Periodic Reporting for period 2 - RECOFIB (A unique commercially viable recombinant manufacturing platform for human fibrinogen (rhFib) & thrombin (rhThr)
Reporting period: 2023-04-01 to 2023-12-31
Fibrinogen and thrombin are two important clotting proteins in our blood that function as 1st line responders to tissue injury. Not only do they prevent blood loss, they also trigger host defense against pathogens and support tissue repair. This multifaceted role is underpinned by the occurrence of different fibrinogen variants in blood. Each of these naturally occurring variants can form an insoluble fibrin matrix after activation by thrombin. However, each variant has distinct functional properties that are unique to that specific variant. All of the human fibrinogen currently used in various heamostsis products on the market is sourced from donor blood/plasma and is a mixture, of the different variants. Extracting individual fibrinogen variants from donor plasma is commercially infeasible, making recombinant production in mammalian cells the only alternative. Furthermore, the use of donor blood as a source of fibrinogen and thrombin has several other disadvantages, namely [1] poor safety profile: risk of blood born disease transmission; [2] low purity resulting in limited stability; [3] limited supply of donor blood/plasma creating a dependence on plasma from the US and sensitive to environmental factors such as the COVID19 pandemic and [4] regional import and export restrictions.
The only way to fully exploit the full healthcare potential of the individual fibrinogen variants and to overcome the disadvantages of using donor blood, we have developed the RECOFIB platform. This recombinant manufacturing platform is based on Chinese Hamster Ovary Cells (CHO) expression technology & proprietary upstream purification processes. The RECOFIB platform aims to bring to market fully intact & functional individual rhFib variants and active rhThr at commercially acceptable cost. With these individual rhFib variants as stand-alone or in combination with rhThr, we plan to develop customized products for [1] bioactive topical haemostats to stop excessive bleeding during surgery (RecoSeel); [2] anti-microbial coatings for medical devices to reduce development of antibiotic resistant biofilms (RecoCoat) and [3] supply of rhFib variants & rhThr as OEM products. Additional benefits of the RECOFIB platform include higher product purity, increased stability, improved safety profile, lower production cost (for rhThr) and reduction of the reliance of donor blood or plasma.
The only way to fully exploit the full healthcare potential of the individual fibrinogen variants and to overcome the disadvantages of using donor blood, we have developed the RECOFIB platform. This recombinant manufacturing platform is based on Chinese Hamster Ovary Cells (CHO) expression technology & proprietary upstream purification processes. The RECOFIB platform aims to bring to market fully intact & functional individual rhFib variants and active rhThr at commercially acceptable cost. With these individual rhFib variants as stand-alone or in combination with rhThr, we plan to develop customized products for [1] bioactive topical haemostats to stop excessive bleeding during surgery (RecoSeel); [2] anti-microbial coatings for medical devices to reduce development of antibiotic resistant biofilms (RecoCoat) and [3] supply of rhFib variants & rhThr as OEM products. Additional benefits of the RECOFIB platform include higher product purity, increased stability, improved safety profile, lower production cost (for rhThr) and reduction of the reliance of donor blood or plasma.
Fibriant established a unique manufacturing platform for recombinant fibrinogen and recombinant thrombin. The main achievement in the 1st year of the RecoFib project are:
1) For two individual fibrinogen variants, rhFib WT (variant 1) and rhFib gamma’ (variant 2), the Lead production clones, were sujected to limited dilution sub-cloning. The production levels and production stability of six sub-clones were confirmed. One sub-clone was selected for each rhFib variant and identified as the Final production clone on which genetic stability testing was performed. The Final production clones for both variants demonstrated stable > 1 gr/L expression levels of non-degraded fully functional human fibrinogen and genetic stability over > 30 population doublings. The rhFib variants 1 and 2 are ready for GMP Master Cell Bank preparation.
2) For 4 other fibrinogen variants expression vectors were generated and transfections were performed. The 1st round clone selection was performed for 2 of these variants and resulted in clones that showed good growth characteristics in small scale fed batches but with expression levels <1 gr/l . A 2nd round of transfection and selection is ongoing.
