Development Of Stable Cell Lines Research Articles

Establishing Primary and Stable Cell Lines from Frozen Wing Biopsies for Cellular, Physiological, and Genetic Studies in Bats.

Bats stand out among mammalian species for their exceptional traits, including the capacity to navigate through flight and echolocation, conserve energy through torpor/hibernation, harbor a multitude of viruses, exhibit resistance to disease, survive harsh environmental conditions, and demonstrate exceptional longevity compared to other mammals of similar size. In vivo studies of bats are challenging for several reasons, such as difficulty in locating and capturing them in their natural environments, limited accessibility, low sample size, environmental variation, long lifespans, slow reproductive rates, zoonotic disease risks, species protection, and ethical concerns. Thus, establishing alternative laboratory models is crucial for investigating the diverse physiological adaptations observed in bats. Obtaining quality cells from tissues is a critical first step for successful primary cell derivation. However, it is often impractical to collect fresh tissue and process the samples immediately for cell culture due to the resources required for isolating and expanding cells. As a result, frozen tissue is typically the starting resource for bat primary cell derivation, but cells in frozen tissue are usually damaged and have low integrity and viability. Isolating primary cells from frozen tissues thus poses a significant challenge. Herein, we present a successfully developed protocol for isolating primary dermal fibroblasts from frozen bat wing biopsies. This protocol marks a significant milestone, as this is the first protocol specifically focused on fibroblast isolation from bat frozen tissue. We also describe methods for primary cell characterization, genetic manipulation of primary cells through lentivirus transduction, and the development of stable cell lines. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Bat wing biopsy collection and preservation Support Protocol 1: Blood collection from bat venipuncture Basic Protocol 2: Isolation of primary fibroblasts from adult bat frozen wing biopsy Support Protocol 2: Primary fibroblast culture and subculture Support Protocol 3: Determination of growth curve and doubling time Support Protocol 4: Cell banking and thawing of primary fibroblasts Basic Protocol 3: Lentiviral transduction of bat primary fibroblasts Basic Protocol 4: Bat stable fibroblast cell line development Support Protocol 5: Bat fibroblast validation by immunofluorescence staining Basic Protocol 5: Chromosome counting.

Context-dependent genomic locus effects on antibody production in recombinant Chinese hamster ovary cells generated through random integration

High-yield production of therapeutic protein using Chinese hamster ovary (CHO) cells requires stable cell line development (CLD). CLD typically uses random integration of transgenes; however, this results in clonal variation and subsequent laborious clone screening. Therefore, site-specific integration of a protein expression cassette into a desired chromosomal locus showing high transcriptional activity and stability, referred to as a hot spot, is emerging. Although positional effects are important for therapeutic protein expression, the sequence-specific mechanisms by which hotspots work are not well understood. In this study, we performed whole-genome sequencing (WGS) to locate randomly inserted vectors in the genome of recombinant CHO cells expressing high levels of monoclonal antibodies (mAbs) and experimentally validated these locations and vector compositions. The integration site was characterized by active histone marks and potential enhancer activities, and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) mediated indel mutations in the region upstream of the integration site led to a significant reduction in specific antibody productivity by up to 30%. Notably, the integration site and its core region did not function equivalently outside the native genomic context, showing a minimal effect on the increase in exogenous protein expression in the host cell line. We also observed a superior production capacity of the mAb expressing cell line compared to that of the host cell line. Collectively, this study demonstrates that developing recombinant CHO cell lines to produce therapeutic proteins at high levels requires a balance of factors including transgene configuration, genomic locus landscape, and host cell properties.

Development of a novel tyrosine-based selection system for generation of recombinant Chinese hamster ovary cells

Efficiently expanding Chinese hamster ovary (CHO) cells, which serve as the primary host cells for recombinant protein production, have gained increasing industrial significance. A significant hurdle in stable cell line development is the low efficiency of the target gene integrated into the host genome, implying the necessity for an effective screening and selection procedure to separate these stable cells. In this study, the genes of phenylalanine hydroxylase (PAH) and pterin 4 alpha carbinolamine dehydratase 1 (PCBD1), which are key enzymes in the tyrosine synthesis pathway, were utilized as selection markers and transduced into host cells together with the target genes. This research investigated the enrichment effect of this system and advanced further in understanding its benefits for cell line development and rCHO cell culture. A novel tyrosine-based selection system that only used PCBD1 as a selection marker was designed to promote the enrichment effect. Post 9 days of starvation, positive transductants in the cell pool approached 100%. Applied the novel tyrosine-based selection system, rCHO cells expressing E2 protein were generated and named CHO TS cells. It could continue to grow, and the yield of E2 achieved 95.95mg/L in a tyrosine-free and chemically-defined (CD) medium. Herein, we introduced an alternative to antibiotic-based selections for the establishment of CHO cell lines and provided useful insights for the design and development of CD medium.

