Producer Cell Lines Research Articles

Characterization of the function of Adenovirus L4 gene products and their impact on AAV vector production

Efficient manufacturing of recombinant adenovirus-associated viral vectors is critical to the successful development of genomic medicines. We attempted to optimize AAV vector production in a producer cell line platform. In this system, helper functions required for AAV replication and production are provided via infection with a replication-competent wild-type Adenovirus. To evaluate strategies for reduction of replication and packaging of adenovirus as well as to understand the interplay of recombinant AAV and the helper virus during AAV vector production, wild-type adenovirus was compared to a mutant (Ad5ts149) containing a temperature-sensitive mutation in the DNA polymerase gene. Infection of a producer cell line with Ad5ts149 at the restrictive temperature reduced recombinant AAV titer and altered the pattern of AAV protein expression. Further investigation revealed that the adenoviral late L4-22K/33K gene products regulated both AAV rep/cap gene transcription and splicing of the rep/cap transcripts. Furthermore, the L4-33K gene products were found to impact AAV production in both the producer cell line and transient transfection platforms. Optimization of Adenovirus L4-22K/33K expression to facilitate efficient expression and splicing of AAV rep/cap transcripts therefore represents a unique opportunity to optimize AAV vector production.

Elevated endoplasmic reticulum pH is associated with high growth and bisAb aggregation in CHO cells.

Chinese hamster ovary (CHO) bioprocesses, the dominant platform for therapeutic protein production, are increasingly used to produce complex multispecific proteins. Product quantity and quality are affected by intracellular conditions, but these are challenging to measure and often overlooked during process optimization studies. pH is known to impact quality attributes like protein aggregation across upstream and downstream processes, yet the effects of intracellular pH on cell culture performance are largely unknown. Recently, advances in protein biosensors have enabled investigations of intracellular environments with high spatiotemporal resolution. In this study, we integrated a fluorescent pH-sensitive biosensor into a bispecifc (bisAb)-producing cell line to investigate changes in endoplasmic reticulum pH (pHER). We then investigated how changes in lactate metabolism impacted pHER, cellular redox, and product quality in fed-batch and perfusion bioreactors. Our data show pHER rapidly increased during exponential growth to a maximum of pH 7.7, followed by a sharp drop in the stationary phase in all perfusion and fed-batch conditions. pHER decline in the stationary phase was driven by an apparent loss of cellular pH regulation that occurred despite differences in redox profiles. Finally, we found protein aggregate levels correlated most closely with pHER which provides new insights into product aggregate formation in CHO processes. An improved understanding of the intracellular changes impacting bioprocesses can ultimately help guide media optimizations, improve bioprocess control strategies, or provide new targets for cell engineering.

Establishment and Characterization of a Stable Producer Cell Line Generation Platform for the Manufacturing of Clinical-Grade Lentiviral Vectors.

Background/Objectives: To date, nearly 300 lentiviral-based gene therapy clinical trials have been conducted, with eight therapies receiving regulatory approval for commercialization. These advances, along with the increased number of advanced-phase clinical trials, have prompted contract development and manufacturing organizations (CDMOs) to develop innovative strategies to address the growing demand for large-scale batches of lentiviral vectors (LVVs). Consequently, manufacturers have focused on optimizing processes under good manufacturing practices (GMPs) to improve cost-efficiency, increase process robustness, and ensure regulatory compliance. Nowadays, the LVV production process mainly relies on the transient transfection of four plasmids encoding for the lentiviral helper genes and the transgene. While this method is efficient at small scales and has also proven to be scalable, the industry is exploring alternative processes due to the high cost of GMP reagents, and the batch-to-batch variability predominantly attributed to the transfection step. Methods: Here, we report the development and implementation of a reliable and clinical-grade envisioned platform based on the generation of stable producer cell lines (SCLs) from an initial well-characterized lentiviral packaging cell line (PCL). Results: This platform enables the production of VSV-G-pseudotyped LVVs through a fully transfection-free manufacturing process. Our data demonstrate that the developed platform will facilitate successful technological transfer to large-scale LVV production for clinical application. Conclusions: With this simple and robust stable cell line generation strategy, we address key concerns associated with the costs and reproducibility of current manufacturing processes.

