Recombinant Lysate Manufacture: Considerations for Contamination Control

Friday, August 1, 2025

Professor Tim Sandle, PhD- Head of GxP Compliance, Quality Risk Management & Sterility Assurance, Kedrion BioPharma, UK

Introduction

Pyrogens in general (including lipoteichoic acids, CpG DNA, viruses, and flagellin), and Gram-negative bacteria that release endotoxin, present a risk for pharmaceutical products that are administered in ways that bypass the body’s natural defenses, especially those that are infused into the bloodstream. This concern exists since endotoxin stimulates host macrophages to release inflammatory cytokines, and it can cause endotoxic shock. The traditional test for endotoxin, across six decades, simulates immunological response within the horseshoe crab by harnessing Factor C as a biosensor. Today, the direction of laboratory travel now has the likely potential to gravitate strongly towards recombinant reagents.

Recombinant Factor C (rFC) is a non-animal-derived reagent used to detect bacterial endotoxins in pharmaceutical products. The reagent was developed in the 1990s;1 however, considerable time has been taken to verify its suitability (successfully against a range of different pharmaceutical products,2 albeit with some experts expressing concerns over the ability to detect autochthonous endotoxins since lipopolysaccharide from different Gram-negative bacteria may possess different numbers and arrangements of acyl chains)3,4 Time has also been needed to gain compendial acceptance. With its application, the binding of endotoxin activates the rFC zymogen, which catalytically hydrolyzes synthetic substrates to form measurable products (a fluorogenic substrate, resulting in the generation of a fluorogenic compound), thus quantifying the endotoxin. The reagent requires the use of an end-point fluorescence-based endotoxin method, using a microplate reader in order to obviate the need for additional amplification. The difference in fluorescence is proportional to the endotoxin concentration in the sample and is used to calculate a final endotoxin result.

An alternative is the Recombinant Cascade Reagent (rCR), an endotoxin detection method that mimics the natural enzymatic cascade of the Limulus Amebocyte Lysate (LAL) assay, and which does not require alternative testing equipment to LAL. With these approaches, the former is described in the European Pharmacopeia (from January 2021), whereas both approaches are permissible as described in the United States Pharmacopeia (from May 2025).

In terms of manufacture, rFC is produced using recombinant DNA technology,5 where the gene encoding the natural Factor C protein from horseshoe crabs is inserted into a host organism, like mammalian cells, to produce the protein in large quantities.6 Based on the manufacturing process, the production method is at risk of contamination in line with other biotechnologically produced materials. Since the vector containing the Factor C gene is introduced into a host cell,7 such as mammalian cells (like CHO DG44 or HEK293) or yeast cells, this step is especially prone to contamination with viruses or mycoplasmas. In particular, host cell transformation carries the risk of contamination with host cell DNA, which can pose production concerns and the risk of financial losses and facility downtime. This article considers these issues.

Path to Recombinant Endotoxin Reagents

In 1911, Florence Seibert developed a test for exogenous pyrogens using Oryctolagus cuniculus rabbits (based on immune system responses between Leporidae and humans. This entered regular use during the 1920s. Latterly, rabbit pyrogen testing, where non-endotoxin pyrogens are a concern, has been replaced by the Monocyte Activation Test (MAT). With endotoxin, by the 1980s, a substantial volume of pyrogen test was being undertaken using an endotoxin-specific test using the reagent extracted from horseshoe crabs (largely Limulus together with species of Tachypleus).8 The endotoxin reagent - a blood cell lysate extracted from the arthropods - was developed by Bang and Levin in the mid-1960s.9

Triggered by a combination of seeking to protect horseshoe crab species and sustain populations (not least due to fatality rates of between 15 and 30% per year from the bleeding process10 with survivors displaying reduced participation in spawning activity and sustained reduction in hemocyanin)11 and to lower production costs (dwindling horseshoe crab populations began to turn a low-cost, high profit reagent into something far more costly albeit that the production of rFC involves an expensive pharmaceutical-grade process), as well as a more stable reagent, the endotoxin test industry has been gradually phasing production and encouraging customers towards adopting a synthetic alternative.

