1. Application of ‘Omics Tools in the Rational Engineering of
Biopharmaceutical Production Cell Lines
Annette Sieron[1], Markus Michael Müller[1], Germán Leparc[2], Barbara Enenkel[1], AnneTolstrup[1], Hitto Kaufmann[1], Jennifer Könitzer[1]
[1] Biopharmaceuticals Process Development & Strategy
[2] Research Germany,Target Discovery Research,Target ValidationTechnologies
Boehringer Ingelheim, Birkendorfer Straße 63, Biberach/Riss, Germany Contact: jennifer.koenitzer@boehringer-ingelheim.com
GENE EXPRESSION PROFILING IN CHO HOSTCELLS
The availability of CHO genome models allows high throughput transcriptome profiling by RNA-seq. The technology is
ideally suited to investigate cells across a wide variety of process conditions. RNA-seq is performed on an Illumina
Genome Analyzer using standard protocols. Reads are analysed with the depicted pipeline, which incorporates STAR for
mapping, Cufflinks for quantification and DESeq for calculating differential gene expression. The CHO-K1 gene model
was refined using ortholog mapping in which the mouse gene annotation was transferred onto CHO by best hit reciprocal
BLAST. Further data analysis is carried out using Spotfire (TIBCO) and the Ingenuity Pathway Analyzer (Qiagen).
GLYCOSYLATION PROFILING INTWO INDEPENDENTCHO-DG44 SUBCLONES
Protein glycosylation is a key product quality feature of biotherapeutics. For therapeutic antibodies, N-linked
glycosylation plays a paramount role in maintaining drug efficacy, safety and half life. Regulatory bodies require the
extensive characterisation of glycosylated proteins. It is well documented that the cell type, media and process
conditions impact antibody glycosylation patterns.
TRANSCRIPTOME PROFILING OF THE
conCERT™ CELL LINE
The conCERT™ cell line is a CHO derivative overexpressing the
human ceramide transporter protein (CERT).
CERT facilitates both lipid and protein transport. conCERT™
cells show enhanced protein secretion, resulting in 1.3 -2x
higher product titers.
Gene expression profiling of ConCERT™ in comparison to non-
CERT expressing CHO cells revealed a strong upregulation of
CERT, the ion channel FXYD2 and the fatty acid transporter
SLC27A5. This profile indicates that ConCERT™ cells
experience a bottle neck in their lipid metabolism, which
identifies an interesting target for rational media engineering.
Gene expression profiling of CHO1 vs. CHO2 revealed, that, while
both cell lines express the same number of genes associated with
mammalian glycosylation (170/300 with RPKM>1 data not shown);
several of those are differentially expressed (cut-offs >1 for log2-
foldchanges & <0.05 for p values).
At Boehringer Ingelheim, we have observed that two independent
DG44 subclones (CHO1 and CHO2) consistently produce different
levels of the fucosylated biantennary glycans A2FG0 and A2FG1,
which either lack or carry a terminal galactose residue. Both species
comprise the majority (85%) of N-linked antibody glycans. CHO1,
however, exhibits a higher fraction (Mean = 67%) of A2FG0 compared
to CHO2 (51%). Accordingly, the A2FG1 fraction is lower in CHO1
(18%) than in CHO2 (34%).
The data is derived from 6 different antibodies and 122 (CHO1) or
125 (CHO2) separate measurements. Sugar patterns were analysed
by HPLC that followed a N-Glycosidase F enzymatic digest and 2-
aminobenzamide fluorescent labelling of released sugars. (Blue
squares: N-acetylglucosamine; green circles: Mannose; red triangles:
Fucose; yellow circles: Galactose.)
Among the differentially expressed genes are several Galactosyltransferases (B3GALT1, B4GALT1, B4GALT3) that
show higher expression in CHO2 than CHO1. SCL35A2, a UDP-galactose solute carrier, and B3GNT2, an N-
acetylglucosaminyltransferase, have lower expression levels in CHO2 than CHO1. In addition, several
sialyltransferases (ST3GAL1-3; ST8SIA6) are also present at lower levels in CHO2 compared to CHO1.
RATIONAL ENGINEERING OFTARGETED INTEGRATION CAPABLE CELLS USINGTHE CHO GENOME & NGSTECHNOLOGIES
The generation of high producers for biopharmaceutical manufacturing is still largely accomplished by random integration of the protein expressing transgene. Chromosomal
positioning effects invariably cause a highly heterogeneous cell population requiring substantial efforts in order to isolate a suitable clone. Targeted integration (TI) into the CHO
genome can overcome this variability. Technologies to facilitate TI, such as recombinases or designer endonucleases, have been available for a long time. With the CHO genome and Next
Generation Sequencing (NGS), it is now also possible to isolate genomic regions that support a high level of transcriptional activity (hot spots).
Single/Low copy integrants: Qp 3-6 passages 6w format
“Hot Spots” can be identified from
CHO high producer clones
characterised by both a low number of
integration sites and transgene copies.
For that purpose, protein expressing CHO clones were generated and screened for copy
number by qPCR, protein expression levels and integration site pattern by Southern Blot. The
average Qp of the best low copy clones is shown. For the identification of the actual
integration site, genomic DNA was isolated. Following an enrichment procedure for the
inserted vector sequence (red), samples were subjected to Illumina or 454 Sequencing. Reads
can be analysed to exclude those to mapping only to the CHO genome (blue) or only to the
vector sequence (red). The reads depicted in green define the vector/genome junction and
thus the integration site.