APOBEC3B expression in 293T viral producer cells drives mutations in chimeric antigen receptors and reduces CAR T cell efficacy

Chimeric antigen receptor (CAR) Tcells are a clinically approved therapy for blood cancers. To produce clinical-grade CAR Tcells, a retroviral or lentiviral vector is used to deliver the CAR and associated genes to patient Tcells. Apolipoprotein B editing enzyme, catalytic polypeptide 3 (APOBEC3) enzymes are known to be upregulated after transfection and retroviral infection and to deaminate cytidine to uracil in nucleic acids, resulting in cytidine-to-thymine mutations in DNA. Here, we hypothesized that APOBEC3 enzymes, induced during the production of CAR Tcells, impact the efficacy of the resulting CAR Tcells. We demonstrated that APOBEC3 family member APOBEC3B was upregulated at the RNA and protein levels after transfection of HEK293T cells with plasmids to make lentivirus, and that APOBEC3 signature mutations were present in the CAR construct. APOBEC3B overexpression in HEK293T cells led to further mutations in the resulting CAR Tcells, and significantly decreased CAR Tcell killing. APOBEC3B knockout in HEK293T cells led to reduced mutations in the CAR construct and significantly increased in CAR Tcell killing. These results suggest that generation of CAR-expressing viruses from producer cell lines deficient in genome-modifying proteins such as APOBEC3B could enhance the quality of CAR Tcell production.

Lentiviral vector packaging and producer cell lines yield titers equivalent to the industry-standard four-plasmid process

Lentiviral vector (LVV)-mediated cell and gene therapies have the potential to cure diseases that currently require lifelong intervention. However, the requirement for plasmid transfection hinders large-scale LVV manufacture. Moreover, large-scale plasmid production, testing and transfection all contribute to operational risk and the high cost associated with this therapeutic modality. Thus, we developed LVV packaging and producer cell lines, which reduce or eliminate the need for plasmid transfection during LVV manufacture. To develop a packaging cell line, lentiviral packaging genes were stably integrated by random integration of linearised plasmid DNA. Then, to develop EGFP- and anti-CD19 chimeric antigen receptor-encoding producer cell lines, transfer plasmids were integrated by transposase-mediated integration. Single cell isolation and testing were performed to isolate the top-performing clonal packaging and producer cell lines. Production of LVV that encode various cargo genes revealed consistency in the production performance of the packaging and producer cell lines compared to the industry-standard four-plasmid transfection method. By reducing or eliminating the requirement for plasmid transfection, while achieving production performance consistent with the current industry standard, the packaging and producer cell lines developed here can reduce costs and operational risks of LVV manufacture, thus increasing patient access to LVV-mediated cell and gene therapies.

Genomic barcoding for clonal diversity monitoring and control in cell-based complex antibody production

Engineered mammalian cells are key for biotechnology by enabling broad applications ranging from in vitro model systems to therapeutic biofactories. Engineered cell lines exist as a population containing sub-lineages of cell clones that exhibit substantial genetic and phenotypic heterogeneity. There is still a limited understanding of the source of this inter-clonal heterogeneity as well as its implications for biotechnological applications. Here, we developed a genomic barcoding strategy for a targeted integration (TI)-based CHO antibody producer cell line development process. This technology provided novel insights about clone diversity during stable cell line selection on pool level, enabled an imaging-independent monoclonality assessment after single cell cloning, and eventually improved hit-picking of antibody producer clones by monitoring of cellular lineages during the cell line development (CLD) process. Specifically, we observed that CHO producer pools generated by TI of two plasmids at a single genomic site displayed a low diversity (< 0.1% RMCE efficiency), which further depends on the expressed molecules, and underwent rapid population skewing towards dominant clones during routine cultivation. Clonal cell lines from one individual TI event demonstrated a significantly lower variance regarding production-relevant and phenotypic parameters as compared to cell lines from distinct TI events. This implies that the observed cellular diversity lies within pre-existing cell-intrinsic factors and that the majority of clonal variation did not develop during the CLD process, especially during single cell cloning. Using cellular barcodes as a proxy for cellular diversity, we improved our CLD screening workflow and enriched diversity of production-relevant parameters substantially. This work, by enabling clonal diversity monitoring and control, paves the way for an economically valuable and data-driven CLD process.