A NOVEL B7H3/NKP30 BISPECIFIC NK CELL ENGAGER FOR CANCER IMMUNOTHERAPY

Abstract Background and Significance Biological therapies harnessing the adaptive immune system have achieved a great success, given the clinical efficacy of CAR-T therapies and CD3 based T cell engagers in treating hematologic tumors, and the application of T cell immune checkpoint inhibitors in various indications. However, the challenges still exist due to the limitation of CAR-T therapies and T cell engagers in treating solid tumors. Side effects such as cytokine release storm and neurotoxicity remain a concern in the selection of CAR-T therapies and CD3-based T cell engagers as therapeutics. For PD1/L1-based immunotherapy, the relatively low objective response rate (ORR), short progression-free survival (PFS) of patients, and drug resistance and recurrence especially for solid malignancy, are the major clinical challenges. In contrast, innate effector cells such as NK cells and macrophages are naturally existing in human body as front line to defeat general pathogens and cancers，which will have a better safety profile. Also, NK cell activation is not limited by the antigen presentation of the MHC on the cell surface which makes it has broader anti-tumor effects. However, the innate effector cell based therapy is facing the limitation of low cell number, poor in vitro activation, and short effective duration in vivo. SunHo has generated a B7H3/NKp30 NK cell engager bispecific antibody which can directly activate and enrich NK cells to the TME when used alone or enhance the efficacy when used in combination NK cell therapy. NKp30 is a potent NK cell activation receptor with wide and persistent expression compared with other NK markers. B7H3 as a tumor associate antigen is also widely expressed on many kinds of tumor cells with great potential for multiple indications, especially for solid tumors. Methods Three anti-NKp30 VHHs were identified from Alpaca immunized library. The VHHs were fused to the N-terminus or C-terminus of an anti-B7H3 mAb heavy chain with different IgG isotypes (IgG1 or IgG4). The binding activity to NK cells was evaluated by FACS. IFNγ level was detected in the NK cell activation assay. The NK cell mediated target cell killing was evaluated using either NK92MI-cd16a or Primary NK cells as effector cells. To evaluate the non-specific self-killing of NK cell without target cell, the B7H3/NKp30 candidates were incubated with NK92MI-cd16a and cell lysis percentage were calculated. NSG mice bearing Hs.746T tumors were used to evaluate the in vivo efficacy of B7H3/NKp30 candidates. Results The B7H3/NKp30 candidates showed good binding activity and better activation to NK cells compared with benchmarks. Notably, the B7H3/NKp30 candidate IAN0982-VHH25 with NKp30 fused to the C-terminal with an IgG1 isotype showed lowest risk of non-specific NK cell killing. In the in vivo study, B7H3/NKp30 candidates in combination with 1x106 NK cells showed excellent anti-tumor activity with TGI over 95%. And we didn’t observe any significant change in body weight of the animals demonstrating good safety profile of the candidates. Moreover, even with lower NK cell amount (5x105), IAN0982-VHH25 showed great anti-tumor efficacy. We have also generated three mini pools of stably transfected cells for IAN0982-VHH25 for the commercial stable cell line development. Conclusion IAN0982-VHH25 is a first-in-class B7H3/NKp30 bispecific NK cell engager for cancer immunotherapy with excellent NK cell activation and in vivo anti-tumor efficacy, and also low risk of non-specific NK cell killing. It is expected to lift the limitations of current adaptive and innate effector cells-based therapies.

A splinkerette PCR-based genome walking technique for the identification of transgene integration sites in CHO cells.

