A Prodomain Fragment from the Proteolytic Activation of Growth Differentiation Factor 11 Remains Associated with the Mature Growth Factor and Keeps It Soluble.

Growth differentiation factor 11 (GDF11), a member of the transforming growth factor ß (TGF-ß) family, plays diverse roles in mammalian development. It is synthesized as a large, inactive precursor protein containing a prodomain, pro-GDF11, and exists as a homodimer. Activation requires two proteolytic processing steps that release the prodomains and transform latent pro-GDF11 into active mature GDF11. In studying proteolytic activation in vitro, we discovered that a 6-kDa prodomain peptide containing residues 60-114, PDP60-114, remained associated with the mature growth factor. Whereas the full-length prodomain of GDF11 is a functional antagonist, PDP60-114 had no impact on activity. The specific activity of the GDF11/PDP60-114 complex (EC50 = 1 nM) in a SMAD2/3 reporter assay was identical to that of mature GDF11 alone. PDP60-114 improved the solubility of mature GDF11 at neutral pH. As the growth factor normally aggregates/precipitates at neutral pH, PDP60-114 can be used as a solubility-enhancing formulation. Expression of two engineered constructs with PDP60-114 genetically fused to the mature domain of GDF11 through a 2x or 3x G4S linker produced soluble monomeric products that could be dimerized through redox reactions. The construct with a 3x G4S linker retained 10% activity (EC50 = 10 nM), whereas the construct connected with a 2x G4S linker could only be activated (EC50 = 2 nM) by protease treatment. Complex formation with PDP60-114 represents a new strategy for stabilizing GDF11 in an active state that may translate to other members of the TGF-ß family that form latent pro/mature domain complexes.

Discovery and investigation of misincorporation of serine at asparagine positions in recombinant proteins expressed in Chinese hamster ovary cells.

Misincorporation of amino acids in proteins expressed in Escherichia coli has been well documented but not in proteins expressed in mammalian cells under normal recombinant protein production conditions. Here we report for the first time that Ser can be incorporated at Asn positions in proteins expressed in Chinese hamster ovary cells. This misincorporation was discovered as a result of intact mass measurement, peptide mapping analysis, and tandem mass spectroscopy sequencing. Our analyses showed that the substitution was not related to specific protein molecules or DNA codons and was not site-specific. We believe that the incorporation of Ser at sites coded for Asn was due to mischarging of tRNA(Asn) rather than to codon misreading. The rationale for substitution of Asn by Ser and not by other amino acids is also discussed. Further investigation indicated that the substitution was due to the starvation for Asn in the cell culture medium and that the substitution could be limited by using the Asn-rich feed. These observations demonstrate that the quality of expressed proteins should be closely monitored when altering cell culture conditions.

Control of misincorporation of serine for asparagine during antibody production using CHO cells.

A recombinant monoclonal antibody produced by Chinese hamster ovary (CHO) cell fed-batch culture was found to have amino acid sequence misincorporation upon analysis by intact mass and peptide mapping mass spectrometry. A detailed analysis revealed multiple sites for asparagine were being randomly substituted by serine, pointing to mistranslation as the likely source. Results from time-course analysis of cell culture suggest that misincorporation was occurring midway through the fed-batch process and was correlated to asparagine reduction to below detectable levels in the culture. Separate shake flask experiments were carried out that confirmed starvation of asparagine and not excess of serine in the medium as the root cause of the phenomenon. Reduction in serine concentration under asparagine starvation conditions helped reduce extent of misincorporation. Supplementation with glutamine also helped reduce extent of misincorporation. Maintenance of asparagine at low levels in 2 L bench-scale culture via controlled supplementation of asparagine-containing feed eliminated the occurrence of misincorporation. This strategy was implemented in a clinical manufacturing process and scaled up successfully to the 200 and 2,000 L bioreactor scales.

Enhancement of transient gene expression and culture viability using Chinese hamster ovary cells overexpressing Bcl-x(L).

Transient gene expression (TGE) provides a method for quickly delivering protein for research using mammalian cells. While high levels of recombinant proteins have been produced in TGE experiments in HEK 293 cells, TGE efforts in the commercially prominent CHO cell line still suffer from inadequate protein yields. Here, we describe a cell-engineering strategy to improve transient production of proteins using CHO cells. CHO-DG44 cells were engineered to overexpress the anti-apoptotic protein Bcl-x(L) and transiently transfected using polyethylenimine (PEI) in serum-free media. Pools and cell lines stably expressing Bcl-x(L) showed enhanced viable cell density and increased production of a glycosylated, therapeutic fusion protein in shake flask TGE studies. The improved cell lines showed fusion protein production levels ranging from 12.6 to 27.0 mg/L in the supernatant compared to the control cultures which produced 6.3-7.3 mg/L, representing a 70-270% increase in yield after 14 days of fed-batch culture. All Bcl-xL-expressing cell lines also exhibited an increase in specific productivity during the first 8 days of culture. In addition to increased production, Bcl-x(L) cell lines maintained viabilities above 90% and less apoptosis compared to the DG44 host which had viabilities below 60% after 14 days. Product quality was comparable between a Bcl-xL-engineered cell line and the CHO host. The work presented here provides the foundation for using anti-apoptosis engineered CHO cell lines for increased production of therapeutic proteins in TGE applications.

