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CHEF1 Expression Technology AdvantageThe CHEF1 expression system has been used extensively to create

clinical production cell lines and has been approved for commercial

production. The CHEF1 integrated expression platform accelerates

development of cell lines for cGMP production by using robust

procedures and reliable raw materials including chemically defined

media, adapted Chinese hamster ovary (CHO) cells, and CHEF1

expression plasmids to produce high levels of recombinant protein

in rapid time-frames.

CHEF1 Expression PlasmidsThe CHEF1 system allows rapid isolation of CHO cell lines

expressing high levels of a variety of recombinant proteins. Based

on the constitutively-expressed Chinese hamster EF-1α regulatory

sequences,1 CHEF1 vector expression levels are typically 6- to

25-fold greater than those obtained with other expression

plasmids, such as those utilizing the Cytomegalovirus (CMV)

promoter. A variety of CHEF1 vectors are available with different

selectable markers that permit multiple transfections of a gene of

interest or genes encoding multi-subunit proteins.

The CHEF1 vectors can also be used to easily co-express proteins

that perform post-translational modifications of the protein of

interest. CHEF1 vectors do not require gene amplification to

achieve high-level expression. Therefore, the CHEF1 system

decreases development timelines by many months in comparison to

the industry standard CHO expression system, reduces regulatory

concerns by eliminating use of methotrexate, and increases speed

to initiation of early clinical trials.

CHEF1® Expression Platform Summary

HOST CELLS

Suspension adapted, serum-free CHO DG44 cells

EXPRESSION SYSTEM

Hamster elongation factor promoter with constitutive,

high-level expression

Rapid cell-line development

NoMTXamplification

Stable cell-line growth and productivity

Up to 4g/L in four months from top clones

EARLY PRECLINICAL MATERIAL

Stableexpressionpoolsinfiveweekscapableofproviding

>10g non-GMP material

Scalable up to 50g from transfection pools or top clones

IND ENABLING CLINICAL MATERIAL

12–16 months from receipt of DNA to delivery

mAb 2.012 Fast Track: 12 months from DNA receipt

to 500g GMP material (see mAb 2.012 brochure)

Standard Development and Manufacturing: 16 months

to release of GMP material (includes engineering run)

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CHEF1® ProprietaryExpression Technology

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CHEF1 Platform TechnologyThe CHEF1 platform technology incorporates CHO DG44 cells

that have been adapted to suspension growth in CMC Biologics’

chemically defined media (CD-CIM1). With the CHEF1 vectors,

CD-CIM1 media, and adapted CHO cells, stable expression pools

can be generated in as little as five weeks, easily providing greater

than 10 grams of protein in bench-scale production models for

pre-clinical studies, purification and analytical development. Since

gene amplification is not needed, transfection pools can proceed

directly into cell line cloning. A relatively large fraction of clones

derived from a CHEF1 transfection express the protein of interest

at high levels,2 thereby reducing the number of clones that need to

be screened to identify high-expressors.

Cell line cloning is completely serum-free and yields clones that

easily progress from growth in tissue-culture plates to shake flasks

in as little as three weeks. Early-stage productivity screens are

highly predictive of future expression potential, enabling rapid cell

line development time lines (see figure above). CHEF1 cell lines are

stable throughout the in-vitro expansion phase required for large-

scale production ensuring reliable manufacturing productivity.

Using CHEF1 vectors and platform technologies, cell lines capable

of 4 grams per liter titers have been obtained in 12 weeks from

transfection to lead clone selection. All cell line development

activities are well characterized and documented for process

transfer to cGMP production. This short timeline allows more rapid

development and transfer of a cell culture production process and

thus more rapid entry into clinical trials.

FIGuRE 1

12-Month Monoclonal AntibodyProduction Timeline

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Receive DNA Research Cell Bank

Material Available for Pre-Clinical Formulation Studies

MCB Available for Production

Full Disposition

Drug Substance Available

CHEF 1 CELL LINE DEvELOPMENT

DOwNSTREAM PROCESS DEvELOPMENT

ANALYTICAL AND FORMuLATION DEvELOPMENT

uPSTREAM PROCESS DEvELOPMENT

cGMP RuN

CHEF1 Licensing TermsA range of packages is available for pre-IND, clinical

development, and commercial licensing terms.

References

1. Allison, Dan United States Patent, Number 5,888,809. March 1999.

2. Running Deer, J. and Allison, D. Biotechnology Progress, 20, pp. 880-889, 2004.