Identification of recombinant gene integrations sites in the Chinese hamster ovary (CHO) cell genome is increasingly important to assure monoclonality. While next-generation sequencing (NGS) is commonly used for the gene integration site analysis, it is a time-consuming and costly technique as it analyzes the entire genome. Hence, simple, easy, and inexpensive methods to analyze transgene insertion sites are necessary. To selectively capture the integration site of transgene in the CHO genome, we applied splinkerette-PCR (spPCR). SpPCR is an adaptor ligation-based method using splinkerette adaptors that have a stable hairpin loop. Restriction enzymes with high frequencies in the CHO genome were chosen using a Python script and used for the in vitro spPCR assay development. After testing on two CHO housekeeping genes with known loci, the spPCR-based genome walking technique was successfully applied to recombinant CHO cells to identify the transgene integration site. Finally, the comparison with NGS methods exhibited that the time and cost required for the analysis can be substantially reduced. Taken together, the established technique would aid the stable cell line development process by providing a rapid and cost-effective method for transgene integration site analysis.

Optimization of recombinant antibody production based on the vector design and the level of metabolites for generation of Ig- producing stable cell lines

BackgroundThe biopharmaceutical industry is significantly growing worldwide, and the Chinese hamster ovary (CHO) cells are used as a main expression host for the production of recombinant monoclonal antibodies. Various metabolic engineering approaches have been investigated to generate cell lines with improved metabolic characteristics for increasing longevity and mAb production. A novel cell culture method based on the 2-stage selection makes it possible to develop a stable cell line with high-quality mAb production. ResultsWe have constructed several design options of mammalian expression vectors for the high production of recombinant human IgG antibodies. Versions for bipromoter and bicistronic expression plasmids different in promoter orientation and cistron arrangements were generated.The aim of the work presented here was to assess a high-throughput mAb production system that integrates the advantages of high-efficiency cloning and stable cell clones to stage strategy selection reducing the time and effort required to express therapeutic monoclonal mAbs.Development of a stable cell line using bicistronic construct with EMCV IRES-long link gave an advantage in high mAb expression and long-term stability. Two-stage selection strategies allowed the elimination of low-producer clones by using metabolic level intensity to estimate the IgG production in the early steps of selection. The practical application of the new method allows to reduce time and costs during stable cell line development.

Programmable RNA-Guided Large DNA Transgenesis by CRISPR/Cas9 and Site-Specific Integrase Bxb1.

The inability to insert large DNA constructs into the genome efficiently and precisely is a key challenge in genomic engineering. Random transgenesis, which is widely used, lacks precision, and comes with a slew of drawbacks. Lentiviral and adeno-associated viral methods are plagued by, respectively, DNA toxicity and a payload capacity of less than 5 kb. Homology-directed repair (HDR) techniques based on CRISPR-Cas9 can be effective, but only in the 1–5 kb range. In addition, long homology arms—DNA sequences that permit construct insertion—of lengths ranging from 0.5 to 5 kb are required by currently known HDR-based techniques. A potential new method that uses Cas9-guided transposases to insert DNA structures up to 10 kb in length works well in bacteria, but only in bacteria. Surmounting these roadblocks, a new toolkit has recently been developed that combines RNA-guided Cas9 and the site-specific integrase Bxb1 to integrate DNA constructs ranging in length from 5 to 43 kb into mouse zygotes with germline transmission and into human cells. This ground-breaking toolkit will give researchers a valuable resource for developing novel, urgently needed mouse and human induced pluripotent stem cell (hiPSC) models of cancer and other genetic diseases, as well as therapeutic gene integration and biopharmaceutical applications, such as the development of stable cell lines to produce therapeutic protein products.

Codon and signal peptide optimization for therapeutic antibody production from Chinese hamster ovary (CHO) cell

• A scheme for the selection of optimized codon algorithm was suggested. • A scheme for the selection of optimized SP combination was suggested. • Codon optimization index (CAI) is an important parameter. • Different Abs require different combinations of SP for optimal production. • Transient expression experiments can facilitate stable cell line development.

Transient light-activated gene expression in Chinese hamster ovary cells

BackgroundChinese hamster ovary (CHO) cells are widely used for industrial production of biopharmaceuticals. Many genetic, chemical, and environmental approaches have been developed to modulate cellular pathways to improve titers. However, these methods are often irreversible or have off-target effects. Development of techniques which are precise, tunable, and reversible will facilitate temporal regulation of target pathways to maximize titers. In this study, we investigate the use of optogenetics in CHO cells. The light-activated CRISPR-dCas9 effector (LACE) system was first transiently transfected to express eGFP in a light-inducible manner. Then, a stable system was tested using lentiviral transduction.ResultsTransient transfections resulted in increasing eGFP expression as a function of LED intensity, and activation for 48 h yielded up to 4-fold increased eGFP expression compared to cells kept in the dark. Fluorescence decreased once the LACE system was deactivated, and a protein half-life of 14.9 h was calculated, which is in agreement with values reported in the literature. In cells stably expressing the LACE system, eGFP expression was confirmed, but there was no significant increase in expression following light activation.ConclusionsTaken together, these results suggest that optogenetics can regulate CHO cell cultures, but development of stable cell lines requires optimized expression levels of the LACE components to maintain high dynamic range.

