Transposases have transformed genetic engineering, yet functional systems remain scarce. In response, an unknown transposase system from Acyrthosiphon pisum was identified by metagenomic screening. Through systematic optimization, we enhanced nuclear localization, transposon architecture, and created a hyperactive transposase variant to boost efficiency. Intriguingly, the combined application of the newly discovered transposase with inverted terminal repeat sequences from a related pea aphid species, Aphis craccivora, further enhanced transposition activity, resulting in the first chimeric transposase system reported so far. We investigated the genomic integration events following transposition in mammalian cells to understand the underlying mechanisms and optimize the efficiency of transgene integration. This optimized system can expedite the generation of recombinant protein-producing Chinese Hamster Ovary (CHO) cell lines, even surpassing the hyperactive piggyBac system with regard to cell-specific productivity. These findings introduce a significant addition to the field of semi-targeted transgene integration technologies, offering substantial potential for enhancing biologics manufacturing.

The use of metabolic selection markers has advanced stable cell line generation, increasing productivity while simultaneously eliminating the need for antibiotic reagents. This study explores the potential of bacterially derived glutamine synthetases (GS) as a novel generation of metabolic selection markers to further enhance CHO cell culture performance. GS-I proteins were extracted from the genomes of enterobacterial and actinomycetes species. Three of these enzymes demonstrated functionality when stably transfected into GS-deficient CHO cells, leading to a 3- to 4-fold increase in antibody titer compared to endogenous GS from Cricetulus griseus. This study indicates that the functionality of bacterial GS enzymes in mammalian cells is determined by solvent accessibility and the geometry of the catalytic binding pocket. Dysfunctional variants showed a less accessible bifunnel. Bacterial GS were evaluated for their bioprocess performance leading to superior stable pool and clone performance. Transcriptome analysis further revealed that regulatory cellular mechanisms were decoupled in a cross-species set-up, reinforcing the suitability of repurposing bacterial enzymes as selection markers in mammalian cell lines. By modulating the selection stringency, an increase in expression performance was achieved without impairing the bioprocess behavior or long-term cell line stability.

Cell line development represents a crucial step in the development process of a therapeutic glycoprotein. Chinese hamster ovary (CHO) cells are the most frequently employed mammalian host cell system for the industrial manufacturing of biologics. The predominant application of CHO cells for heterologous recombinant protein expression lies in the relative simplicity of stably introducing ectopic DNA into the CHO host cell genome. Since CHO cells were first used as expression host for the industrial production of biologics in the late 1980s, stable genomic transgene integration has been achieved almost exclusively by random integration. Since then, random transgene integration had become the gold standard for generating stable CHO production cell lines due to a lack of viable alternatives. However, it was eventually demonstrated that this approach poses significant challenges on the cell line development process such as an increased risk of inducing cell line instability. In recent years, significant discoveries of new and highly potent (semi)-targeted transgene integration systems have paved the way for a technological revolution in the cell line development sector. These advanced methodologies comprise the application of transposase-, recombinase- or Cas9 nuclease-mediated site-specific genomic integration techniques, which enable a scarless transfer of the transgene expression cassette into transcriptionally active loci within the host cell genome. This review summarizes recent advancements in the field of transgene integration technologies for CHO cell line development and compare them to the established random integration approach. Moreover, advantages and limitations of (semi)-targeted integration techniques are discussed, and benefits and opportunities for the biopharmaceutical industry are outlined.