Quantitative proteomics reveals cellular responses to individual mAb expression and tunicamycin in CHO cells

Chinese hamster ovary (CHO) cells are popular in the pharmaceutical industry for their ability to produce high concentrations of antibodies and their resemblance to human cells in terms of protein glycosylation patterns. Current data indicate the relevance of CHO cells in the biopharmaceutical industry, with a high number of product commendations and a significant market share for monoclonal antibodies. To enhance the production capabilities of CHO cells, a deep understanding of their cellular and molecular composition is crucial. Genome sequencing and proteomic analysis have provided valuable insights into the impact of the bioprocessing conditions, productivity, and product quality. In our investigation, we conducted a comparative analysis of proteomic profiles in high and low monoclonal antibody–producing cell lines and studied the impact of tunicamycin (TM)-induced endoplasmic reticulum (ER) stress. We examined the expression levels of different proteins including unfolded protein response (UPR) target genes by using label-free quantification techniques for protein abundance. Our results show the upregulation of proteins associated with protein folding mechanisms in low producer vs. high producer cell line suggesting a form of ER stress related to specific protein production. Further, Hspa9 and Dnaja3 are notable candidates activated by the mitochondria UPR and play important roles in protein folding processes in mitochondria. We identified significant upregulation of Nedd8 and Lgmn proteins in similar levels which may contribute to UPR stress. Interestingly, the downregulation of Hspa5/Bip and Pdia4 in response to tunicamycin treatment suggests a low-level UPR activation.Key points• Proteome profiling of recombinant CHO cells under mild TM treatment.• Identified protein clusters are associated with the unfolded protein response (UPR).• The compared cell lines revealed noticeable disparities in protein expression levels.

Role of the CTCF Binding Site in Human T-Cell Leukemia Virus-1 Pathogenesis.

During HTLV-1 infection, the virus integrates into the host cell genome as a provirus with a single CCCTC binding protein (CTCF) binding site (vCTCF-BS), which acts as an insulator between transcriptionally active and inactive regions. Previous studies have shown that the vCTCF-BS is important for maintenance of chromatin structure, regulation of viral expression, and DNA and histone methylation. Here, we show that the vCTCF-BS also regulates viral infection and pathogenesis in vivo in a humanized (Hu) mouse model of adult T-cell leukemia/lymphoma. Three cell lines were used to initiate infection of the Hu-mice, i) HTLV-1-WT which carries an intact HTLV-1 provirus genome, ii) HTLV-1-CTCF, which contains a provirus with a mutated vCTCF-BS which abolishes CTCF binding, and a stop codon immediate upstream of the mutated vCTCF-BS which deletes the last 23 amino acids of p12, and iii) HTLV-1-p12stop that contains the intact vCTCF-BS, but retains the same stop codon in p12 as in the HTLV-1-CTCF cell line. Hu-mice were infected with mitomycin treated or irradiated HTLV-1 producing cell lines. There was a delay in pathogenicity when Hu-mice were infected with the CTCF virus compared to mice infected with either p12 stop or WT virus. Proviral load (PVL), spleen weights, and CD4 T cell counts were significantly lower in HTLV-1-CTCF infected mice compared to HTLV-1-p12stop infected mice. Furthermore, we found a direct correlation between the PVL in peripheral blood and death of HTLV-1-CTCF infected mice. In cell lines, we found that the vCTCF-BS regulates Tax expression in a time-dependent manner. The scRNAseq analysis of splenocytes from infected mice suggests that the vCTCF-BS plays an important role in activation and expansion of T lymphocytes in vivo. Overall, these findings indicate that the vCTCF-BS regulates Tax expression, proviral load, and HTLV pathogenicity in vivo.

Adeno-associated virus perfusion enhanced expression: A commercially scalable, high titer, high quality producer cell line process

With safety and efficacy demonstrated over hundreds of clinical trials in the last 30 years, along with at least six recent global marketing authorizations achieved since 2017, recombinant adeno-associated viruses (rAAVs) have been established asthe leading therapeutic gene transfer vector for rare, monogenic diseases. Significant advances in manufacturing technology have been made in the last few decades to address challenges with GMP production of rAAV products, although yield, cost, scalability, and quality remain a challenge. With transient transfection processes established as a manufacturing platform for multiple commercial AAV products, there remains significant yield, cost, robustness, and scalability constraints that need to be resolved to enable a reliable supply of rAAV products for global patient access. The development of stable producer cell lines for rAAV products has enabled scalability and, in some cases, improvements in productivity. Herein we describe a novel AAV perfusion-enhanced expression (APEX) process, resulting in higher maximum cell densities in the production bioreactor with a 3- to 6-fold increase in volumetric productivity. This process has been successfully demonstrated across multiple serotypes in large scale cell culture with titers approaching 1×1012 GC/mL. The APEX production platform marks a significant leap forward in the efficient and effective manufacturing of rAAV vector products.