3) Lead production clones for human recombinant thrombin were generated and demonstrated high level expression of > 1 Mil IU/L. Limited dilution sub-cloning was performed and eight rhThr sub-clones were selected for confirmation of expression levels and expression stability. One sub-clone was identified as designated Final production clone, and genetic stability testing was initiated.
4) Pilot upstream production (USP) runs at different scales (3 and 10 L) were performed with the Final production clones for the rhFib variants WT and Gamma’ and for rhThr. Growth characteristics such as cell density, cell viability, glucose consumption were measured. Clarified harvest was prepared and end-of batch production levels were determined.
5) Analytical methods for quality control of rhFib material produced and characterization of functional properties were developed and qualified as fit-for-use. Focus was on methods measuring the product Identity, Quantity, Potency, Purity and Safety.
1) For two individual fibrinogen variants, rhFib WT (variant 1) and rhFib gamma’ (variant 2), the Lead production clones, were sujected to limited dilution sub-cloning. The production levels and production stability of six sub-clones were confirmed. One sub-clone was selected for each rhFib variant and identified as the Final production clone on which genetic stability testing was performed. The Final production clones for both variants demonstrated stable > 1 gr/L expression levels of non-degraded fully functional human fibrinogen and genetic stability over > 30 population doublings. The rhFib variants 1 and 2 are ready for GMP Master Cell Bank preparation.
2) For 4 other fibrinogen variants expression vectors were generated and transfections were performed. The 1st round clone selection was performed for 2 of these variants and resulted in clones that showed good growth characteristics in small scale fed batches but with expression levels <1 gr/l . A 2nd round of transfection and selection is ongoing.
3) Lead production clones for human recombinant thrombin were generated and demonstrated high level expression of > 1 Mil IU/L. Limited dilution sub-cloning was performed and eight rhThr sub-clones were selected for confirmation of expression levels and expression stability. One sub-clone was identified as designated Final production clone, and genetic stability testing was initiated.
4) Pilot upstream production (USP) runs at different scales (3 and 10 L) were performed with the Final production clones for the rhFib variants WT and Gamma’ and for rhThr. Growth characteristics such as cell density, cell viability, glucose consumption were measured. Clarified harvest was prepared and end-of batch production levels were determined.
5) Analytical methods for quality control of rhFib material produced and characterization of functional properties were developed and qualified as fit-for-use. Focus was on methods measuring the product Identity, Quantity, Potency, Purity and Safety.
The global fibrinogen market, currently entirely based on plasma derived fibrinogen is showing strong growth world-wide. The RECOFIB platform has the potential to disrupt this existing market with its new recombinant production process because it not only it provides a huge improvement of the existing haemostat and wound closure products but it also creates new , innovative product opportunities in other therapeutic areas. Furthermore, RECOFIB also facilitates global marketing of fibrinogen/thrombin products without the regional regulatory limitations that apply to plasma-derived products. The main results beyond the state of the art are:
• Recombinant production creates the opportunity to produce individual fibrinogen variants with unique functional properties. This enables customization and improvement of heamostasis products by using fibrinogen variants with optimal clotting properties and creates the opportunity to develop products outside the traditional haemostasis and wound closure field.
• Recombinant production of rhFib and rhThr results in improved safety profile, higher purity and stability, consistency and control of production, as compared to plasma fibrinogen and thrombin.
• Manufacturers and R&D companies often have to make use of low purity plasma-derived fibrinogen which is not stable in a liquid form. Recombinant fibrinogen has demonstrated increased stability in a liquid formulation enabling the development of a ready-for-use RecoSeel product that requires short preparation time (time-to-table) facilitating the use in acute care situations.
• Recombinant production creates the opportunity to produce individual fibrinogen variants with unique functional properties. This enables customization and improvement of heamostasis products by using fibrinogen variants with optimal clotting properties and creates the opportunity to develop products outside the traditional haemostasis and wound closure field.
• Recombinant production of rhFib and rhThr results in improved safety profile, higher purity and stability, consistency and control of production, as compared to plasma fibrinogen and thrombin.
• Manufacturers and R&D companies often have to make use of low purity plasma-derived fibrinogen which is not stable in a liquid form. Recombinant fibrinogen has demonstrated increased stability in a liquid formulation enabling the development of a ready-for-use RecoSeel product that requires short preparation time (time-to-table) facilitating the use in acute care situations.