Bioprocessing Safety Essentials: Endotoxin, Mycoplasma, and Sterility Testing

Endotoxin Testing

Endotoxin control is mandated throughout bioprocessing, beginning with raw materials such as water-for-injection (WFI), media components, and excipients. USP <85>, EP 2.6.14, and JP 4.01 all define the current gold-standard assay, limulus amoebocyte lysate (LAL), requiring validated methods (gel-clot, turbidimetric, or chromogenic) with established sensitivity to ensure results below specified endotoxin limits. EMA guidelines recommend routine in-process monitoring to verify that purification steps consistently remove endotoxins to acceptable levels. Historically, the Limulus Amebocyte Lysate (LAL) assay has been the gold standard for endotoxin testing. While sensitive and well-established, the LAL assay presents several challenges including animal dependency (lysate from horseshoe crabs), process matrices interferences, and variability. To address these limitations, recombinant Factor C (rFC) assays are increasingly replacing LAL in bioprocessing. rFC utilizes a recombinant version of the horseshoe crab clotting enzyme, eliminating the need for animal-derived lysate.

Benefits of rFC include:

• Sustainability and ethics: No horseshoe crabs are harvested, making the assay environmentally friendly
• Matrix robustness: rFC assays are often less susceptible to interference from complex media and excipients, reducing the need for extensive sample preparation
• Consistency and reproducibility: Recombinant production ensures lot-to-lot consistency and reliable performance across different sites and manufacturing campaigns

Regulatory agencies, including the FDA and EMA, now recognize rFC as an acceptable alternative to LAL, provided equivalence is demonstrated through method validation. For modern bioprocessing, adopting rFC supports sustainable operations while maintaining the stringent endotoxin control required for product safety.

Mycoplasma Testing

Cell culture–based processes are highly vulnerable to mycoplasma, which can enter via contaminated raw materials (e.g., bovine serum, trypsin) or operator/environmental exposure in upstream operations. USP <63> and EP 2.6.7 require that biologics manufactured using mammalian cells be tested for freedom from mycoplasma prior to batch release (6,7). Classical compendial testing includes 28-day broth and agar culture methods combined with indicator cell assays (DNA staining). However, FDA and EMA guidances permit validated nucleic acid amplification techniques (NATs) as rapid alternatives, allowing earlier detection during upstream processing and reducing risk of large-scale contamination. Bioprocessing best practice applies mycoplasma testing not only to the final drug substance, but also to master cell banks (MCB), working cell banks (WCB), and in-process harvest materials.

Sterility Rapid Testing

Sterility assurance spans the entire bioprocessing chain. USP <71>, EP 2.6.1, and JP 4.06 mandate sterility testing of final drug products using either membrane filtration (preferred for clear solutions and large volumes) or direct inoculation (used for small-volume or oil-based products), with a 14-day incubation period (10–12). For raw materials like WFI and culture media, compendial microbiological limits (USP <61>, <62>) are applied upstream (13,14). In downstream processing, aseptic filtration, cleanroom controls, and validated sterilizing-grade filters (0.22 μm) are required to maintain sterility assurance. Advanced therapy medicinal products (ATMPs) and short shelf-life biologics are increasingly adopting rapid microbiological methods (RMMs), provided they are validated as equivalent or superior to compendial sterility tests (15,16).