Quasi-perfusion studies for intensified lentiviral vector production using a continuous stable producer cell line

Quasi-perfusion culture was employed to intensify lentiviral vector (LV) manufacturing using a continuous stable producer cell line in an 8-day process. Initial studies aimed to identify a scalable seeding density, with 3, 4, and 5 x 104 cells cm-2 providing similar specific productivities of infectious LV. Seeding at 3 x 104 cells cm-2 was selected, and the quasi-perfusion was modulated to minimise inhibitory metabolite accumulation and vector exposure at 37 °C. Similar specific productivities of infectious and physical LV were achieved at 1, 2, and 3 vessel volumes per day (VVD), with 1 VVD selected to minimise downstream processing volumes. The optimised process was scaled 50-fold to 1,264 cm2 flasks, achieving similar LV titres. However, scaling up beyond this to a 6,320 cm2 multilayer flask reduced titres, possibly from suboptimal gas exchange. Across three independent processes in 25 cm2 to 6,320 cm2 flasks, reproducibility was high with a coefficient of variation of (7.7 ± 2.9)% and (11.9 ± 3.0)% for infectious and physical LV titres, respectively. The optimised flask process was successfully transferred to the iCELLis™ Nano (Cytiva) fixed-bed bioreactor, with quasi-perfusion at 1 VVD yielding 1.62 x 108 TU.

Generation of stable suspension producer cell lines for serum-free lentivirus production.

The production of lentiviral vectors (LVs) pseudotyped with the vesicular stomatitis virus envelope glycoprotein (VSV-G) is limited by the associated cytotoxicity of the envelope and by the production methods used, such as transient transfection of adherent cell lines. In this study, we established stable suspension producer cell lines for scalable and serum-free LV production derived from two stable, inducible packaging cell lines, named GPRG and GPRTG. The established polyclonal producer cell lines produce self-inactivating (SIN) LVs carrying a WAS-T2A-GFP construct at an average infectious titer of up to4.64×107TU mL-1in a semi-perfusion process in a shake flask and can be generated in less than two months. The derived monoclonal cell lines are functionally stable in continuous culture and produce an average infectious titer of up to 9.38×107TU mL-1in a semi-perfusion shake flask process. The producer clones are able to maintain a productivity of>1×107TUmL-1day-1for up to 29 consecutive days in a non-optimized 5L stirred-tank bioreactor perfusion process, representing a major milestone in the field of LV manufacturing. As the producer cell lines are based on an inducible Tet-off expression system, the established process allows LV production in the absence of inducers such as antibiotics. The purified LVs efficiently transduce human CD34+ cells, reducing the LV quantities required for gene and cell therapy applications.

Harnessing producer cell lines and process intensification to address the AAV process productivity conundrum

Harnessing producer cell lines and process intensification to address the AAV process productivity conundrum

Abstract 2714: Developing an mRNA encoded therapeutic antibody platform to simplify manufacturing and reduce time to clinic

