CHO Expression Data

Horizon's GS Null CHO K1 cells are in the process of being independently evaluated in over 15 organisations globally. Some of these evaluations have given us permission to share their cell line development data relating to the expression of recombinant proteins, including monoclonal antibodies and bispecifics. Although at this stage considerable optimisation is still being performed on the culture process, we are consistently receiving reports of expression exceeding expectations. While some of the data provided below utilises MSX to improve the stringency of selection with individual vectors, the CHO SOURCE platform combining Horizon's GS null CHO K1 cell line with an expression vector from ProteoNic does not require MSX during selection, nor does it require any amplification steps commonly associated with alternative metabolic selection systems such as the Methotrexate / DHFR selection approaches.

CHO Licensing

COMPARISON WITH MSX BASED GS SYSTEM

After being transfected with a GS selection vector containing a monoclonal antibody and placed under metabolic selection in glutamine free media, the cells expressed significantly higher titres of recombinant protein than the reference system comprising MSX treated wild type CHO K1 cells. Data shown is generated in a 5L Bioreactor and represents titre from stable pools of cells.

msx gs comparison

STABLE POOL EXPRESSION

As part of an evaluation of different vector technologies, the cells were transfected with a construct expressing a monoclonal antibody in a vector containing UnicTM elements from ProteoNic.

Titres in excess of 2g/L of a biosimilar IgG were achieved at stable pool stage.

Stable pool expression

CLONAL EXPRESSION

Four hundred clones from the stable pool above were screened and assessed for productivity. Three clones expressed at >5g/L.

This is currently an un-optimised process, with further advances expected.

Clonal expression

STABLE POOL EXPRESSION

As part of an evaluation of HD-BIOP3, a Biopharmaceutical company in China developed stable pools using a vector provided by DNA 2.0. They assessed the impact of using bulk selection compared with generating minipools through applying selection to 500 cells. Together with this, they evaluated the impact of using MSX with this vector.

Data shown is after 5 days of shake culture.

 

STABLE POOL EXPRESSION

As part of an evaluation of HD-BIOP3, a Biopharmaceutical company in the USA developed stable pools using a vector provided by DNA 2.0, together with increasing concentrations of MSX.

Data shown is after Protein A purification.

Evaluation of Horizon HD-BIOP3 CHO cells

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Cell Line Derivation

Cell Line Derivation

CHO cell line derivation step 1 CHO cell line derivation step 2 CHO cell line derivation step 3 CHO cell line derivation step 4 CHO cell line derivation step 5 CHO cell line derivation step 6
  • Cells originated from ECACC CHOK1 line
  • Banked under cGMP conditions
  • Exon 6 of GS gene (required for enzymatic activity) removed by rAAV mediated recombination
  • Adapted to suspension growth in commercially available chemically defined, animal component free media
  • No off target mutations
  • Genetic testing
  • Sterility testing
  • Comprehensive testing for adventitious agents
  • Redacted cell line history available as part of the cell line evaluation
  • Full history available with a full commercial license.

CHO Licensing options  Recommended culturing conditions MRHA Scientific Advice

INITIAL ACQUISITION OF CHO K1 CELLS

Illustration of different steps for the acquisition, characterization and banking of parental CHO K1 stock cells. Yellow boxes illustrate sequential cell handling steps and dark blue boxes cell banking stages. Orange boxes indicate off-line testing and assessment steps.Acquisition, characterization and banking of parental CHO K1 stock cells

DELETION OF EXON 6 FROM THE FIRST ALLELE OF THE GS GENE 

Schematic illustration of the steps taken to delete the first allele of exon 6 on the GS gene in CHO K1 cells. Green boxes illustrate sequential cell handling steps and dark blue boxes cell banking stages. Orange boxes indicate off-line testing and assessment steps.

A full description of the targeting strategy is shown below.

Deletion the first allele of exon 6 on the GS gene in CHO K1 cells

TARGETING STRATEGY
GS knockout generation.

(A) Illustration of rAAV/GS DNA construct to target deletion of exon 6 of the hamster GS gene. The GS left and right homologies correspond to genomic DNA sequences flanking exon 6 of the hamster GS gene, loxP sequences correspond to recognition sequences for the bacteriophage Cre recombinase enzyme

(B) Illustration of production of vector plasmid particles for CHO cell transduction.

(C) Deletion of chromosomal loci.  Recombination between left and right homology regions in rAAV vector (A) and CHO chromosomal DNA (B) results in deletion of intervening chromosomal DNA (GS Exon 6) and insertion of Neor flanked by two loxP sites (C). Neor is then removed using the Cre/LoxP system.

DELETION OF EXON 6 FROM THE SECOND ALLELE OF THE GS GENE 

Schematic illustration of steps to delete allele 2 of the GS gene. Pink boxes illustrate sequential cell handling steps and dark blue boxes cell banking stages. Orange boxes indicate off-line testing and assessment steps.

The same targeting strategy that was used to remove exon 6 from the first allele was used.

CELL LINE VALIDATION

Cell line validation

 

MOLECULAR BIOLOGICAL VALIDATION OF KNOCKOUT

Knockout of exon 6 was confirmed by PCR and sequencing.

Knockout of EXON 6

FUNCTIONAL VALIDATION OF KNOCKOUT

When transferred to media lacking glutamine, the wild type CHO cells remain viable whilst the GS-/- cells are unable to survive. However, when transferred to media supplemented with 4mM glutamine, both cell lines grow at comparable rates.

Functional validation of knockout

Suspension adaptation and banking 

A frozen vial of GS null HD-BIOP3 cells was transferred to LakePharma Inc. (CA, USA) for adaptation to suspension growth in chemically defined, animal component free media (this work was carried out between 4th August 2014 and 8th July 2015). Following transfer back to Horizon, the cells were sent to Wuxi Apptec for banking under GMP and testing for adventitious agents in line with ICH Q5.

Full details of this are available as part of the cell line history report.

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