Standardized, Scalable And Efficient: Producing Recombinant Factor C to Quality Standards

Tim Sandle- Head of GxP Compliance and Quality Risk Management, Bio Products Laboratory Limited.

Introduction

The shift in the endotoxin reagent industry away from reagents sourced from the horseshoe crab towards reagents produced using recombinant technology has been occurring along a rapid and, most probably, inescapable trajectory. This is in the form of recombinant factor C (rFC) based on the gene sequence of the horseshoe crab and fluorimetric detection (or, in the case of one manufacturer, replicating the entire cascade through the use of recombinant protein technology with the exclusion of the glucan-triggered factor). Concerns over the variability of animal-derived lysate (influenced by the age, sex, location, and season of horseshoe crab harvest)¹ and reducing the risk of animal mortality^2,3 as well as boosting sustainability,⁴ have been the leading drivers propelling this change.⁵ The basis for the use of recombinant reagent is to provide a reagent that reacts in the same way as the natural cascade within the North American (Limulus) and Asian (Tachypleus) horseshoe crabs.

By way of a marker, in 2020 this author wrote in American Pharmaceutical Review: “This somewhat gradual process is attributable to a clash between drivers (seeking to conserve horseshoe crabs, albeit with the mortality rate an imprecise and sometimes underreported figure) and points of hesitancy (regulatory uncertainty, lack of standardization, imprecise validation requirements and so on).”⁶ The text was written upon publication of the European Pharmacopeia monograph describing the use of recombinant factor C. Further progress has now been made with each of the main endotoxin reagent manufacturers (as well as some emergent entrants) now offering a recombinant factor C product. To serve the needs of the pharmaceutical and healthcare sectors, factor C needs to be standardized, produced to scale, purified, characterized, and of the appropriate quality. This article looks at the variables that affect these criteria.

Evolution of Recombinant Factors as Test Reagents

This path towards a recombinant product has its origins in advances in our understanding of the LAL test reaction. In 1986 researchers demonstrated an important component in the clotting cascade that is responsible for the LAL test reaction (and for the immune response of the horseshoe crab).⁷ This was called factor C proenzyme and it was experimentally demonstrated that factor C was the only biosensor that detected endotoxins, thus establishing the cascade reaction. With the clotting reaction, activated factor C activates factor B, which in turn activates a pro-clotting enzyme to form a corresponding clotting enzyme. The clotting enzyme hydrolyzes a specific site of a coagulogen molecule and a coagulin gel is formed, and thus the lysate is coagulated. For the LAL/TAL test, endotoxin is measured by measuring the lysate coagulation reaction. In an unmodified form of the lysate, another factor (factor G) was shown to also generate a cascade response in the presence of beta-glucans.⁸

The modified recombinant assay is based on a simple single reaction that involves the cleaving of a fluorogenic substrate by factor C. The simplified assay does not require the remaining proteins that make up the LAL cascade, such as factor B, factor G, and proclotting enzyme. Hence, alongside the simplicity is greater assay specificity. Other endotoxin test assays use recombinant protein technology to replicate the complete pathways from the point of endotoxin detection to create reagents that are closer to horseshoe crab lysate.

Recombinant Reagent Manufacturing Process

In 1997 a bacterial endotoxin test based on recombinant factor C (rFC) was demonstrated experimentally and reported.⁹ Following this, in 2004, a working reagent with commercial potential was developed. This was rFC, as an animal-free alternative to the endotoxin sensor protein inside of Limulus amoebocyte lysate (LAL). As well as having the same functionality as horseshoe crab lysate, recombinant factor C does not recognize the factor G pathway; hence the product can detect endotoxin more selectively.¹⁰ Since these initial developments numerous studies have compared LAL and rFC in terms of bacterial endotoxin detection.^11-14 Generally, the outcome of such studies has been favorable, although method validation for each type of sample (rather than verification) is advised.