Abstract The manufacturing of therapeutic antibodies requires expensive production, often challenging purification, lengthy stability optimization and complex protein characterization that despite continual improvement, keeps the cost of treatment in the clinic high. Alternatively, leveraging gene-based approaches such as mRNA to express therapeutics circumvents many of the manufacturing challenges and instead rely on in situ production of antibodies within a patient, improving the developability and cost of sophisticated, disease-modifying antibodies. Crucially, recent work demonstrates that therapeutic antibodies translated in vivo from mRNA can be readily detected within hours following infusion into pre-clinical models and can persist up to several days or weeks. Peak levels of circulating mRNA-encoded antibodies are comparable to infused recombinant equivalents dosed to patients and have been shown to be within favorable therapeutic ranges in phase I trials. Here we outline an mRNA-LNP based platform to encode and deliver therapeutic antibodies in vivo that can overcome costly and challenging manufacturing and that demonstrates both robust PK/PD kinetics and potency. We describe how to effectively encode a standard-of-care anti-HER2 antibody - Trastuzumab - using LNP-encapsulated modified mRNA and validate using in vitro as well as in vivo efficacy models. Initial in vitro characterization of mRNA encoded antibodies demonstrates robust translation in producer cell lines, reproducible expression kinetics, retention of antigen specificity and HC:LC integrity when analyzed by ELISA and WB. To evaluate whether Trastuzumab-encoding mRNA-produced protein is detectable in vivo, antibody levels were measured in mouse serum after IV administration of a single dose of mRNA formulated LNPs. Peak plasma concentrations of mRNA-encoded trastuzumab were benchmarked against circulating recombinant Trastuzumab in parallel groups, and pharmacokinetics interrogated. Of note, the majority of mRNA encoded antibody was likely produced in the liver as shown by bioluminescence imaging. To demonstrate the efficacy of the platform, the potency of mRNA-encoded Trastuzumab isolated from producer cell lines will be assessed using in vitro cytotoxicity assays with human PBMCs and HER2+ tumor lines to dissect ADCC or direct tumor growth inhibition. Additionally, to determine whether mRNA-encoded Trastuzumab is able to retain anti-tumor activity in vivo, a mouse tumor xenograft model will be established and the mRNA antibody-dependent effects on tumor volume, growth and morbidity-free survival will be examined. Overall, the data demonstrate that mRNA-encoded therapeutic antibodies could provide an effective, alternate strategy for solid tumor treatment and may unlock a strategy to deliver lead biologics at reduced cost and with improved developability. Citation Format: Roxana Redis, Dan Rocca, Rachel Pooley, Matthew Benson, Namrata Jayanth, Eva Oswald, Alexander Hale, Louise Brackenbury, Justin Bryans. Developing an mRNA encoded therapeutic antibody platform to simplify manufacturing and reduce time to clinic [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2714.

Abstract 1483: Sugar analogs inhibit KSHV replication by blocking N-glycosylation and inducing the unfolded protein response

Abstract Background: Kaposi´s Sarcoma (KS) herpesvirus (KSHV) is one of the seven known human oncogenic viruses. KSHV infection precedes the development of KS, an AIDS-associated malignancy. While the incidence of AIDS-KS globally has seen a decline, this endothelial cancer is refractory to treatment. The standard of care for AIDS-KS has gone virtually unchanged for over 20 years and effective treatments against the oncovirus KSHV have yet to be developed. 2-DG (2-deoxy-D-glucose), 2-FDG (2-fluoro-deoxy-D-glucose), and2-DFM (2-deoxy-2-fluoro-D-mannose) are sugar analogs. 2-DG and 2-FDG are D-glucose analogs and strong glycolysis inhibitors. 2-DG is also a D-mannose analog so that can inhibit N-glycosylation. In this work we aimed to further characterize and compare the inhibition of the three drug analogs on KSHV replication and pathogenesis. Design/Methods: We determined the ability of each drug to inhibit KSHV reactivation and infection by flow cytometry using the KSHV producer cell line iSLK.219 Virus titers were measured by de novo infection of naïve AdHEK293 cells and quantifying the number of AdHEK293 infected cells. We further characterized sugar analog inhibition assessing viral glycoprotein expression by western blot and quantifying virion production by qPCR. To show that N-glycosylation impairment is the main contributor of virus replication inhibition, we carried out D-mannose rescue experiments and analyzed activation of the unfolded protein response (UPR). When UPR cannot be resolved, it leads to either apoptosis or autophagy. For this reason, we analyzed the cellular fate of reactivated sugar analog-treated cells by western blot using apoptotic and autophagic markers. Additionally, we calculated the cell death index using the IncuCyte® machine. Results: All three sugar analogs negatively affected KSHV replication and virion infectivity, but 2-DFM displayed the strongest inhibition. Viral glycoproteins K8.1 and gB were down-regulated and virion production was diminished especially with 2-FDG and 2-DFM sugar analogs. D-mannose could rescue viral glycoprotein expression and virion infectivity, but could not reverse the number of reactivated cells, indicating that N-glycosylation is the main target of the drugs. In line with this, all analogs triggered UPR and even overcame KSHV-induced suppression of the PERK pathway. Finally, 2-DG, 2-FDG, and 2-DFM played a protective role in reactivated cells, displaying the autophagic marker instead of the apoptotic signature observed in the untreated cells. Conclusion: Collectively, this work identifies the non-toxic inhibition of N-glycosylation by sugar analogs as an important viral target and reinforces the potential of 2-DG as an antiviral for KSHV and other enveloped viruses. We also found that 2-DFM is a more potent and target-oriented compound and a promising therapeutic antiviral agent against an oncogenic virus. Citation Format: Mariana Schlesinger, Christian McDonald, Anuj Ahuja, Carolina Alvez-Canete, Zelmira Nunez Del Prado, Julian Naipauer, Theodore Lampidis, Enrique Mesri, Noula Shembade. Sugar analogs inhibit KSHV replication by blocking N-glycosylation and inducing the unfolded protein response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1483.