Monoclonal antibodies (mAb) and other biological drugs are affected by enzymatic polysorbate (PS) degradation that reduces product stability and jeopardizes the supply of innovative medicines. PS represents a critical surfactant stabilizing the active pharmaceutical ingredients, which are produced by recombinant Chinese hamster ovary (CHO) cell lines. While the list of potential PS-degrading CHO host cell proteins (HCPs) has grown over the years, tangible data on industrially relevant HCPs are still scarce. By means of a highly sensitive liquid chromatography-tandem mass spectrometry method, we investigated seven different mAb products, resulting in the identification of 12 potentially PS-degrading hydrolases, including the strongly PS-degrading lipoprotein lipase (LPL). Using an LPL knockout CHO host cell line, we were able to stably overexpress and purify the remaining candidate hydrolases through orthogonal affinity chromatography methods, enabling their detailed functional characterization. Applying a PS degradation assay, we found nine mostly secreted, PS-active hydrolases with varying hydrolytic activity. All active hydrolases showed a serine-histidine-aspartate/glutamate catalytical triad. Further, we subjected the active hydrolases to pH-screenings and revealed a diverse range of activity optima, which can facilitate the identification of residual hydrolases during bioprocess development. Ultimately, we compiled our dataset in a risk matrix identifying PAF-AH, LIPA, PPT1, and LPLA2 as highly critical hydrolases based on their cellular expression, detection in purified antibodies, active secretion, and PS degradation activity. With this work, we pave the way toward a comprehensive functional characterization of PS-degrading hydrolases and provide a basis for a future reduction of PS degradation in biopharmaceutical drug products.

Molecular biological methods have emerged as inevitable tools to accompany the process of cell line development for the generation of stable and highly productive manufacturing cell lines in the biopharmaceutical industry. PCR-based methods are especially useful for screening and characterization of cell lines due to their low cost, scalability, precision and propensity for multidimensional read-outs. In this study, the diverse applications of droplet digital PCR (ddPCR) as a molecular biological tool for cell line development are demonstrated. Specifically, it is shown that ddPCR can be used to enable precise, sensitive and reproducible absolute quantification of genomically integrated transgene copies during cell line development and cell bank characterization. Additionally, an amplitude multiplexing approach is applied to simultaneously run multiple assays on different genetic targets in a single reaction and advance clonal screening by measuring gene expression profiles to predict the assembly and homogeneity of difficult-to-express (DTE) proteins. The implementation of ddPCR-based assays during cell line development allows for early screening at a transcriptional level, particularly for complex, multidomain proteins, where balanced polypeptide chain ratios are of primary importance. Moreover, it is demonstrated that ddPCR-based genomic characterization improves the robustness, efficiency and comparability of absolute transgene copy number quantification, an essential genetic parameter that must be demonstrated to regulatory authorities during clinical trial and market authorization application submissions to support genetic stability and consistency of the selected cell substrate.

Despite advances in genetic characterization of Chinese hamster ovary (CHO) cell lines regarding identification of integration sites using next generation sequencing, e.g. targeted locus amplification sequencing (TLA-seq), the concatemer structure of the integrated vectors remains elusive. Here, the entire integration locus of two CHO manufacturing cell lines was reconstructed combining CRISPR/Cas9 target enrichment, nanopore sequencing and the Canu de novo assembly pipeline. An IgG producing CHO cell line integrated 3 vector copies, which were near full-length and contained all relevant vector elements such as transgenes and their promoters on each of the vector copies. In contrast, a second CHO cell line producing a bivalent bispecific antibody integrated 7 highly fragmented vector copies in different orientations leading to head-to-head and tail-to-tail fusions. The size of the vector fragments ranged from 3.0 to 11.4 kbp each carrying 1-3 transgenes. The breakpoints of the genome-vector and vector-vector junctions were validated using Sanger sequencing and Southern blotting. A compari-son to TLA-seq data confirmed the genomic breakpoints, but most of the breakpoints of the vector-vector fusions were missed by TLA-seq. For the first time, the complete transgene locus of CHO manufacturing cell lines could be deciphered. Strikingly, the application of the nanopore long-read sequencing technology led to novel insights into the complexity of genomic transgene integrations of CHO manufacturing cell lines generated via random integration.

