COMPLEX PROTEINS

PLASMA PROTEINS

Human blood plasma as source for biotherapeutics is very limited and safety concerns still persist, leading to a growing need for recombinant Plasma Proteins. In addition, glycosylation patterns of most Plasma Proteins are very complex and represent a huge challenge to recombinant protein expression. With a comprehensive portfolio of glyco-optimized human cell lines, the CAP™ Go expression system paves the way for recombinant Plasma Proteins.

Recombinant human C1 Inhibitor (C1-INH)

C1-Inhibitor deficiency patients suffer from an inherited form of angioedema which is currently predominantly being treated with intravenous injections of C1 Inhibitor purified from human serum. CEVEC´s CAP™ Go derived C1 Inhibitor showed in a pivotal pharmacokinetic rat study a serum half-life matching Berinert®, one of the two currently available, plasma derived treatments. This result, in combination with excellent specific activities of CAP™ Go derived C1 Inh and commercially attractive production yields, paves the way to develop a safer and more economic therapy for acute and prophylactic HAE indications.

CAP™Go derived human C1 Inh matches PK of plasma purified C1 Inh Berinert

A Specific inhibitory activity of rhC1 inhibitor produced in CAP™ Go.2 was compared with a commercially available preparation of serum C1 Inh (Berinert).

B Residual C1 Inh was measured in the serum of rats injected with conventional recombinant hC1 Inh, rhC1 Inh produced in CAP™ Go.2 cells or Berinert.

C Serum half life of conventional recombinant hC1 Inh and C1 Inh produced in different CAP™ Go cell lines in comparison to Berinert.

ENZYMES

Today, various alkaline phosphatase (AP) isoforms are under development for the treatment of acute and chronic inflammatory diseases such as Rheumatoid arthritis. Additionally, chronic degenerative diseases such as Alzheimer’s disease or Amyotrophic lateral sclerosis (ALS or Lou Gehrig’s disease) are being considered promising therapeutic indications for alkaline Phosphatases. Currently, ongoing clinical trials are being conducted with purified proteins from either bovine intestinal tissues or with non-naturally occurring reassembled recombinant versions of these molecules. The molecule developed by CEVEC, a recombinant placental human AP (rhPLAP) with a high, specific activity and a prolonged serum-half life, is expected to become the preferred alternative for these approaches. CEVEC plans to partner the further preclinical & clinical development of the molecule.

Placental alkaline Phophatase

Expression of recombinant human rhPLAP in CAP™ Go.1 or CAP™ Go.3 cells results in glyoproteins with increased sialylation of N-glycans and significantly increased serum half-life.

A IEF gel analysis indicates a similar degree of sialylation of rhPLAP from CAP™ Go.1 and CAP™ Go.3. Conventional rhPLAP is significantly less sialylated.

B rhPLAP pharmacokinetic studies comparing rhPLAP from CAP™ Go.1 or CAP™ Go.3 cells to conventional rhPALP in a rat animal study reveal an outstanding serum half-life of the material produced in CAP™ Go.1 and CAP™ Go.3 cells.

CYTOKINES

Cytokines are small signaling molecules often involved in immunomodulation, oxidant stress or cell maturation processes like erythropoiesis. Comprising proteins, peptides and glycoproteins, they represent a heterogenous class of regulators.

Comparison of cytokine production in CAP™ Go cells with other expression systems

Figure legend left: rhHGF
High protein titer of recombinant human Hepatocyte Growth Factor, a plasma protein with 1 O-linked and 3 N-linked glycans, expressed in CAP™ Go cells. By single cell cloning yields could be improved 5-fold.

Figure legend right: rhEPO
rhEPO transient or stable expressed in CAP™ Go cells display similar pattern of N-glycosylation as rhEPO stably expressed in CHO cells (SDS-PAGE after PNGaseF digestion).

ION CHANNELS

Ion Channels are pore-forming and ion permeable membrane proteins which are able to produce and maintain a resting membrane potential as well as control ion flux and regulatory signaling.

Mammalian cells expressing functional Ion Channels are a valuable cell-based assay tool for drug screening.

CAP™ Go cells are easy to handle, single cell suspension cultures which show outstanding sealing properties in automated patch clamp assays.

Ion Channel production in CAP-T cells compared to HEK 293 cells

Transient expression of KV1.5 reporter gene in CAP-T and HEK293 cells. The expression of the ion channel was detected in HEK293 by immunofluorescence labelling (a+b) using a monoclonal antibody (green). Transfected CAP-T (C) and HEK293 cells (D) were also analyzed for KV1.5 currents by automated patch-clamp using a Port-á-Patch (Nanion) and compared to mock transfected cells (A+B). Data by courtesy of Evotec (CCS Cell Culture service GmbH), Hamburg, Germany.

Virus Envelope Proteins

Virus Envelope Proteins are difficult to express highly glycosylated proteins, e.g. the HIV-1 envelope protein gp120 has 25 N-linked glycosylation sites. In a project conducted in cooperation with TCF GmbH and the University Medical Center of the Johannes Gutenberg-University Mainz, Department of Dermatology, the following results could be found:

gp120 titer > 30 mg/L = 5.6 pcd
No degradation or aggregation in the CAP™Go-derived material
CAP™ Go-derived gp120 appears to be more homogenously glycosylated than CHO derived material

Expression in CAP™Go Cells

A Comparison of CAP™ cell vs. CHO-cell derived gp120

B Viable cell density of CAP™ cells expressing gp120 during a 10 d fed-batch and expression of gp120 virus envelope over time

HIGH MOLECULAR MASS MULTIMER PROTEIN COMPLEXES

Expression of high molecular Multimeric Protein complexes e.g. Fibrinogen (340 kD trimeric protein 2x(a+b+g)) in CAP™ Go cells results in high expression titers with significantly less degradation than in HEK293 cell derived Fibrinogen.

From pool generation to selected single cell clones

A Expression of CAP™ cell derived Fibrinogen expression of Pool vs. SCC

B Comparison of CAP™ cell vs. HEK-cell derived Fibrinogen with reducing conctions

C Comparison of CAP™ cell vs. HEK-cell derived Fibrinogen with non-reducing conctions