Simplifying stable CHO cell line generation with high probability of monoclonality by using microfluidic dispensing as an alternative to fluorescence activated cell sorting.

Single cell cloning is a critical step for cell line development (CLD) for therapeutic protein production, with proof of monoclonality being compulsorily sought in regulatory filings. Among the different single cell deposition technologies, we found that fluorescence activated cell sorting (FACS) offers high probability of monoclonality and can allow selective enrichment of the producer cells. However, FACS instruments are expensive and resource-intensive, have a large footprint, require highly skilled operators and take hours for setup, thereby complicating the cell line generation process. With the aim of finding an easy-to-use alternative to FACS, we identified a flow cytometry-based microfluidic cell dispenser, which presents a single cell sorting solution for biopharmaceutical CLD. The microfluidic cell dispenser is small, budget-friendly, easy-to-use, requires lower-cost consumables, permits flow cytometry-enabled multiparametric target cell enrichment and offers fast and gentle single cell dispensing into multiwell plates. Following comprehensive evaluation, we found that single cell deposition by the microfluidic cell dispenser resulted in >99% probability of monoclonality for production cell lines. Moreover, the clonally derived producer cell lines generated from the microfluidic cell dispenser demonstrated comparable or improved growth profiles and production capability compared to the FACS derived cell lines. Taken together, microfluidic cell dispensing can serve as a cost-effective, efficient and convenient alternative to FACS, simplifying the biopharmaceutical CLD platform with significant reductions in both scientist time and running costs.

Tuning capsid formation dynamics in recombinant adeno-associated virus producing synthetic cell lines to enhance full particle productivity.

Recombinant adeno-associated virus (rAAV) is widely used as an in vivo delivery vector for gene therapy. It is used in a very large dose, and the large quantities required for broad applications present manufacturing challenges. We have developed a synthetic biology platform of constructing cell lines integrated with essential viral genes which can be induced to produce rAAV without plasmid transfection or virus transduction. Through iterative design-construct-characterization cycles, we have showcased the potential of this synthetic cell production system. Systems characterization of the dynamics of viral transcripts and proteins as well as virus assembly and packaging revealed that the expression level and balance of viral genome and capsid protein are keys to not only the productivity but also the full particle content, an important product quality attribute. Boosting cap gene expression by sequential transfection and integration of multiple copies of the cap gene elevated the rAAV titer to levels on a par with traditional plasmid transfection and virus infection. However, overexpression of the cap gene shifted the balance and kinetics of the genome and capsid. We independently tuned the dynamics of genome amplification and capsid protein synthesis by modulating the induction concentration as well as the time profile, and significantly enhanced full particle content while maintaining a high productivity. This strategy of constructing an inducible stable producer cell line is readily adaptable to rAAV vectors of different serotypes and payloads. It can greatly facilitate scalable production of gene therapy vectors.

A complete workflow for single cell mtDNAseq in CHO cells, from cell culture to bioinformatic analysis.

Chinese hamster ovary (CHO) cells have a long history in the biopharmaceutical industry and currently produce the vast majority of recombinant therapeutic proteins. A key step in controlling the process and product consistency is the development of a producer cell line derived from a single cell clone. However, it is recognized that genetic and phenotypic heterogeneity between individual cells in a clonal CHO population tends to arise over time. Previous bulk analysis of CHO cell populations revealed considerable variation within the mtDNA sequence (heteroplasmy), which could have implications for the performance of the cell line. By analyzing the heteroplasmy of single cells within the same population, this heterogeneity can be characterized with greater resolution. Such analysis may identify heterogeneity in the mitochondrial genome, which impacts the overall phenotypic performance of a producer cell population, and potentially reveal routes for genetic engineering. A critical first step is the development of robust experimental and computational methods to enable single cell mtDNA sequencing (termed scmtDNAseq). Here, we present a protocol from cell culture to bioinformatic analysis and provide preliminary evidence of significant mtDNA heteroplasmy across a small panel of single CHO cells.

Development of Stable Packaging and Producer Cell Lines for the Production of AAV Vectors.

