Optimization of Heavy Chain and Light Chain Signal Peptides for High Level Expression of Therapeutic Antibodies in CHO Cells

Translocation of a nascent protein from the cytosol into the ER mediated by its signal peptide is a critical step in protein secretion. The aim of this work was to develop a platform technology to optimize the signal peptides for high level production of therapeutic antibodies in CHO cells. A database of signal peptides from a large number of human immunoglobulin (Ig) heavy chain (HC) and kappa light chain (LC) was generated. Most of the HC signal peptides contain 19 amino acids which can be divided into three domains and the LC signal peptides contain 22 amino acids. The signal peptides were then clustered according to sequence similarity. Based on the clustering, 8 HC and 2 LC signal peptides were analyzed for their impacts on the production of 5-top selling antibody therapeutics, namely, Herceptin, Avastin, Remicade, Rituxan, and Humira. The best HC and LC signal peptides for producing these 5 antibodies were identified. The optimized signal peptides for Rituxan is 2-fold better compared to its native signal peptides which are available in the public database. Substitution of a single amino acid in the optimized HC signal peptide for Avastin reduced its production significantly. Mass spectrometry analyses revealed that all optimized signal peptides are accurately removed in the mature antibodies. The results presented in this report are particularly important for the production of these 5 antibodies as biosimilar drugs. They also have the potential to be the best signal peptides for the production of new antibodies in CHO cells.     

Avian antimicrobial peptides: the defense role of beta-defensins

Avian antimicrobial peptides, classified as beta-defensins, have been identified from bloods of chicken, turkey, and ostrich; epithelial cells of chicken and turkey; and king penguin stomach contents. Beta-defensins are a family of antimicrobial peptides characterized by six cysteine residues forming beta-defensin motifs that are also found in bovine, ovine, pig, and human. These peptides are active against a wide range of microorganisms including Gram-positive and Gram-negative bacteria, fungi, and yeast. Analysis of evolutionary relationships of vertebrate beta-defensins showed that there might be a common ancestral gene between avian and other mammalian peptides. This ancient gene may have been passed down and evolved from species older than the oldest living birds, forming a beta-defensin-like precursor molecule. This review describes potential applications of these peptides in health care products.     

Characterisation of a novel high-purity, double virus inactivated von Willebrand Factor and Factor VIII concentrate (Wilate)

This study summarises the biochemical and functional properties of a new generation plasma-derived, double virus inactivated von Willebrand Factor/Factor VIII (VWF/FVIII) concentrate, Wilate, targeted for the treatment of both von Willebrand disease (VWD) and haemophilia A. The manufacturing process comprises two chromatographic steps based on different performance principles, ensuring a high purity of the concentrate (mean specific activity in 15 consecutive production batches: 122 IU FVIII:C/mg total protein) and, thus, minimising the administered protein load to the patient (specification: < or = 15 mg total protein per 900 IU Wilate). The optimised solvent/detergent (S/D) treatment and prolonged terminal dry-heat (PermaHeat) treatment of the lyophilised product at a specified residual moisture (RM) provide two mechanistically independent, effective and robust virus inactivation procedures for enveloped viruses and one step for non-enveloped viruses. These process steps are aggressive enough to inactivate viruses efficiently, but yet gentle enough to maintain the structural integrity and function of the VWF and FVIII molecules, as proven by state-of-the-art assays covering the diverse features of importance. The VWF multimeric pattern is close to the one displayed by normal plasma, with a consistent content of more than 10 multimers, but a relatively lower portion of the very high multimers. The multimeric triplet structure is normal, underlining the gentle and effective manufacturing process, which does not require the addition of protein stabilisers at any step. The balanced activity ratio of VWF to FVIII is close to that of plasma from healthy subjects, rendering Wilate suitable also for the safe and effective treatment of patients with VWD.     

