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Protein-Product Separation Technologies for Biocatalytic Reactions Utilized in Commercial API Manufacture

January 25, 2023

The ETC is seeking seeking information on scalable solutions that separate product from protein from typical biocatalytic reaction streams and are capable of operating with the following considerations: 
  • Compatible with aqueous / organic solvent mixtures
  • Can tolerate insoluble solids
  • Can tolerate enzyme loadings up to 50 wt% relative to starting material.
  • Aspirationally, the separation technology results in product with < 1 wt% residual protein and has > 90% product recovery
We are interested in both established technologies and novel approaches which could be adapted to conventional small-molecule manufacturing trains without significant facility modification. Ultimately, we seek approaches that maximize product recovery and purity, and enable efficient, robust application and development from kg to tonne production scales for API synthesis.
The efficient, scalable, protein-product separation remains a key challenge for the successful commercial implementation of biocatalysis. Many biocatalytic processes are performed in conventional chemical reactors using one or more unpurified, recombinantly produced enzymes (which contain both the desired enzyme and residual host cell proteins from the expression host). Existing protein-product separation strategies suffer from numerous challenges including volumetric inefficiency, poor protein rejection, and high product loss. Most approaches must be developed as fit-for-purpose solutions that are tuned for the specific chemistry, scale, and manufacturing infrastructure. They are often insufficiently robust and challenging to develop due to a lack of appropriate scale-down models. As a result, protein-product separation operations require significant engineering resources to develop, introduce substantial risk and process variability, and can negatively impact process productivity and yield.

Download the Request for Information and submit your response.

RFI ISSUED:  January 25, 2023

QUESTIONS on RFI DUE to ETC:  March 1, 2023

RFI RESPONSES DUE to ETC:  May 1, 2023

Q&A - Note see FAQ document for answers to common questions

  1. Is it necessary to have protein-product separation demonstrated with organic synthesis reactor connected to the separator? Can we just have a feed vessel having an appropriate mixture of enzymes and product (s) along with residual host cell proteins (HCPs)? Can we then circulate the feed solution of protein mixtures and the product through the protein-product separation device and back to the feed vessel in a recycling mode till > 90 wt% product is recovered? We will employ a suitable temperature.

    • The separation can be demonstrated using a simulated end of reaction stream. Ideal strategies will be volumetrically and time efficient to mininmize processing time at scale and minimize added unit operations. Recirculating strategies are a potential approach to address these challenges.

  2. Can you specify the molecular weight range of the enzymes and HCPs that are involved in such processes? Would you like to specify particular enzymes and products along with other model proteins representing host cell proteins whose separation can then be studied? Mentioning the solvent at the same time is helpful. Could we employ bovine serum albumin (BSA) as a model for HCPs? Can you specify an enzyme for the proposed study? Can you specify the preferred organic solvent for such an enzyme and specify organic-aqueous ratio in the solvent mixture along with the API needed to be removed and recovered? Hopefully the enzyme, proteins and the API would be easily available from the marketplace.​​

    • ​​For purposes of this RFI, the enzyme can be assumed to be obtained from a lyophilized clarified cellular lysate which was recombinantly produced by high-density fermentation in a typical GRAS species such as e. coli. The API MW can be assumed to be < 1 kDa. Depending on the separation approach utilized, BSA could be an acceptable simulant, however given the high molecular weight it may not be representative of the underlying residual host cell proteins. For high-density fermentation, the majority of protein content will be the enzyme of interest. It is anticipated that materials necessary to support development can be obtained through normal commercial channels.
  3. Will there be any host cells in the feed solution to be used? Provide an example of insoluble solids e.g., a salt/compound precipitated.

    • The feed solution would utilize an an unpurified enzyme which was produced by high density fermentation and clarified prior to lyopholization. Depending on the reaction conditions (pH, temperature, solvent, etc.) protein (either host cell or desired species) may precipitate.

  4.  Do you expect such a process for protein-product separation run continuously as if it were coupled to a reactor in a continuous API manufacturing system that is of current interest? Do you on the other hand expect this to be a batch process of sorts so that the development will focus on treating (1) a batch volume obtained after the reaction is over or (2) as the batch reaction is going on?

    • The methodology should be applicable to batch processes. However compatibility with continuous processes would be great. 

  5. What is the molecular weight range of APIs under consideration 200-1,000 Da?

    •  This is an acceptable range.

  6. Can you specify selected APIs that could be used for any proposed study?

    • Should the project demonstration involve sequential demonstrations with one API first, then another API? Or is demonstration with one API enough?

    • One Demonstration with a model API (for development purposes, it should be commercially available, safe, and easily assayed) that is representative of molecular weight and physical properties of a typical small-molecule API is sufficient. The ETC does not have a particular preferred substrate 

  7. What is the preferred scale of operation? Are two different scales enough to demonstrate scalability? Do we need two temperatures of operation, say, 15oC and at say 60oC? Do we need to demonstrate at 4oC and 80oC?

    • The ETC seeks to demonstrate applicability of the technology at laboratory  scales but should have a technical feasibility of scaling up to metric-tonnes based on established scaling principles.  The expected operational range of the envisioned approach should be discussed. If a technical justification fo the operational range cannot be provided a plan for assessing these ranges would be beneficial. If the operation will occur concurrently with the biocatalytic transformation, it should enable operation undre the conditoins specified in Table 2 of the RFI.

  8. Do we need to demonstrate with two organic solvents and conclude if we can show via other studies that the system can handle the organic solvents mentioned in the RFP? What are these two solvents?

    • The proposal need not demonstrate compatibility with an extensive range of solvents, however a compatibility assessment against a range of cosolvents or solvents utilized in biocatalytic transformations.

  9. How long does it have to run during the final demonstration?

    • The final demonstration should deliver material with the targeted protein levels and product recovery specified in the RFI. While durability is important, the RFI should focus on demonstration of the approach across individual batches rather than continuous operation. We favor approaches which are robust while minimizing operational time (plant time).

  10. Is it useful if we explore two different separation techniques for two different APIs?

    • We are interested in techniques which can be broadly applied to different API’s. It is not necessary to demonstrate multiple approaches, however demonstration of multiple solvent systems/API’s during development will be important.

  11. How much time do you allow for the project? Two years, three years?

    • The project duration should be proposed by the 3rd party, based upon the time they believe they need to complete the project. Projects in ETC typically last 1-3 years with options to execute additional work through new SOWs after the completion of the initial SOW

  12. Do you expect that the development should be scaled up and commercialized by the proposer and / or the industrial collaborator of the proposal?

    • The ETC seeks to demonstrate applicability of the technology at tsclaboratory scales but should have a technical feasibility of scaling up to metric-tonnes based on established scaling principles.

  13. The RFI for protein-product separation states that commercially available and supported systems are desired. Our technology relies on the self-assembly of magnetic particles to entrap enzymes, which can then be separated more easily. While the production of our magnetic particles are commercially viable at the scales desired in the RFI, they are currently only used for internal projects and not sold commercially. Would this be an acceptable stage of development for the RFI?

    • Approaches with commercial viability are acceptable. Where possible, it would be beneficial for the applicant to provide brief details discussing amenability to scale-up of existing processes or line of sight towards commercial supply.  If possible, the review would benefit from details on current and planned production capabilities or outsourcing.

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