# Lysis-free extraction of biopharmaceuticals from the periplasm of Clean Genome E. coli

> **NIH NIH R44** · SCARAB GENOMICS, LLC · 2020 · $737,950

## Abstract

Thirty percent of recombinant therapeutic proteins are made in E. coli and include important vaccine
components such as carrier proteins. Further, burgeoning new immunotherapies that use proteins such as
single chain antibodies and virus-like particles are enjoying increasing success. To meet the production needs
of these expanding areas, Scarab Genomics proposes to extend the versatility of its Clean Genome® E.coli
production system to include a method by which recombinant therapeutic proteins delivered into the cell
periplasm can be extracted directly into the culture medium for easy purification. Combined with Scarab’s
extended fermentation platform, which enables high-density growth of Clean Genome E. coli and the constant
production of target protein, a simplified recombinant protein extraction method will expand Scarab’s platform
and facilitate continuous production, as mandated by the FDA. The development of an extraction method will
reduce production time and effort, greatly simplify purification of therapeutic proteins such as the carrier protein
CRM197 and dramatically reduce total production costs. Preliminary experiments using the carrier proteins
CRM197, Haemophilus protein D and Pseudomonas exoprotein A indicate that subtle changes to the
production host strain combined with a proprietary extraction buffer can induce up to 90% recovery of
recombinant periplasmic protein. Further, recombinant therapeutic proteins released into the medium were
stable for more than two weeks further emphasizing the versatility of the system. In Phase 1, fermentation and
extraction conditions for the test carrier protein CRM197 will be improved and reagents needed to optimize
expression and extraction will be developed. The first aim of Phase 2 proposes to determine whether deletions
of key transpeptidase enzymes that are important in the stability of the periplasm, and which have been shown
to enhance the release of recombinant proteins from the periplasm, can improve extraction. Similarly, the
influence of peptidoglycan-modifying enzymes expressed from the production plasmid will be examined in the
context of the extraction method. The second aim will examine the expression and extraction of four target
proteins in extended fermentation to test the extraction system at production scale. In addition to the carrier
proteins Haemophilus protein D and Pseudomonas exoprotein A, a single chain antibody and a single-chain
variable fragment will be examined. The antibody fragments will also be subjected to purification to confirm that
extraction of recombinants into culture medium simplifies purification, as was evident for carrier proteins. The
third aim proposes to devise a method for removing cells and cell debris from the extracted solution with the
goal of providing continuous chromatographic systems with clarified material for a truly continuous process.

## Key facts

- **NIH application ID:** 9961488
- **Project number:** 5R44AI143170-03
- **Recipient organization:** SCARAB GENOMICS, LLC
- **Principal Investigator:** FREDERICK R BLATTNER
- **Activity code:** R44 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $737,950
- **Award type:** 5
- **Project period:** 2019-01-01 → 2022-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9961488

## Citation

> US National Institutes of Health, RePORTER application 9961488, Lysis-free extraction of biopharmaceuticals from the periplasm of Clean Genome E. coli (5R44AI143170-03). Retrieved via AI Analytics 2026-05-27 from https://api.ai-analytics.org/grant/nih/9961488. Licensed CC0.

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