The pathway for this came about once genes encoding the proteins in the horseshoe crab clotting cascade were sequenced, starting with the zymogen Factor C and then with other cascade proteases (Factor B and Factor G).12 This allowed for their production via recombinant protein production, starting with a Factor C reagent, followed by the recombinant cascade with Factor G removed (to avoid β-Glucan-induced false positivity), hence leaving the cascade composed of Factor C, Factor B, and proclotting enzyme. The first presentation of the suitability of the recombinant cascade was in 2015, eighteen years after Factor C. Subsequently, there is less published information about the recombinant cascade, which accounts for the hesitancy in Europe to, as yet, adopt it.13

The recombinant reagent centers on the polypeptide Factor C. The manufactured rFC is a 123 to 13kDa-sized molecule, created through genetic engineering.14 Factor C has an endotoxin (lipopolysaccharide) receptor (present in Heavy Chain Fragment Factor C with Lectin Domain) to which endotoxin binds to activate its catalytic site.15 The molecule can be produced in a single-chain form or a double-chain form (made up of one larger chain and one smaller chain connected by disulfide bonds).16 Full-length rFC is more efficacious in terms of its endotoxin specificity, as ascertained by lipopolysaccharide binding assays.17 This was subsequently enhanced by the purification (by sepharose chromatography) of rFC to enhance its binding affinity to lipopolysaccharide18 and to ensure sufficient protease activity.

Recombinant protein expression occurs following the insertion of the target gene into the genome of host cells, enabling the production of high quantities of the protein of interest (in this case, Factor C) following its subsequent isolation from cell lysates.19 The first iterations of recombinant reagent manufacture began with modifying the pathways of yeast (such as Saccharomyces cerevisiae and Pichia pastoris), using the mangrove-dwelling horseshoe crab Carcinoscorpius rotundicauda, as the homologous source, to produce the recombinant reagent (success coming at Singapore University in 1997, by Ding and colleagues).20 Subsequent iterations have demonstrated a general similarity in performance between Factor C and LAL.21 By similarity, this should not be taken to mean that one reagent is superior to the other (peer-reviewed studies do not tend to show this).22 There has been insufficient data published as to whether rFC, compared with LA, L matches the MAT, in terms of the IL-6 levels produced by a human monocyte cell line.

The use of yeast gravitated towards bacteria (Escherichia coli)23 and then towards mammalian cells (CHO DG44 (CCHO ) or HEK293 (CHEK))24,25 or insect cells (such as Sf9) in combination with baculoviruses. Later processes have included genetic material cloned from L. polyphemus. Interestingly, at rFC made from this different source is generally consistent in terms of its reactivity to endotoxin and similarity to LAL (unlike LAL reagents from different manufacturers, which can vary widely in their sensitivities as well as the occurrence of lot-to-lot variances between products from the same manufacturer).27,28 The mammalian and insect cell lines approaches are post-translational modifications of transfected cells.

Contamination: Types and Control Strategies

Recombinant DNA technology utilizes a series of procedures to join (recombine) segments of two or more different DNA molecules. When put into cell culture, a recombinant DNA molecule multiplies itself to form a colony of daughter cells that secretes the desired protein. These cells become “factories” for the production of the protein coded for by the inserted DNA.29

The cell culturist has two types of contamination to be concerned with: contamination of cell cultures with microbiological organisms, viruses, and bacteria (in the form of mycoplasma and related genera), 30, and the contamination of one cell line with another.31 Neither type can be wholly eliminated, only controlled and managed, with a focus on minimizing the possibility of occurrence through good practices.32 As well as these biological sources, other types of contamination can also occur (albeit less frequently) in the form of chemical contamination (like deposits of disinfectants or detergents on glassware; residues, impurities, and toxins in water, media,  or sera).33

Contamination consequences for a high-value product, as with recombinant endotoxin test reagents, can be costly in terms of time and money for the production facility. A control strategy is best centered on risk assessment, perhaps using Hazard Analysis Critical Control Points (HACCP) approaches. Here, it is necessary to determine critical process parameters (CPP) and the specific critical control points (CCPs) in the manufacturing process. Risk assessments should focus on 34

  • Selecting and testing source material for the absence of known detectable viruses and mycoplasma;
  • Testing the capacity of the manufacturing process to inactivate or remove viruses;
  • Testing the product at appropriate time points and process stages for detectable viruses.
  • Assessing the suitability of the various test methods, like qPCR, for both viruses and mycoplasma.