A large number of companies observe polysorbate (PS) degradation and associated (sub-)visible particle formation in biological drug formulations, which compromise the stability of the drug product, ultimately posing a risk toward delivering innovative medicines to patients. The main culprits of PS degradation are hydrolytic host cell proteins (HCPs) originating from the production cell lines, which are mostly Chinese hamster ovary (CHO) cell derived. Here, a small portion of particularly difficult-to-remove HCPs-mainly lipases-cause hydrolytic cleavage of PS resulting in the accumulation of free fatty acid aggregates/particles. One possible mitigation strategy is the removal of such critical HCPs in the production cell line. Multigene regulation can be achieved via microRNAs (miRNAs) thereby serving as a smart tool to reduce the expression of different target genes using a single miRNA. To enable a tailored gene regulation of multiple specific target lipases self-designed and non-naturally occurring artificial miRNAs (amiRNA) can be designed. Based on micro-conserved regions in the mRNA sequence of two sets of target HCPs, we provide a proof-of-concept for a simultaneous multi-lipase knockdown in CHO cells using single amiRNAs. By this, we were not only able to reduce PS degradation but laid the foundation to expand this tool to other areas of cell line phenotype engineering.

Biotechnology and BioengineeringVolume 119, Issue 3 p. 671-675 ISSUE INFORMATIONFree Access Biotechnology and Bioengineering: Volume 119, Number 3, March 2022 First published: 07 February 2022 https://doi.org/10.1002/bit.27825AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Volume119, Issue3March 2022Pages 671-675 RelatedInformation

In recent years, acceleration of development timelines has become a major focus within the biopharmaceutical industry to bring innovative therapies faster to patients. However, in order to address a high unmet medical need even faster further acceleration potential has to be identified to transform "speed-to-clinic" concepts into "warp-speed" develop-ment programs. Recombinant Chinese hamster ovary (CHO) cell lines are the predominant expression system for monoclonal antibodies (mAbs) and are routinely generated by random transgene integration (RTI) of the genetic in-formation into the host cell genome. This process, however, exhibits considerable challenges such as the requirement for a time-consuming clone screening process to identify a suitable clonally derived manufacturing cell line. Hence, RTI represents an error prone and tedious method leading to long development timelines until availability of Good Manufacturing Practice (GMP)-grade drug substance (DS). Transposase-mediated semi-targeted transgene integration (STI) has been recently identified as a promising alternative to RTI as it allows for a more rapid generation of high-performing and stable production cell lines. In this report, we demonstrate how a STI technology was leveraged to develop a very robust DS manufacturing process based on a stable pool cell line at unprecedented pace. Application of the novel strategy resulted in the manufacturing of GMP-grade DS at 2,000 L scale in less than three months paving the way for a start of Phase I clinical trials only six months after transfection. Finally, using a clonally derived production cell line, which was established from the parental stable pool, we were able to successfully implement a process with an increased mAb titer of up to 5 g per liter at the envisioned commercial scale (12,000 L) within eight months.

Genetically modified CHO cell lines are traditionally used for the production of biopharmaceuticals. However, an in-depth molecular understanding of the mechanism and exact position of transgene integration into the genome of pharmaceutical manufacturing cell lines is still scarce. Next-generation sequencing (NGS) holds great promise for strongly facilitating the understanding of CHO cell factories, as it has matured to a powerful and affordable technology for cellular genotype analysis. Targeted Locus Amplification (TLA) combined with NGS allows for robust detection of genomic positions of transgene integration and structural genomic changes occurring upon stable integration of expression vectors. TLA was applied to generate comparative genomic fingerprints of several CHO production cell lines expressing different monoclonal antibodies. Moreover, high producers resulting from an additional round of transfection of an existing cell line (supertransfection) were analyzed to investigate the integrity and the number of integration sites. Our analyses enabled detailed genetic characterization of the integration regions with respect to the number of integrates and structural changes of the host cell's genome. Single integration sites per clone with concatenated transgene copies could be detected and were in some cases found to be associated with genomic rearrangements, deletions or translocations. Supertransfection resulted in an increase in titer associated with an additional integration site per clone. Based on the TLA fingerprints, CHO cell lines originating from the same mother clone could clearly be distinguished. Interestingly, two CHO cell lines originating from the same mother clone were shown to differ genetically and phenotypically despite their identical TLA fingerprints. Taken together, TLA provides an accurate genetic characterization with respect to transgene integration sites compared with conventional methods and represents a valuable tool for a comprehensive evaluation of CHO production clones early in cell line development.

