Amino acid and glucose metabolism in fed-batch CHO cell culture affects antibody production and glycosylation.

Fed-batch Chinese hamster ovary (CHO) cell culture is the most commonly used process for IgG production in the biopharmaceutical industry. Amino acid and glucose consumption, cell growth, metabolism, antibody titer, and N-glycosylation patterns are always the major concerns during upstream process optimization, especially media optimization. Gaining knowledge on their interrelations could provide insight for obtaining higher immunoglobulin G (IgG) titer and better controlling glycosylation-related product quality. In this work, different fed-batch processes with two chemically defined proprietary media and feeds were studied using two IgG-producing cell lines. Our results indicate that the balance of glucose and amino acid concentration in the culture is important for cell growth, IgG titer and N-glycosylation. Accordingly, the ideal fate of glucose and amino acids in the culture could be mainly towards energy and recombinant product, respectively. Accumulation of by-products such as NH4(+) and lactate as a consequence of unbalanced nutrient supply to cell activities inhibits cell growth. The levels of Leu and Arg in the culture, which relate to cell growth and IgG productivity, need to be well controlled. Amino acids with the highest consumption rates correlate with the most abundant amino acids present in the produced IgG, and thus require sufficient availability during culture. Case-by-case analysis is necessary for understanding the effect of media and process optimization on glycosylation. We found that in certain cases the presence of Man5 glycan can be linked to limitation of UDP-GlcNAc biosynthesis as a result of insufficient extracellular Gln. However, under different culture conditions, high Man5 levels can also result from low α-1,3-mannosyl-glycoprotein 2-ß-N-acetylglucosaminyltransferase (GnTI) and UDP-GlcNAc transporter activities, which may be attributed to high level of NH4+ in the cell culture. Furthermore, galactosylation of the mAb Fc glycans was found to be limited by UDP-Gal biosynthesis, which was observed to be both cell line and cultivation condition-dependent. Extracellular glucose and glutamine concentrations and uptake rates were positively correlated with intracellular UDP-Gal availability. All these findings are important for optimization of fed-batch culture for improving IgG production and directing glycosylation quality.

A multi-pronged investigation into the effect of glucose starvation and culture duration on fed-batch CHO cell culture.

In this study, omics-based analysis tools were used to explore the effect of glucose starvation and culture duration on monoclonal antibody (mAb) production in fed-batch CHO cell culture to gain better insight into how these parameters can be controlled to ensure optimal mAb productivity and quality. Titer and N-glycosylation of mAbs, as well as proteomic signature and metabolic status of the production cells in the culture were assessed. We found that the impact of glucose starvation on the titer and N-glycosylation of mAbs was dependent on the degree of starvation during early stationary phase of the fed-batch culture. Higher degree of glucose starvation reduced intracellular concentrations of UDP-GlcNAc and UDP-GalNAc, but increased the levels of UDP-Glc and UDP-Gal. Increased GlcNAc and Gal occupancy correlated well with increased degree of glucose starvation, which can be attributed to the interplay between the dilution effect associated with change in specific productivity of mAbs and the changed nucleotide sugar metabolism. Herein, we also show and discuss that increased cell culture duration negatively affect the maturation of glycans. In addition, comparative proteomics analysis of cells was conducted to observe differences in protein abundance between early growth and early stationary phases. Generally higher expression of proteins involved in regulating cellular metabolism, extracellular matrix, apoptosis, protein secretion and glycosylation was found in early stationary phase. These analyses offered a systematic view of the intrinsic properties of these cells and allowed us to explore the root causes correlating culture duration with variations in the productivity and glycosylation quality of monoclonal antibodies produced with CHO cells.

RUBY® - a tetravalent (2+2) bispecific antibody format with excellent functionality and IgG-like stability, pharmacology and developability properties.