Mycoplasma

Mycoplasmas are a type of bacteria, the smallest free-living organisms. In cell culture, these bacteria are undesirable, yet common contaminants, and present a difficult-to-detect risk factor for human and animal cell lines. They can also spread relatively quickly. Mycoplasmas can alter infected cells at the molecular level and trigger visible changes in cell morphology and growth characteristics.35 A common source of mycoplasma contamination comes from the personnel working with the cell cultures. This places importance on good aseptic technique as well as controlling the sources that can generate aerosols. Controls around people include:

  • Effective face mask control.
  • Mask replacement.
  • Good growing.

Moreover, to minimize contamination of the cell bank, no other living or infectious material (such as virus, cell lines, or cell strains) should be handled simultaneously in the same area.36

The main problem presented by mycoplasmas is with their ability to adhere to cells (hence, adherence is the major virulence factor). Mycoplasmas have developed various genetic systems enabling their attachment to host tissues as well as a highly plastic set of variable surface proteins. This is through the versatility of their surface coat and size variation – this also provides mycoplasmas with mechanisms for immune system avoidance.37

As well as avoidance, effort needs to be put into screening. Traditionally, cultural methods were deployed; these days, advances in mycoplasma detection provide greater accuracy in the form of the development of PCR assays. The PCR test is based on the detection of 16S and, sometimes, 23S rRNA molecules of the most common species of mycoplasma contaminating cell cultures.38

Control of Mycoplasma

 A starting point for control is to avoid beginning the process with contaminated cells; hence, purchasing cultures from reputable suppliers is necessary. Thorough washing and disinfecting of personnel's hands is key to working as cleanly as possible. Even when reliable sources are used, it is good practice to keep incoming cultures in quarantine until proven to be mycoplasma-free, and immediately discard contaminated cultures (if possible). .39

Where cell cultures have been contaminated, attempts can be made to remove mycoplasmas without damaging human or animal cells. However, such processes are complex, and they are not always effective. Antibiotic treatment is the most conventional and probably the most common method, but this too has met with variable success.40 Although overuse of antibiotics is not recommended, for precious cell cultures, the continuous use of appropriate antibiotics that are effective against all mycoplasma species is recommended. Among the other methods that can be selected are filtration and the application of ultraviolet light.

Where the continuation cannot be addressed, then a reliable decontamination step should be undertaken before disposing of the culture (and hence to avoid an opportunity for further contamination).

Viruses

Viral contamination can affect raw materials, cell culture processes, bioreactor contamination, and downstream processing. For cell culture, as ICH Q5A (R2) points out,41 this includes viruses that are composed of DNA (such as herpes viruses) or RNA (such as hepatitis viruses) encapsulated by a protein coat. As an example, verivirus is a non-enveloped virus with a single-stranded RNA genome belonging to the Calicivirus family. Where contamination occurs, levels in infected CHO cells can exceed 109 viral particles/mL. Another example is Sf-Rhabdovirus, which can infect insect cell lines.

Increased viral contamination risks occur when 42

  • Changes in critical process parameters that alter the safety profile take place;
  • Failures of virus detection systems to detect low levels of viruses.
  • Data errors, for example, with the extrapolation of viral inactivation data;
  • New and emerging viral risks.

The most common control mechanisms are effective HVAC systems and effective decontamination. Such as the use of biocides, including ethanol, sodium hypochlorite, and benzalkonium chloride.

Where viruses are present, viral clearance can be attempted. Examples of inactivation methods are 43

Solvent/detergent inactivation.

  • Low pH inactivation.
  • Heat (pasteurization), microwave heating, irradiation, and high-energy light.

These methods are generally effective against enveloped viruses. However, some will have a limited effect on non-enveloped viruses. For these types of viruses, the focus is more on their removal:

  • Affinity steps (like Protein A) and chromatographic separations can be optimized for virus clearance.

» Some anionic chromatography mechanisms are effective in the removal of viruses in the downstream purification processes of biopharmaceuticals.