Chinese hamster ovary (CHO) cells are known not to express appreciable levels of the sialic acid residue N-glycolylneuraminic acid (NGNA) on monoclonal antibodies. However, we actually have identified a recombinant CHO cell line expressing an IgG with unusually high levels of NGNA sialylation (>30%). Comprehensive multi-OMICs based experimental analyses unraveled the root cause of this atypical sialylation: (1) expression of the cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) gene was spontaneously switched on, (2) CMAH mRNA showed an anti-correlated expression to the newly discovered Cricetulus griseus (cgr) specific microRNA cgr-miR-111 and exhibits two putative miR-111 binding sites, (3) miR-111 expression depends on the transcription of its host gene SDK1, and (4) a single point mutation within the promoter region of the sidekick cell adhesion molecule 1 (SDK1) gene generated a binding site for the transcriptional repressor histone H4 transcription factor HINF-P. The resulting transcriptional repression of SDK1 led to a downregulation of its co-expressed miR-111 and hence to a spontaneous upregulation of CMAH expression finally increasing NGNA protein sialylation.

Biotechnology and BioengineeringVolume 117, Issue 1 p. 1-4 ISSUE INFORMATIONFree Access Biotechnology and Bioengineering: Volume 117, Number 1, January 2020 First published: 10 December 2019 https://doi.org/10.1002/bit.27027AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinked InRedditWechat Volume117, Issue1January 2020Pages 1-4 RelatedInformation

Although most therapeutic monoclonal antibodies (mAbs) can routinely be produced in the multigram per litre range, some mAb candidates turn out to be difficult-to-express (DTE). In addition, the class of more complex biological formats is permanently increasing and mammalian expression systems like Chinese hamster ovary (CHO) cell lines can show low performance. Hence, there is an urgent need to identify any rate limiting processing step during cellular synthesis. Therefore, we assessed the intracellular location of the DTE antibody mAb2 by fluorescence and electron microscopy (EM) and revealed an accumulation of the antibody, which led to an aberrant morphology of the endoplasmic reticulum (ER). Analysis of underlying cellular mechanisms revealed that neither aggregation nor antibody assembly, but folding represented the reason for hampered secretion. We identified that the disulfide bridge formation within the antibody light chain (LC) was impaired due to less recognition by protein disulfide isomerase (PDI). As a consequence, the DTE molecule was degraded intracellularly by the ubiquitin proteasome system via ER-associated degradation (ERAD). This study revealed that with the continuous emergence of DTE therapeutic protein candidates, special attention needs to be drawn to optimization processes to ensure manufacturability.

With the advance of complex biological formats such as bispecific antibodies or fusion proteins, mammalian expression systems often show low performance. Described determining factors may be accumulation or haltering of heterologous proteins within the different cellular compartments disturbing transport or secretion. In case of the investigated bispecific antibody (bsAb)-producing Chinese hamster ovary (CHO) cell line neither impaired transcription nor decreased translation processes were identified and thus satisfactorily explained its low production capacity. Hence, we established a streamlined confocal microscopy-based methodology for CHO production cells investigating the distribution of the recombinant protein within the respective organelles of the secretory pathway and visualised the structure of the endoplasmic reticulum (ER) to be affected pinpointing towards an intra-ER bottleneck putatively hampering or limiting efficient secretion. The ER displayed not only a heavily altered morphology in comparison to a high immunoglobulin G (IgG)-producing cell line with a possibly inflated or overloaded structure, but the recombinant protein was also completely absent in the Golgi apparatus. Notably, the results obtained using an automated microscopy approach suggest the possible application of this methodology in cell line development and engineering.