Despite the large number of existing bispecific antibody (bsAb) formats, the generation of novel bsAbs is still associated with development and bioprocessing challenges. Here, we present RUBY, a novel bispecific antibody format that allows rapid generation of bsAbs that fulfill key development criteria. The RUBYTM format has a 2 + 2 geometry, where two Fab fragments are linked via their light chains to the C-termini of an IgG, and carries mutations for optimal chain pairing. The unique design enables generation of bsAbs with mAb-like attributes. Our data demonstrate that RUBY bsAbs are compatible with small-scale production systems for screening purposes and can be produced at high yields (>3 g/L) from stable cell lines. The bsAbs produced are shown to, in general, contain low amounts of aggregates and display favorable solubility and stress endurance profiles. Further, compatibility with various IgG isotypes is shown and tailored Fc gamma receptor binding confirmed. Also, retained interaction with FcRn is demonstrated to translate into a pharmacokinetic profile in mice and non-human primates that is comparable to mAb controls. Functionality of conditional active RUBY bsAbs is confirmed in vitro. Anti-tumor effects in vivo have previously been demonstrated, and shown to be superior to a comparable mAb, and here it is further shown that RUBY bsAbs penetrate and localize to tumor tissue in vivo. In all, the RUBY format has attractive mAb-like attributes and offers the possibility to mitigate many of the development challenges linked to other bsAb formats, facilitating both high functionality and developability.

Defining lncRNAs Correlated with CHO Cell Growth and IgG Productivity by RNA-Seq.

How the long non-coding RNA (lncRNA) genome in recombinant protein producing Chinese hamster ovary (CHO) cell lines relates to phenotype is not well described. We therefore defined the CHO cell lncRNA transcriptome from cells grown in controlled miniature bioreactors under fed-batch conditions using RNA-Seq to identify lncRNAs and how the expression of these changes throughout growth and between IgG producers. We identify lncRNAs including Adapt15, linked to ER stress, GAS5, linked to mTOR signaling/growth arrest, and PVT1, linked to Myc expression, which are differentially regulated during fed-batch culture and whose expression correlates to productivity and growth. Changes in (non)-coding RNA expression between the seed train and the equivalent day of fed-batch culture are also reported and compared with existing datasets. Collectively, we present a comprehensive lncRNA CHO cell profiling and identify targets for engineering growth and productivity characteristics of CHO cells.

The fission yeast heterochromatin protein Rik1 is required for telomere clustering during meiosis.

Telomeres share the ability to silence nearby transcription with heterochromatin, but the requirement of heterochromatin proteins for most telomere functions is unknown. The fission yeast Rik1 protein is required for heterochromatin formation at centromeres and the mating-type locus, as it recruits the Clr4 histone methyltransferase, whose modification of histone H3 triggers binding by Swi6, a conserved protein involved in spreading of heterochromatin. Here, we demonstrate that Rik1 and Clr4, but not Swi6, are required along with the telomere protein Taz1 for crucial chromosome movements during meiosis. However, Rik1 is dispensable for the protective roles of telomeres in preventing chromosome end-fusion. Thus, a Swi6-independent heterochromatin function distinct from that at centromeres and the mating-type locus operates at telomeres during sexual differentiation.

Dynamics of immature mAb glycoform secretion during CHO cell culture: An integrated modelling framework.

Ensuring consistent glycosylation-associated quality of therapeutic monoclonal antibodies (mAbs) has become a priority in pharmaceutical bioprocessing given that the distribution and composition of the carbohydrates (glycans) bound to these molecules determines their therapeutic efficacy and immunogenicity. However, the interaction between bioprocess conditions, cellular metabolism and the intracellular process of glycosylation remains to be fully understood. To gain further insight into these interactions, we present a novel integrated modelling platform that links dynamic variations in mAb glycosylation with cellular secretory capacity. Two alternative mechanistic representations of how mAb specific productivity (qp ) influences glycosylation are compared. In the first, mAb glycosylation is modulated by the linear velocity with which secretory cargo traverses the Golgi apparatus. In the second, glycosylation is influenced by variations in Golgi volume. Within our modelling framework, both mechanisms accurately reproduce experimentally-observed dynamic changes in mAb glycosylation. In addition, an optimisation-based strategy has been developed to estimate the concentration of glycosylation enzymes required to minimise mAb glycoform variability. Our results suggest that the availability of glycosylation machinery relative to cellular secretory capacity may play a crucial role in mAb glycosylation. In the future, the modelling framework presented here may aid in selecting and engineering cell lines that ensure consistent mAb glycosylatio.