» The effectiveness is dependent upon the resin and binding mode.

  • Membrane chromatography is used in large-scale biopharmaceutical manufacturing processes.

» This type of process will remove impurities as well as viruses.

» The filter capacity must be known and analytics used to assess risk.

» This method works by removing viruses by adsorptive removal, such as ion-exchange membrane adsorbers with ligand–virus binding properties (with a positive surface charge).

» In validating the process, factors that can influence the efficiency of virus removal include protein concentration, product characteristics (e.g., hydrophobicity), and the nature and amount of impurities present.

  • Nanofiltration.

»  Viruses are removed from the product through size exclusion.

» The effective virus removal of large viruses (> 50 nm) is possible using both large- and small-pore-size filters.

» For the removal of small viruses (~ 25 nm), there are several process-dependent factors that require careful optimization.

With all forms of virus clearance, validation is necessary, and before embarking on a validation exercise, the facility needs to decide upon the level of virus clearance required (as per logarithmic reduction).

As with mycoplasma detection, PCR is often the method of testing choice.44 The PCR process makes a very large number of copies of short sections of DNA from a very small sample of genetic material. This process is called “amplifying”. The DNA of viruses is composed of thousands of different genes that code for their proteins. The latest qPCR methods can detect as few as 10 copies of viruses like MMV genomic DNA per PCR reaction.45

Quality Control

Ever since scientists first began growing human cells in lab dishes in 1952, they have focused on improving the chemical soup that feeds the cells and helps regulate their growth.46 Acclimatization is especially important from the outset, since artificially-grown cells experience altered cell states within three days as they adapt to their new environment. Hence, the cell culture process and the industrialization to produce recombinant products continue on a development curve.47 This includes optimizing the surfaces that cells interact with. This is in relation to creating a more precise system for growing cells, which offers both theoretical and practical advantages.

Other good quality control practices include:

  • Cell harvesting and the condition of harvested cells.
  • Cell storage.
  • Freezing and storage of cells.
  • Recovery of cells.
  • Effective preparation of master cell banks and working stocks.
  • Cryoprotectant and assessments of viability and plating efficiency after freezing.

Purification is important in order to maximize the yield of the recombinant reagent.48 This is partly achieved through achieving manufacturing consistency. Each manufacturing process from different providers of recombinant endotoxin test reagents will differ, even formulations are ostensibly the same. Recombinant proteins, even with the same amino-acid sequences, exhibit different activity and stability depending on their different post-translational modifications, especially as seen with differing glycosylation patterns. Furthermore, recombinant products contain polysorbates, which may cause false-negative results.49 This means the performance of reagents will differ, and laboratory users should be encouraged to compare recombinant reagents from multiple vendors against their product portfolio prior to making a selection. Variations that are manifest by different reagents include different binding efficacies to lipopolysaccharide, residual protease activity (as expressed by milliabsorbance units per unit time), and susceptibility to interference (from pH, salt, detergents, chelating agents, and proteins). Moreover, recombinant regens can behave differently to the inhibitory components found in pharmaceutical preparations (which can influence the dilution path needed to counter the inhibitory influence). .50

Summary

This article has looked at the recent advances in recombinant reagents for endotoxin detection. In recent years, protein fragments of Factor C, which have reliably been proven to detect endotoxins and hence serve as an effective biomarker for several decades, have been transitioned into an effective commercial reagent. More recently, other parts of the blood clotting cascade reaction of the horseshoe crab have been synthesized. Through these advancements, not only is an animal-free product created an issue that had adversely affected quality control laboratories for decades - that of batch-to-batch fluctuations in the sensitivity of natural lysates - is reduced.

However, all recombinant technologies rely on cell lines to produce the protein required. It is important that production techniques are deployed to effectively minimize any impurities inherent in the production processes. In addition, the process needs to prevent the introduction of contaminants external to the manufacturing process, in particular, viruses and mycoplasma. Failure to do so leads to an unacceptable batch, and repeated occurrences can result in a manufacturing shutdown. The minimization of these microbial agents must be central to the production contamination control strategy, supported by rigorous testing of sufficient sensitivity.

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