Identification of functional elements of the GDP-fucose transporter SLC35C1 using a novel Chinese hamster ovary mutant

The GDP-fucose transporter SLC35C1 critically regulates the fucosylation of glycans. Elucidation of its structure-function relationships remains a challenge due to the lack of an appropriate mutant cell line. Here we report a novel Chinese hamster ovary (CHO) mutant, CHO-gmt5, generated by the zinc-finger nuclease technology, in which the Slc35c1 gene was knocked out from a previously reported CHO mutant that has a dysfunctional CMP-sialic acid transporter (CST) gene (Slc35a1). Consequently, CHO-gmt5 harbors double genetic defects in Slc35a1 and Slc35c1 and produces N-glycans deficient in both sialic acid and fucose. The structure-function relationships of SLC35C1 were studied using CHO-gmt5 cells. In contrast to the CST and UDP-galactose transporter, the C-terminal tail of SLC35C1 is not required for its Golgi localization but is essential for generating glycans that are recognized by a fucose-binding lectin, Aleuria aurantia lectin (AAL), suggesting an important role in the transport activity of SLC35C1. Furthermore, we found that this impact can be independently contributed by a cluster of three lysine residues and a Glu-Met (EM) sequence within the C terminus. We also showed that the conserved glycine residues at positions 180 and 277 of SLC35C1 have significant impacts on AAL binding to CHO-gmt5 cells, suggesting that these conserved glycine residues are required for the transport activity of Slc35 proteins. The absence of sialic acid and fucose on Fc N-glycan has been independently shown to enhance the antibody-dependent cellular cytotoxicity (ADCC) effect. By combining these features into one cell line, we postulate that CHO-gmt5 may represent a more advantageous cell line for the production of recombinant antibodies with enhanced ADCC effect.     

Deletion of cscR in Escherichia coli W improves growth and poly-3-hydroxybutyrate (PHB) production from sucrose in fed batch culture

Sucrose has several advantages over glucose as a feedstock for bioprocesses, both environmentally and economically. However, most industrial Escherichia coli strains are unable to utilize sucrose. E. coli W can grow on sucrose but stops growing when sucrose concentrations become low. This is undesirable in fed-batch conditions where sugar levels are low between feeding pulses. Sucrose uptake rates were improved by removal of the cscR gene, which encodes a protein that represses expression of the sucrose utilization genes at low sucrose concentrations. Poly-3-hydroxybutyrate (PHB) was used as a model compound in order to assess the effect of improved sugar utilization on bio-production. In the cscR knockout strain, production from sucrose was improved by 50%; this strain also produced 30% more PHB than the wild-type using glucose. This result demonstrates the feasibility of utilizing sucrose as an industrial feedstock for E. coli-based bioprocesses in high cell density culture.     

Native-feather degradation by Fervidobacterium islandicum AW-1, a newly isolated keratinase-producing thermophilic anaerobe

A native-feather-degrading thermophilic anaerobe was isolated from a geothermal hot stream in Indonesia. Isolate AW-1, identified as a member of the species Fervidobacterium islandicum, was shown to degrade native feathers (0.8%, w/v) completely at 70 degrees C and pH 7 with a maximum specific growth rate (0.14 h(-1)) in Thermotoga- Fervidobacterium(TF) medium. After 24 h of culture, feather degradation led to an increase in free amino acids such as histidine, cysteine and lysine. Moreover, nutritionally essential amino acids such as tryptophan and methionine, which are rare in feather keratin, were also produced as microbial metabolites. A homomultimeric membrane-bound keratinolytic protease (>200 kDa; 97 kDa subunits) was purified from a cell extract of F. islandicum AW-1. The enzyme exhibited activity toward casein and soluble keratin optimally at 100 degrees C and pH 9, and had a half-life of 90 min at 100 degrees C. The enzyme showed higher specific activity for the keratinous substrates than other proteases and catalyzed the cleavage of peptide bonds more rapidly following the reduction of disulfide bridges in feather keratin by 10 mM dithiothreitol. Therefore, the enzyme from F. islandicum AW-1 is a novel, thermostable keratinolytic serine protease.     