With the recombinant reagent (either rFC or a wider pathway), a recombinant protein is an artificially produced (and often purified) protein, generated from the appropriate gene from one of the four known living species of horseshoe crab. The recombinant process occurs when recombinant DNA encoding a protein is introduced into a host organism to express foreign proteins. This process requires the use of specialized expression vectors together with restructuring by foreign coding sequences.^14,15 Since the resultant reagent is not derived from horseshoe crab amoebocytes it cannot be called LAL or TAL.

The first recombinant factor C reagents were produced based on biosynthesis using yeasts.¹⁶ In the early stages the manufacturer proved variable, in trying to produce rFC as an approximate 132 kDa molecule in the form of a zymogen/proenzyme (a biologically inactive substance that is metabolized into an enzyme). The first larger-scale rFC was based on the cloned cDNA sequence of factor C from the mangrove horseshoe crab Carcinoscorpius rotundicauda and expressed initially in insect cells with other methods subsequently developed, demonstrating the appropriate productivity, bioactivity, and physicochemical characteristics.¹⁷ High-throughput screening, design-of-experiments, and other analytical advancements paved the way for recombinant factor C (and for replicating other parts of the clotting pathway) production to scale.

Given that the protein secretory pathway is an important pathway for eukaryotic cells, therefore, based on protein synthesis capabilities, eukaryotic cell lines are used widely for the process of producing recombinant proteins. Mammalian cell systems for recombinant protein production offer diverse advantages. These include good protein folding, the absence of many potentially immunogenic post-translational modification products, good secretion capacity, and a medium to high product yield. The important considerations required during cell-line development include a selection of an expression host, vectors, and transfection, and cell-line selection.

Recombinant protein production begins with expression vector engineering and transfection into a host system. This step is followed by the steps of:¹⁸

• Cell selection
• Medium selection (defining the essential nutrients required for optimal cell growth and target protein productivity is very important)
• Cloning
• Screening
• Evaluation

The objective of manufacturing is the standardized production of the same rFC protein through the use of a bioreactor. This requires achieving a certain level of quality and scalability. Important criteria for an optimal cell line include:¹⁹

• High cell density in suspension culture.
• Key cultivation parameters, such as media composition, pH, agitation, aeration, temperature, cell density, the concentration of inducers, induction time, and feeding strategies.
• Suitable bioreactor methods, with batch, fed-batch (where nutrients are fed during cultivation), and continuous or perfusion culture (where the culture is fed through the medium in order to streamline both waste removal and nutrient supply), are available for bulk production of recombinant proteins.

» The type of bioreactor is also very important. The types of models available include stirred tank bioreactors, airlift bioreactors, membrane bioreactors, bubble column bioreactors, hollow fiber bioreactors, and fixed bed and fluidized bed bioreactors.

» Operational parameters for bioreactors include Agitation power settings, maintaining the oxygen transfer coefficient, consistency of continual mixing times, agitation impeller speed, control of the heat transfer rate (where heat and volume are inversely proportional), control of the gas volumetric flow rate (which requires control of pressure), and the maintenance of a constant gas superficial velocity.

• The production of high protein yield.
• Additional use of cell retention devices (such as tangential flow filtration or spin filters) when the perfusion culture technique is used.
• Suitability for use in transfection (to study and modulate gene expression).
• Cell survival in serum-free media.
• Controls to lower the risk of viral infection.

To enhance production, high-throughput devices can be used for bioprocess optimization. As with other areas of biopharmaceutical manufacturing, advances have been made with sterile, disposable single-use systems, continuous upstream processing, continuous chromatography, and integrated continuous bioprocessing. Efficiency can also be delivered at the outset by applying Quality by Design and through manufacturing by applying process analytical technologies to achieve the desired quality of the product and the target yield. Continuous bioprocessing is also being applied for both upstream and downstream manufacturing, which allows for a smaller production footprint.