Combination primer polymerase chain reaction for multi-site mutagenesis of close proximity sites.

We describe a rapid and efficient polymerase chain reaction procedure for multi-site-directed mutagenesis for cases in which the sites to be mutated are in close proximity. The combination primer polymer chain reaction method is based on a multi-site directed mutagenesis protocol together with a splicing by overlapping extension polymerase chain reaction protocol. several different combinations of multiple mutations were successfully performed with this method and are reported in this study.

Elk3 from hamster--a ternary complex factor with strong transcriptional repressor activity.

Elk3 belongs to the Ets family of transcription factors, which are regulated by the Ras/mitogen-activated protein kinase-signaling pathway. In the absence of Ras, this protein is a strong inhibitor of transcription and may be directly involved in regulation of growth by downregulating the transcription of genes that are activated during entry into G1. We have isolated the Cricetulus griseus Elk3 gene from the Chinese hamster ovary (CHO) cell line and investigated the transcriptional potential of this factor. Transient transfections revealed that, in addition to its regulation of the c-fos promoter, Elk3 from CHO cells seems to inhibit other promoters controlling expression of proteins involved in G1/S phase progression; Cyclin D1 and DHFR. As has been described for the Elk3 homologs Net (Mouse) and Sap-2 (Human), the results of the present study further indicate that hamster Elk3 is a target of the Ras-Raf-MAPK pathway, and cotransfections with constitutively active H-ras relieves its negative transcriptional activity. No cells stably expressing exogenous Elk3 could be obtained, possibly due to an unspecified toxic or growth retarding effect. These findings support a possible role for Elk3 in growth regulation and reveal a high degree of homology for this protein across species.

Pan-HER, an Antibody Mixture Simultaneously Targeting EGFR, HER2, and HER3, Effectively Overcomes Tumor Heterogeneity and Plasticity.

PURPOSE: Accumulating evidence indicates a high degree of plasticity and compensatory signaling within the human epidermal growth factor receptor (HER) family, leading to resistance upon therapeutic intervention with HER family members. EXPERIMENTAL DESIGN/RESULTS: We have generated Pan-HER, a mixture of six antibodies targeting each of the HER family members EGFR, HER2, and HER3 with synergistic pairs of antibodies, which simultaneously remove all three targets, thereby preventing compensatory tumor promoting mechanisms within the HER family. Pan-HER induces potent growth inhibition in a range of cancer cell lines and xenograft models, including cell lines with acquired resistance to therapeutic antibodies. Pan-HER is also highly efficacious in the presence of HER family ligands, indicating that it is capable of overcoming acquired resistance due to increased ligand production. All three target specificities contribute to the enhanced efficacy, demonstrating a distinct benefit of combined HER family targeting when compared with single-receptor targeting. CONCLUSIONS: Our data show that simultaneous targeting of three receptors provides broader efficacy than targeting a single receptor or any combination of two receptors in the HER family, especially in the presence of HER family ligands. Pan-HER represents a novel strategy to deal with primary and acquired resistance due to tumor heterogeneity and plasticity in terms of HER family dependency and as such may be a viable alternative in the clinic.

Efficient expression from one CMV enhancer controlling two core promoters.

The human CMV promoter/enhancer is one of the strongest promoters for recombinant protein expression in mammalian cells, making the promoter very popular for production of recombinant antibodies. We used an antibody vector design where the antibody heavy and light chain genes were transcribed from a promoter complex consisting of two promoters arranged divergently with the 5' ends of the promoters in close proximity. However, when two identical CMV promoters constituted this promoter complex, the antibody expression observed was lower than expected based on the strength of the individual promoters. To optimize expression we prepared truncated promoter complexes where only one CMV enhancer controlled the initiation of transcription from two divergent minimal CMV core promoters. Antibody expression from the truncated promoter complexes was analyzed both when transiently transfected and upon stable site-specific integration into a CHO DG44 derived cell line. The data showed that it was possible for one enhancer to drive the expression of two core promoters. However, efficient expression from both divergent core promoters was seen only when the unique region upstream of the CMV enhancer was removed. Notably, a 12-fold increase in expression was found from the best of the truncated promoter complexes after stable site-specific integration when compared to the full-length double CMV promoter complex.