Metabolic comparison of cryopreserved and normal cells from <Emphasis Type="Italic">Tabernaemontana divaricata</Emphasis> suspension cultures

A cryopreservation protocol for Tabernaemontana divaricata suspension cell cultures (6 Div BW 101) was established. Cells were precultured in MS medium supplemented with 0.5 and 0.33 M mannitol for 2 or 3 days following with incubation in MS media with a mixture of 1 M sucrose, 0.5 M glycerol, 0.5 M DMSO, and 0.04 M L-proline as cryoprotectant in an ice bath for 20 min. The cells were transferred into 2 ml cryogenic vials and then, the vials were put into the cryogenic container prior to placing at a −80 °C freezer for 4 h followed by rapid immersion in liquid nitrogen. The cells were transferred without washing a MS medium solidified with 7% (w/v) agarose. Cells that were precultured 3 days after subculturing in MS medium supplemented with 0.5 M mannitol for 3 days, showed growth recovery. Metabolic profiling of control and cryopreserved Tabernaemontana divaricata cells was performed by ¹H-NMR spectroscopy combined with PCA, GC, and HPLC. Differences of metabolic accumulation were found in the level of several amino acids, carbohydrates, and fumaric acid. However, the levels of the main alkaloid precursor tryptamine did not change.     

NDK Interacts with FtsZ and Converts GDP to GTP to Trigger FtsZ Polymerisation - A Novel Role for NDK

Introduction

Nucleoside diphosphate kinase (NDK), conserved across bacteria to humans, synthesises NTP from NDP and ATP. The eukaryotic homologue, the NDPK, uses ATP to phosphorylate the tubulin-bound GDP to GTP for tubulin polymerisation. The bacterial cytokinetic protein FtsZ, which is the tubulin homologue, also uses GTP for polymerisation. Therefore, we examined whether NDK can interact with FtsZ to convert FtsZ-bound GDP and/or free GDP to GTP to trigger FtsZ polymerisation.

Methods

Recombinant and native NDK and FtsZ proteins of Mycobacterium smegmatis and Mycobacterium tuberculosis were used as the experimental samples. FtsZ polymersation was monitored using 90° light scattering and FtsZ polymer pelleting assays. The γ³²P-GTP synthesised by NDK from GDP and γ³²P-ATP was detected using thin layer chromatography and quantitated using phosphorimager. The FtsZ bound ³²P-GTP was quantitated using phosphorimager, after UV-crosslinking, followed by SDS-PAGE. The NDK-FtsZ interaction was determined using Ni²⁺-NTA-pulldown assay and co-immunoprecipitation of the recombinant and native proteins in vitro and ex vivo, respectively.

Results

NDK triggered instantaneous polymerisation of GDP-precharged recombinant FtsZ in the presence of ATP, similar to the polymerisation of recombinant FtsZ (not GDP-precharged) upon the direct addition of GTP. Similarly, NDK triggered polymerisation of recombinant FtsZ (not GDP-precharged) in the presence of free GDP and ATP as well. Mutant NDK, partially deficient in GTP synthesis from ATP and GDP, triggered low level of polymerisation of MsFtsZ, but not of MtFtsZ. As characteristic of NDK’s NTP substrate non-specificity, it used CTP, TTP, and UTP also to convert GDP to GTP, to trigger FtsZ polymerisation. The NDK of one mycobacterial species could trigger the polymerisation of the FtsZ of another mycobacterial species. Both the recombinant and the native NDK and FtsZ showed interaction with each other in vitro and ex vivo, alluding to the possibility of direct phosphorylation of FtsZ-bound GDP by NDK.

Conclusion

Irrespective of the bacterial species, NDK interacts with FtsZ in vitro and ex vivo and, through the synthesis of GTP from FtsZ-bound GDP and/or free GDP, and ATP (CTP/TTP/UTP), triggers FtsZ polymerisation. The possible biological context of this novel activity of NDK is presented.