Pharmaceutical Bioprocessing

Commercial Bioprocessing Made Easy

Production of life saving biotherapeutics to enable testing, expansion and commercialization is challenging. Modifying current mammalian expression platforms where Chinese hamster ovary (CHO) cells are most commonly used is key to increasing efficiency. Cas-CLOVER is the clean alternative to CRISPR/Cas9 for bioprocessing. It provides very high-efficiency gene editing, no detectable off-targets & simple and accessible licensing terms.

For Animal Health bioprocessing specifically, Demeetra is also licensing the piggyBac transposase, a highly validated system.

The Clean Alternative

Demeetra is introducing the Cas-CLOVER technology for commercial bioprocessing as well as newly edited GS knockout CHO cell lines. Cas-CLOVER differs from CRISPR/Cas9 in that it is a dimeric nuclease system lacking detectable off-target mutagenesis. The specificity of Cas-CLOVER enables multiple rounds of targeting at one locus to increase indel frequency without introducing the risk of unwanted off-target mutations.

Demeetra AgBio, Inc.

GS CHO knockout: Cas-CLOVER Validation

Glutamine Synthetase (GS) expression is essential for CHO cell viability. Knockouts for GS result in cell death unless replaced by an exogenous stably integrated GS gene that is proportional to the expression of a protein of interest (for example, a monoclonal antibody), and this system is exceptionally useful in rapidly selecting high expressing pools or clones.

Shown here are indel frequencies of 18% and 43% at the CHO GS locus for one and two rounds of targeting, respectively. These on-target frequencies in CHO cells are higher than reported for ZFN and comparable to those of CRISPR/Cas9.

Demeetra AgBio, Inc.

Simple and Accessible Licenses

GS null CHO cells and gene editing technologies have traditionally been accessible only through cost-prohibitive licensing terms that may require high upfront fees, milestones, and royalties. Demeetra is providing simple licenses structures with accessible economic terms for Cas-CLOVER and GS CHO knockouts for commercial bioprocessing as well as proof-of-concept services.

CRISPR/Cas9 vs. Cas-CLOVER

Compared with CRISPR/Cas9, Cas-CLOVER offers:

  • A cleaner gene editing alternative for commercial bioprocessing
  • A highly specific system showing no detectable off-targets
  • Similar indel frequencies
  • Special license structures for CDMOs
  • Evaluation licenses for Cas-CLOVER and GS CHO knockouts - separately or together - to convert into commercial licenses
  • Issued patents that allow clear freedom to operate

CRISPR-Congress-Poster-Cas-CLOVER-1

PiggyBac for Animal-Health Bioprocess

Commercial Bioprocessing Made Easy

  • Titer-↑ titers (↓ future Cost of Goods)
  • Efficiency-Better use of existing resources
  • Speed-Faster to market

Transient Gene Expression

Transient gene expression in cells allows for the generation of small quantities of material. Although this can be rapid, scaling up to provide gram quantities of material can be prohibitive. Switching to stable gene expression is preferred for generating these large quantities of protein.

Reduced Time To Produce Protein
Still, the generation of clonal cell lines (i.e., CHO or HEK293) can be both times consuming and labor-intensive. More recently, stable CHO cell pools have been utilized, which can reduce the time to produce protein but maybe generally less stable than clonal lines and may also have lower expression titers.

Validated For Use

PiggyBac has been validated for use in the generation of antibodies from CHO cell pools and individual clones. In these studies, published by Eli Lilly and others, antibody titers are as high as 7.6g/L were obtained from a heterogenous CHO cell population. Titers were improved for each of the four antibodies tested in comparison with control pools.

Fold Improvement
On average, the fold increase for the three mono-functional antibodies was approximately 4-fold, and for the bi-functional antibody tested was nearly 12-fold (specific productivity ranged from 4.2-9.3-fold improvement with the piggyBac pools one and outperformed competing Leap-In technology (2).

PiggyBac Outperforms Leap-in Transposon

Scale-up Characterization

No significant differences were observed in growth and viability for piggyBac vs. control CHO cells (above), while productivity improvements are maintained (below) (3)

Citations

  • Rajendra et al. (2016) Generation of Stable Chinese Hamster Ovary Pools Yielding Antibody Titers of up to 7.6 g/L Using the piggyBac Transposon System. Biotechnol. Prog., Vol. 32, No. 5
  • Balasubramanian et al. (2018) Generation of high expressing Chinese Hamster Ovary cell pools using the Leap-In transposon system. Biotechnol J. 2018 Oct;13(10):e1700748
  • Rajendra et al. (2017) Bioreactor scale-up and protein product quality characterization of piggyBac transposon derived CHO pools. Biotechnol Prog. Mar;33(2):534-540
  • Rajendra et al. (2017) Evaluation of piggyBac-mediated CHO pools to enable material generation to support GLP toxicology studies. Biotechnol Prog. 33(6):1436-1448