Process Problems

Proteins are sensitive to their surrounding chemical environment. Changes in pH, ionic strength, and oxidative status can each impact protein stability. During purification, care needs to be taken with the stabilization of the target protein and to facilitate tag exposure. This leads to the control parameters for the bioreactor being important. This extends to operating parameters like temperature, pH, agitation, aeration, dissolved oxygen, carbon dioxide, and hydrodynamic shear forces. Aspects that are more likely to go wrong and adversely affect the process are temperature shifts and gas exchange.

Other things can go wrong during the manufacturing process. These include weak growth of the host; inclusion body formation; protein inactivity; contamination of cell lines (particularly from prions, and oncogenic DNA); and failure to obtain any protein at all.²⁰

Types of Contamination

Microbial contamination includes viral and mycoplasma infection of cell lines. Viruses are of concern as common contaminants and because they are more difficult to detect than other microbial contaminants. Viral contamination is controlled by:

• The selection of suitable raw materials.
• Testing cell banks and in-process materials.
• Putting in place control measures to remove or to inactivate potential undetected viral contaminants during the purification stages.

Raw materials should have a low risk of containing endogenous or adventitious viruses and intermediate manufacturing steps should be assessed to ensure the in-process material is free from detectable viruses. Different viruses will pose different risks to specific cell lines. Techniques like PCR are optimal for virus screening, provided the required primers and probes are available.

Mycoplasma contamination is a widespread and reoccurring problem for many cell culture systems in life science research and the pharmaceutical industry. Mycoplasmas are bacteria of a size 0.15–0.3 μm in diameter and they can grow to high titers in culture media without exhibiting typical bacterial contamination signs such as turbidity. The deleterious effects on cultured cells include fusing with and/or invading the host cell, inducing severe cytopathic effects, causing abnormal cell growth, altering the host’s gene expression profile (i.e., oncogenes, tumor suppressor genes, cytokines, and signaling regulators), outcompeting the host for culture nutrients, including sugars, heavy metals, or amino and fatty acids; causing slowed proliferation, and chromosomal aberrations. As a result, mycoplasma testing and the maintenance of contamination-free cell cultures are essential for manufacturing.

Ironically, one of the contaminants can be endotoxin. Bacterial endotoxin can readily contaminate water, aqueous solutions, and buffers, and therefore suitable controls are required over the quality of water and reagents used during manufacturing. The main challenge posed by endotoxin is its binding affinity to different protein surfaces.²¹ To treat the prepared protein, commonly used techniques for removing endotoxin contaminants are ultrafiltration and ion exchange chromatography.

One source of non-microbial contamination can relate to bioreactors, where metal ions such as iron, chromium, and nickel can exist as leachable under different formulation buffers and pH conditions. With non-microbial contamination, the primary challenge is from the lack of any standardized assay with sufficient sensitivity to cover all possible contaminants of the end product. Data signaling a concern is often provided retrospectively, such as from a customer complaint indicating a potential aberrant release or from a stability result.

Process Optimization and Scale-Up

Manufacturers will also invest heavily in process optimization, devising strategies to increase product yield. When scaling up between development and larger-scale manufacturing, control of parameters is especially important. Successful scale-up is measured by sufficient cell growth, maintaining cell viability, and the achieved protein production (the titer). Given the scale of the endotoxin testing industry and the expected expanding demand for recombinant endotoxin test reagents, consistently manufacturing high-purity recombinant products to scale represents an ongoing priority

Conclusion

The current focus with endotoxin testing is on the transition away from animal-derived lysates and toward recombinant factor C reagents. The steps this takes are still transitional in terms of compendial development, validation versus verification requirements, and regulatory approval; nonetheless, the eventual replacement of horseshoe crab lysate with a recombinant product is inevitable. In the long term, recombinant lysate might be replaced by biosensor technology, including optical, electrochemical, photoelectrochemical, and electrochemiluminescence solutions.²² For the immediate future, recombinant factor C (and recombinant forms of the endotoxin reactive pathway) is here to stay, and the focus needs to be as much on reliable methods of manufacture as it is on reagent qualification.

References

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