Today, recombinant adeno-associated virus (rAAV) vectors represent the vector systems which are mostly used for in vivo gene therapy for the treatment of rare and less-rare diseases. Although most of the past developments have been performed by using a transfection-based method and more than half of the authorized rAAV-based treatments are based on transfection process, the tendency is towards the use of stable inducible packaging and producer cell lines because their use is much more straightforward and leads in parallel to reduction in the overall manufacturing costs. This article presents the development of HeLa cell-based packaging/producer cell lines up to their use for large-scale rAAV vector production, the more recent development of HEK293-based packaging and producer cell lines, as well as of packaging cell lines based on the use of Sf9 cells. The production features are presented in brief (where available), including vector titer, specific productivity, and full-to-empty particle ratio.

Continuous manufacturing of lentiviral vectors using a stable producer cell line in a fixed-bed bioreactor

Continuous manufacturing of lentiviral vectors (LVs) using stable producer cell lines could extend production periods, improve batch-to-batch reproducibility, and eliminate costly plasmid DNA and transfection reagents. A continuous process was established by expanding cells constitutively expressing third-generation LVs in the iCELLis™ Nano fixed-bed bioreactor. Fixed-bed bioreactors provide scalable expansion of adherent cells and enable a straightforward transition from traditional surface-based culture vessels. At 0.5 VVD (vessel volumes per day), the short half-life of LVs resulted in a low total infectious titre at 1.36 x 104 TU cm-2. Higher perfusion rates increased titres, peaking at 7.87 x 104 TU cm-2 at 1.5 VVD. The supernatant at 0.5 VVD had a physical-to-infectious particle ratio of 659, whereas this was 166 ± 15 at 1, 1.5, and 2 VVD. Reducing the pH from 7.20 to 6.85 at 1.5 VVD improved the total infectious yield to 9.10 x 104 TU cm-2. Three independent runs at 1.5 VVD and a culture pH of 6.85 showed low batch-to-batch variability, with a coefficient of variation of 6.4% and 10.0% for total infectious and physical LV yield, respectively. This study demonstrated the manufacture of high-quality LV supernatant using a stable producer cell line that does not require induction.

P134 Characterization for the similarity assessment between proposed biosimilar SB17 and ustekinumab reference product using state-of-the-art analytical method

Abstract Background SB17 is being developed as a proposed biosimilar to ustekinumab reference product (RP), a human monoclonal antibody (IgG1 kappa immunoglobulin) that binds to the common p40 subunit of cytokines interleukin (IL)-23 and IL-12. Binding to this subunit prevents interaction with their receptor, resulting in modulation the immune system responses which play a key role in the inflammatory disease. The objective of this study was to demonstrate structural, physicochemical, and biological similarity between ustekinumab RP and SB17 using various state-of-the-art analytical methods. Methods Comprehensive analytical characterization was conducted with side-by-side comparison of SB17 with the EU and US ustekinumab RP using various analytical methods. Comparisons included purity, product-related impurity, charge heterogeneity, primary structure, post-translational modification, higher order structure, quantity, Fab-related biological activities (potency and binding activity), and Fc-related biological activities. Results Based on the analytical similarity assessment, the structural, physicochemical, and biological characterization results demonstrated that SB17 is highly similar to the EU and US ustekinumab RP. In the structural aspects, it was confirmed that there is no clinically meaningful difference between post-translational modification profiles and higher order structures of SB17 compared to the ustekinumab RP. Product-related impurities in the form of aggregates were also confirmed to be similar. SB17 has a lower of acidic and basic peaks compared with ustekinumab RP, due to the difference in producing cell line. Ustekinumab RP is expressed in an Sp2/0 cell line, whereas SB17 is expressed in CHO cell line. However, the difference between SB17 and ustekinumab RP in the charge variants is not considered clinically meaningful since equivalent biological activity was observed. In case of mechanism of action (MoA)-related biological activities, SB17 is highly similar to the EU and US ustekinumab RP with respect to overall critical and non-critical quality attributes analyzed. Moreover, similarity or lack of activity in Fc-related biological activities was also confirmed. Based on these results, SB17 is expected to have highly similar safety and efficacy as compared to ustekinumab RP. Conclusion In summary, the overall analytical characterization and similarity assessment results show that SB17 is highly similar to the EU and US ustekinumab RP in terms of structural, physicochemical, biophysical, biological attributes.