Protein aggregation remains a major area of focus in the production of monoclonal antibodies. Improving the intrinsic properties of antibodies can improve manufacturability, attrition rates, safety, formulation, titers, immunogenicity, and solubility. Here, we explore the potential of predicting and reducing the aggregation propensity of monoclonal antibodies, based on the identification of aggregation-prone regions and their contribution to the thermodynamic stability of the protein. Although aggregation-prone regions are thought to occur in the antigen binding region to drive hydrophobic binding with antigen, we were able to rationally design variants that display a marked decrease in aggregation propensity while retaining antigen binding through the introduction of artificial aggregation gatekeeper residues. The reduction in aggregation propensity was accompanied by an increase in expression titer, showing that reducing protein aggregation is beneficial throughout the development process. The data presented show that this approach can significantly reduce liabilities in novel therapeutic antibodies and proteins, leading to a more efficient path to clinical studies.

ABSTRACT In recent years, coherent with growing biologics portfolios also the number of complex and thus difficult‐to‐express (DTE) therapeutic proteins has increased considerably. DTE proteins challenge bioprocess development and can include various therapeutic protein formats such as monoclonal antibodies (mAbs), multi‐specific affinity scaffolds (e.g., bispecific antibodies), cytokines, or fusion proteins. Hence, the availability of robust and versatile Chinese hamster ovary (CHO) host cell factories is fundamental for high‐yielding bioprocesses. MicroRNAs (miRNAs) have emerged as potent cell engineering tools to improve process performance of CHO manufacturing cell lines. However, there has not been any report demonstrating the impact of beneficial miRNAs on industrial cell line development (CLD) yet. To address this question, we established novel CHO host cells constitutively expressing a pro‐productive miRNA: miR‐557. Novel host cells were tested in two independent CLD campaigns using two different mAb candidates including a normal as well as a DTE antibody. Presence of miR‐557 significantly enhanced each process step during CLD in a product independent manner. Stable expression of miR‐557 increased the probability to identify high‐producing cell clones. Furthermore, production cell lines derived from miR‐557 expressing host cells exhibited significantly increased final product yields in fed‐batch cultivation processes without compromising product quality. Strikingly, cells co‐expressing miR‐557 and a DTE antibody achieved a twofold increase in product titer compared to clones co‐expressing a negative control miRNA. Thus, host cell engineering using miRNAs represents a promising tool to overcome limitations in industrial CLD especially with regard to DTE proteins. Biotechnol. Bioeng. 2017;114: 1495–1510. © 2017 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.

The development of biopharmaceutical production cell lines typically starts with generation of heterogeneous populations of cells, from which then single cell clones are established. Several regulatory guidelines require that production cell lines are clonal, and the actual demonstration of clonality has been increasingly demanded by regulatory authorities over the last years. Here, the authors describe the relative contribution of flow cytometry mediated deposition of single cells in multiwell plates and subsequent imaging to assurance of clonality in a state of the art approach to single cell generation. Within the flow cytometry step, two unit operations are evaluated separately, doublet discrimination during event selection for deposition and droplet deposition accuracy. The imaging procedure is evaluated for the accuracy of detection of non-clonal populations. By employing mixing experiments of cell populations, the authors demonstrate that doublet discrimination is highly efficient, and that an appropriately set up flow cytometry system already can generate >99.5% true single cell clones. The efficiency of the described imaging process depends on several factors, reaching an optimal detection rate of non-clonal wells of about 99.8%. Our results demonstrate that one well characterized cloning step generate biopharmaceutical production cell lines with a probability of clonality of >99.99%.