline for supernatant harvest (Figures 1 and 2).  Studies of murine, rat and hamster cell lines routinely reveal cells containing endogenous retrovirus particles (Figures 3 and 4a-d.).  In situations where contamination events have occurred, TEM is found to be extremely useful in making initial investigative diagnoses which can narrow the field for a positive identification of the contaminant.  On several occasions bunyavirus, reovirus, adenovirus, and toxoplasma contaminants have been initially seen by TEM (Figures 5 and 6) and later confirmed by specific tests.

Visualisation of Virus Particles

TEM assists in the provision of data in clearance studies and as part of batch release testing of fermenter bulk harvests.  Samples are tested by TEM at stages throughout a production process.  It is also used in the examination of master virus seed stocks (MVSS) and working virus seed stocks (WVSS).  Methodologies employed include negative stain techniques,1-11 thin sectioning of virus pellets3,5,7 and of re-suspended virus/agar matrices.10,11 

Negative staining methods have been the ‘gold standard’ for virus identification (Figures 7 and 8), however, detection limits using this type of method are not suitable for the generation of clearance data.  As part of a regulatory submission, the FDA, CBER recommend that bulk harvests used in the production of biologicals be examined using a thin section transmission electron microscopy technique.13,14  While thin sectioning of virus pellets, or material containing virus particles, are common, many exhibit problems in relation to lack of homogeneity or consistency.

Our laboratory employs a re-suspension method which produces homogeneous sample/agar matrices enabling consistent, accurate and repeatable enumeration of virus particles in fermenter samples and culture supernatants (Table 1).10,11  Samples are ultra-centrifuged, re-suspended in a known volume, mixed with agar and processed to resin. The samples are then sectioned, stained and examined.  Particles are counted from known area sizes (and volume) and titres calculated. On some occasions, at the post-ultracentrifugation stage, no visible pellet could be discerned. The re-suspension method is particularly beneficial when there is insufficient debris present to form a visible pellet.  The ability to add a known volume for re-suspension and formation of sample/agar matrix enables samples to be processed to resin, sectioned and counted is advantageous.  Validation studies indicate that thin sectioning of re-suspended samples is superior to other thin sectioning methodologies.

 

TEM is a powerful tool that complements the battery of techniques available in the laboratory for determining biosafety of biologics and vaccines. While molecular biology methods such as qPCR are extremely sensitive and specific, it is this very specificity that may cause studies to miss detection of viruses not targeted by probes used. Consequently, TEM methodology, will continue to provide essential support in biosafety and diagnostic virology testing throughout the biologics and vaccine production stages.

Author

Euan W. Milne
Electron Microscopy Manager
SGS Life Science Services

References  

1. Watson, D.H. (1962) Electron-Micrographic Particle Counts of Phosphotungstate-Sprayed Virus.  Biochim. Biophys. Acta, 61:321-331.

2. Monroe, J.H. and Brandt, P.M. (1970) Rapid Semi-quantitative Method for Screening Large Numbers of Virus Samples by Negative Staining Electron Microscopy. Applied Microbiology. 20:2;259-262.
3. Miller, M.F., Allen, P.T. and Dmochowski, L. (1973) Quantitative Studies of Oncornaviruses in Thin Sections.  J. Gen. Virol. 21:57-58.
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8. Gelderblom, H.R. and Biel, S. (1999) Electron microscopy of viruses.  In: Virus Culture; A practical approach. Chapter 5, pp.111-148.  ed. A.J. Cann. Oxford University Press.
9. Gelderblom, H.R. (2001) Electron microscopy in diagnostic virology. BIOforum International 5:64-67.

10. Milne, E.W. (2003) Electron Microscopy: Current Techniques Used in Product Safety. BioProcessing Journal. 2:2;65-68.

11. Reid, G.G., Milne, E.W., Coggins, L.W., Wilson, N.W., Smith, K.T. and Shepherd A.J. (2003) Comparison of electron microscopic techniques for enumeration of endogenous retrovirus in mouse and Chinese hamster cell lines used for production of biologics.  J.Virol.Meth. 108:91-96.
12. Food and Drug Administration (FDA), Center for Biologics Evaluation and Research (CBER). Points to consider in the characterisation of cell lines used to produce biologicals (1993), 17 May 1993.
13. FDA, CBER. Points to Consider in the Manufacture and Testing of Monoclonal Antibody Products for Human Use.  Feb 1997.
14. FDA Guidance for Industry. Characterisation and Qualification of Cell Substrates and Other Biological Materials Used in the Production of Viral Vaccines for Infectious Disease Indications. Feb 2010.
15. European Pharmacopoeia 7.0. General Chapter 5.2.3. Cell substrates for the production of vaccines for human use.  01/2011:050203.
16. ICH Harmonised Tripartite Guideline Viral Safety Evaluation of Biotechnology Products Derived from Cell Lines of Human or Animal Origin Q5A (R1), 23 September 1999.
17. The European Medicines Agency (EMEA), ICH Topic Q5A (R1), Step 5. Note for guidance on quality of biotechnological products: viral safety evaluation of biotechnology products derived from cell lines of human or animal origin (CPMP/ICH/295/95), October 1997.
18. WHO – Recommendations for the evaluation of animal cell cultures as substrates for the manufacture of biological medicinal products and for the characterisation of cell banks. Proposed replacement TRS 878 Annex 1 (2010).