Transmission Electron Microscopy testing (TEM testing) is a powerful imaging technique used to visualize the ultrastructure of cells and viruses at the nanometer scale, providing detailed insights into their morphology and composition.

TEM provides high-resolution imaging that can detect and identify potential contaminants, ensuring the integrity and safety of biological materials (e.g., cell lines, virus seeds, bulk harvests) used in production. TEM testing enables visualization of virus particles, determination of particle size and morphology, identification of virus particle location, and quantification.

TEM applications for evaluation of biologic products

TEM for adventitious agents testing

TEM testing enables the detection of aggregates and contaminants, including adventitious agents (e.g., parvovirus, polyomavirus, picornavirus, reovirus, adenovirus) and retroviral particles (e.g., type-A and type-C), in bulk harvest samples, cell banks, or virus seed stocks. This assessment is critical for ensuring viral safety. As outlined in the ICH guideline Q5A (R2), TEM should be utilized on the cell substrate used in biologics production and on unprocessed bulk harvest samples to detect and quantify VLPs and other endogenous viruses or adventitious agents.

For cell line qualification, retrovirus testing should be conducted on the MCB, and cells cultured up to or beyond the Limit of In Vitro Cell Age (LIVCA), also referred to as the End of Production Cell Bank (EOPCB). This includes evaluating retroviral particles via TEM. According to the ICH guideline Q5A (R2), if a cell line is not known to produce retroviral particles, TEM analysis should be performed on the cells, and a PCR-based RT assay should be conducted on the clarified supernatant. If either TEM or RT results are positive, an assay to detect infectious retroviruses in permissible cells must follow.

For cell lines known to produce retroviral particles (e.g., CHO, NS0, Sp2/0), RT activity is anticipated, eliminating the need for a PCR-based RT assay. However, TEM testing must be performed to identify the type of retroviral particle present. This determination is essential for assessing whether the particles are infectious (type-C) or non-infectious (cytoplasmic type-A and type-R). Therefore, infectivity assays should be performed using relevant permissive cells (e.g., Mus dunni for general murine retrovirus detection, SC-1 cells for ecotropic murine retroviruses detection).

For bulk harvests, the manufacturing process’s ability to remove and/or inactivate rodent retroviruses from products must be evaluated. This involves estimating quantitatively the overall levels of virus reduction achieved by the production process and comparing it to the virus levels present in the unprocessed bulk. To perform this comparison, TEM is used to estimate the amount of virus in the unprocessed bulk. For CHO cells e.g., a safety margin of < 10^-4 particles/dose is considered acceptable for RVLPs for mAbs or other recombinant proteins if in vitro testing fails to identify the presence of such infectious retroviruses.

TEM for RVLP quantification in CHO derived products

CHO cells are commonly used to produce mAbs and other recombinant proteins due to their safety profile. Many cell lines, including CHO cells, contain partial or complete retroviral sequences because retroviruses integrate their genomes into the host cell genome. These sequences are remnants of retroviral infections that occurred at the germ line level and were passed to subsequent generations.

Such sequences can be expressed, resulting in defective RVLPs. Although RVLPs have never been transmitted to patients via CHO-derived mAbs or other recombinant drugs, nor have they been linked to human disease, regulatory agencies require manufactures to quantify RVLPs in unprocessed bulk harvests. This quantification monitors RVLP production over sequential lots, demonstrating upstream process consistency. Once the manufacturing process is scaled up for commercialization, RVLPs must be quantified in the bulk harvest.

Regulatory authorities require data from at least three cell culture campaigns, lots, or batches of bulk harvests for submission. In accordance with ICH guideline Q5A (R2), this data must be submitted as part of the marketing application or registration package.

The number of RVLPs in the bulk harvest also sets expectations for viral clearance levels in downstream processing, which must be shown to substantially remove and/or inactivate more RVLPs than there are present in the bulk harvest. Learn more.

TEM for virus & vector characterization

According to ICH guideline Q5A (R2) Annex 6, numerous new product types are manufactured using characterized cell banks of human or animal origin (i.e., avian, mammalian, or insect sources). Annex 6 covers both helper-virus dependent and independent genetically engineered viral vectors and viral vector-derived products, which can undergo virus clearance based on their physicochemical properties. These products encompass VLPs, protein subunits produced from baculovirus/insect cells, nanoparticle-based vaccines, and viral-vector products such as AAVs.

According to Annex 6, TEM testing should be performed to test for retroviruses and other endogenous viruses on the MCB and LIVCA, as well as in the virus seeds. If retroviruses are detected, subsequent steps should include the quantification of potential retroviral particles in the unprocessed bulk harvest from at least three lots/batches to establish the target level for viral clearance. Annex 6 emphasizes that ensuring the viral safety and contamination control of new product types requires a comprehensive program that includes material sourcing, virus testing at appropriate manufacturing steps, and the removal and/or inactivation of adventitious agents and helper viruses.

TEM for comprehensive analysis of AAV products

The British Pharmacopoeia on “Advanced Therapy Medicinal Products Guidance” specifies that during rAAV characterization, TEM should be used to identify other contaminants and impurities, such as broken rAAV capsids, non-rAAV debris, and non-encapsidated DNA. TEM testing is crucial for quantifying rAAV capsid populations (e.g., the distribution of full, partially full, and empty rAAV capsid particles) and ensuring capsid purity (by identifying impurities e.g., inactive product variants and aggregates) and integrity (e.g., intact, and damaged capsids, aggregates).

The EMA Guideline on the Quality, Non-clinical and Clinical Aspects of Gene Therapy Medicinal Products and the FDA Guidance for Industry: CMC Information for Human Gene Therapy INDs both specify the critical role of TEM testing in the characterization of viral vectors (e.g., AAVs, lentiviruses, retroviruses, adenoviruses, baculoviruses).

According to the EMA guideline, controlling the numbers of empty particles, aggregates, and replication-competent vectors is essential for ensuring the safety and efficacy of gene therapy products. TEM testing must be used for characterizing the capsid population and structural integrity.

Similarly, the FDA guidance highlights the necessity of TEM testing for the analysis of viral vectors. It underscores the importance of measuring typical product-related impurities, including defective interfering particles, non-infectious particles, and empty capsid particles. TEM offers a robust method for assessing these impurities and reporting ratios, such as full-empty capsid particles, thereby ensuring that the viral vectors meet all regulatory requirements.

For GMP-compliant commercialization of gene therapy products like AAVs, regulatory authorities mandate comprehensive assessment of the viral capsids. By integrating TEM testing as recommended by British Pharmacopoeia, EMA, and FDA, manufacturers can fully characterize their viral vector products, ensuring they are free from critical impurities and possess the required structural integrity for safe and effective gene therapy applications. Learn more .

TEM techniques for biologic characterization

Negative stain TEM (nsTEM) and positive stain TEM (psTEM) are two techniques used in TEM testing for biologics, including viral and retroviral characterization. A third technique, cryogenic TEM (cryoTEM, also known as cryo-EM), further complements these methods. Choosing the appropriate technique depends on the specific requirements of the TEM testing application and the desired level of details needed for accurate characterization.