E2F-1 overexpression increases viable cell density in batch cultures of Chinese hamster ovary cells.

The cell density is an inherent constraint in commercial mammalian cell cultures. Here, we describe a cell engineering strategy utilizing the overexpression of the E2F-1 cell cycle transcription factor in CHO DG44 cells that produce a monoclonal antibody in serum-free, suspension culture. Stable pools and cell lines expressing E2F-1 were isolated that attained viable cell densities 20% higher than control cell lines and continued proliferation for an additional day in batch culture. There were no significant changes in antibody production, apoptosis, and cell cycle compared to control cells, nor were the growth effects evident in fed-batch conditions. Overall, E2F-1 overexpression postponed entry into stationary phase in mammalian cells, but perhaps novel E2F-1 variants or combination cell cycle engineering strategies will be necessary to realize significant growth benefits in commercial applications.

Engineering potent long-acting variants of the Wnt inhibitor DKK2.

Wnt signaling pathways are required for a wide variety of biological processes ranging from embryonic development to tissue repair and regeneration. Dickkopf-2 (DKK2) is classically defined as a canonical Wnt inhibitor, though it may play a role in activating non-canonical Wnt pathways in the context of endothelial network formation after acute injury. Here we report the discovery of a fusion partner for a DKK2 polypeptide that significantly improves the expression, biochemical properties and pharmacokinetics (PK) of the DKK2 polypeptide. Specifically, human serum albumin (HSA) was identified as a highly effective fusion partner. Substitution of selected amino acid residues in DKK2 designed to decrease heparan sulfate binding by HSA-DKK2 variants, further improved the PK properties of the molecule in rodents. The HSA-DKK2 variants were monomeric, as thermally stable as wild type, and active as measured by their ability to bind to and prevent phosphorylation of the Wnt coreceptor LRP6. Our engineering efforts resulted in potent long-lived variants of the canonical Wnt inhibitor DKK2, applicable for Wnt pathway manipulation either by systematic delivery or focused administration at sites of tissue injury.

Directed evolution of mammalian anti-apoptosis proteins by somatic hypermutation.

Recently, researchers have created novel fluorescent proteins by harnessing the somatic hypermutation ability of B cells. In this study, we examined if this approach could be used to evolve a non-fluorescent protein, namely the anti-apoptosis protein Bcl-x(L), using the Ramos B-cell line. After demonstrating that Ramos cells were capable of mutating a heterologous bcl-x(L) transgene, the cells were exposed to multiple rounds of the chemical apoptosis inducer staurosporine followed by rounds of recovery in fresh medium. The engineered B cells expressing Bcl-x(L) exhibited progressively lower increases in apoptosis activation as measured by caspase-3 activity after successive rounds of selective pressure with staurosporine treatment. Within the B-cell genome, a number of mutated bcl-x(L) transgene variants were identified after three rounds of evolution, including a mutation of Bcl-x(L) Asp29 to either Asn or His, in 8 out of 23 evaluated constructs that represented at least five distinct Ramos subpopulations. Subsequently, Chinese hamster ovary (CHO) cells engineered to overexpress the Bcl-x(L) Asp29Asn variant showed enhanced apoptosis resistance against an orthogonal apoptosis insult, Sindbis virus infection, when compared with cells expressing the wild-type Bcl-x(L) protein. These findings provide, to our knowledge, the first demonstration of evolution of a recombinant mammalian protein in a mammalian expression system.

Mcl-1 overexpression leads to higher viabilities and increased production of humanized monoclonal antibody in Chinese hamster ovary cells.

Bioreactor stresses, including nutrient deprivation, shear stress, and byproduct accumulation can cause apoptosis, leading to lower recombinant protein yields and increased costs in downstream processing. Although cell engineering strategies utilizing the overexpression of antiapoptotic Bcl-2 family proteins such as Bcl-2 and Bcl-x(L) potently inhibit apoptosis, no studies have examined the use of the Bcl-2 family protein, Mcl-1, in commercial mammalian cell culture processes. Here, we overexpress both the wild type Mcl-1 protein and a Mcl-1 mutant protein that is not degraded by the proteasome in a serum-free Chinese hamster ovary (CHO) cell line producing a therapeutic antibody. The expression of Mcl-1 led to increased viabilities in fed-batch culture, with cell lines expressing the Mcl-1 mutant maintaining approximately 90% viability after 14 days when compared with 65% for control cells. In addition to enhanced culture viability, Mcl-1-expressing cell lines were isolated that consistently showed increases in antibody production of 20-35% when compared with control cultures. The quality of the antibody product was not affected in the Mcl-1-expressing cell lines, and Mcl-1-expressing cells exhibited 3-fold lower caspase-3 activation when compared with the control cell lines. Altogether, the expression of Mcl-1 represents a promising alternative cell engineering strategy to delay apoptosis and increase recombinant protein production in CHO cells.