Single-batch production of recombinant human polyclonal antibodies.

We have previously described the development and implementation of a strategy for production of recombinant polyclonal antibodies (rpAb) in single batches employing CHO cells generated by site-specific integration, the Sympress I technology. The Sympress I technology is implemented at industrial scale, supporting a phase II clinical development program. Production of recombinant proteins by site-specific integration, which is based on incorporation of a single copy of the gene of interest, makes the Sympress I technology best suited to support niche indications. To improve titers while maintaining a cost-efficient, highly reproducible single-batch manufacturing mode, we have evaluated a number of different approaches. The most successful results were obtained using random integration in a new producer cell termed ECHO, a CHO DG44 cell derivative engineered for improved productivity at Symphogen. This new expression process is termed the Sympress II technology. Here we describe proof-of-principle data demonstrating the feasibility of the Sympress II technology for single-batch rpAb manufacturing using two model systems each composed of six target-specific antibodies. The compositional stability and the batch-to-batch reproducibility of rpAb produced by the ECHO cells were at least as good as observed previously using site-specific integration technology. Furthermore, the new process had a significant titer increase.

Identification of the site of human mannan-binding lectin involved in the interaction with its partner serine proteases: the essential role of Lys55.

Mannan-binding lectin (MBL) is an oligomeric lectin that binds neutral carbohydrates on pathogens, forms complexes with MBL-associated serine proteases (MASP)-1, -2, and -3 and 19-kDa MBL-associated protein (MAp19), and triggers the complement lectin pathway through activation of MASP-2. To identify the MASP binding site(s) of human MBL, point mutants targeting residues C-terminal to the hinge region were produced and tested for their interaction with the MASPs and MAp19 using surface plasmon resonance and functional assays. Mutation Lys(55)Ala abolished interaction with the MASPs and MAp19 and prevented formation of functional MBL-MASP-2 complexes. Mutations Lys(55)Gln and Lys(55)Glu abolished binding to MASP-1 and -3 and strongly inhibited interaction with MAp19. Conversely, mutation Lys(55)Arg abolished interaction with MASP-2 and MAp19, but only weakened interaction with MASP-1 and -3. Mutation Arg(47)Glu inhibited interaction with MAp19 and decreased the ability of MBL to trigger the lectin pathway. Mutant Arg(47)Lys showed no interaction with the MASPs or MAp19, likely resulting from a defect in oligomerization. In contrast, mutation Arg(47)Ala had no impact on the interaction with the MASPs and MAp19, nor on the ability of MBL to trigger the lectin pathway. Mutation Pro(53)Ala only had a slight effect on the interaction with MASP-1 and -3, whereas mutations at residues Leu(49) and Leu(56) were ineffective. In conclusion, the MASP binding site of MBL involves a sequence stretch centered on residue Lys(55), which may form an ionic bond representing the major component of the MBL-MASP interaction. The binding sites for MASP-2/MAp19 and MASP-1/3 have common features but are not strictly identical.

Characterization of the oligomer structure of recombinant human mannan-binding lectin.

Mannan-binding lectin (MBL) belongs to a family of proteins called the collectins, which show large differences in their ultrastructures. These differences are believed to be determined by different N-terminal disulfide-bonding patterns. So far only the bonding pattern of two of the simple forms (recombinant rat MBL-C and bovine CL-43) have been determined. Recombinant MBL expressed in human cells was purified, and the structure of the N-terminal region was determined. Preliminary results on human plasma-derived MBL revealed high similarity to the recombinant protein. Here we report the structure of the N-terminal part of recombinant human MBL and present a model to explain the oligomerization pattern. Using a strategy of consecutive enzymatic digestions and matrix-assisted laser desorption ionization mass spectrometry, we succeeded in identifying a number of disulfide-linked peptides from the N-terminal cysteine-rich region. Based on these building blocks, we propose a model that can explain the various oligomeric forms found in purified MBL preparations. Furthermore, the model was challenged by the production of cysteine to serine mutants of the three N-terminally situated cysteines. The oligomerization patterns of these mutants support the proposed model. The model indicates that the polypeptide dimer is the basic unit in the oligomerization.