Abstract 563: A biparatopic agonist antibody for OX40 that exhibits superior activity without secondary crosslinking

Abstract The development of agonistic antibodies that activate T-cell co-stimulatory pathways represents a therapeutic strategy with significant clinical potential. However, challenges remain for the translation from in vitro efficacy to clinical success. OX40 and other tumor necrosis factor receptor (TNFR) superfamily members are notorious for requiring high-order receptor clustering in order to achieve full activity. For monoclonal antibodies, this high-order clustering is generally achieved through secondary cross-linking strategies. In vivo, this secondary cross-linking is often supplied through internal immune effector cells via Fc engagement. Bispecific and biparatopic antibodies represent an emerging class of drug molecules that enable unique mechanisms of action relative to their monoclonal counterparts. Here, we describe the use of our bispecific platform for the generation of large panels of biparatopic antibodies which enabled high-throughput screening for the discovery of an array of OX40 agonistic molecules. We have optimized these multivalent antibodies that exceed the potency of the OX40 ligand in NFkB activation without the need for secondary cross-linking. These agonist antibodies have additionally been characterized using primary T cell assays to monitor the kinetics of growth proliferation and cytokine secretion, outperforming cross-linked antibodies currently being tested in clinical trials. In co-culture systems, these agonist antibodies were effective in inhibiting immuno-suppressive properties of Tregs. Our lead OX40 agonist antibody has been optimized for activity and developability and has entered stable cell line development to further support pre-clinical activities. Citation Format: Bonnie Hammer, Mandar Bawadekar, Matthew Bissen, John Painter, Lauren Lehmann, Francis Qufei Li, Lucas Bailey, Bryan Glaser, Roland Green. A biparatopic agonist antibody for OX40 that exhibits superior activity without secondary crosslinking [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 563.

Highly sensitive detection of mutations in CHO cell recombinant DNA using multi-parallel single molecule real-time DNA sequencing.

High-fidelity replication of biologic-encoding recombinant DNA sequences by engineered mammalian cell cultures is an essential pre-requisite for the development of stable cell lines for the production of biotherapeutics. However, immortalized mammalian cells characteristically exhibit an increased point mutation frequency compared to mammalian cells in vivo, both across their genomes and at specific loci (hotspots). Thus unforeseen mutations in recombinant DNA sequences can arise and be maintained within producer cell populations. These may affect both the stability of recombinant gene expression and give rise to protein sequence variants with variable bioactivity and immunogenicity. Rigorous quantitative assessment of recombinant DNA integrity should therefore form part of the cell line development process and be an essential quality assurance metric for instances where synthetic/multi-component assemblies are utilized to engineer mammalian cells, such as the assessment of recombinant DNA fidelity or the mutability of single-site integration target loci. Based on Pacific Biosciences (Menlo Park, CA) single molecule real-time (SMRT™) circular consensus sequencing (CCS) technology we developed a rDNA sequence analysis tool to process the multi-parallel sequencing of ∼40,000 single recombinant DNA molecules. After statistical filtering of raw sequencing data, we show that this analytical method is capable of detecting single point mutations in rDNA to a minimum single mutation frequency of 0.0042% (<1/24,000 bases). Using a stable CHO transfectant pool harboring a randomly integrated 5 kB plasmid construct encoding GFP we found that 28% of recombinant plasmid copies contained at least one low frequency (<0.3%) point mutation. These mutations were predominantly found in GC base pairs (85%) and that there was no positional bias in mutation across the plasmid sequence. There was no discernable difference between the mutation frequencies of coding and non-coding DNA. The putative ratio of non-synonymous and synonymous changes within the open reading frames (ORFs) in the plasmid sequence indicates that natural selection does not impact upon the prevalence of these mutations. Here we have demonstrated the abundance of mutations that fall outside of the reported range of detection of next generation sequencing (NGS) and second generation sequencing (SGS) platforms, providing a methodology capable of being utilized in cell line development platforms to identify the fidelity of recombinant genes throughout the production process.

Strategic deployment of CHO expression platforms to deliver Pfizer's Monoclonal Antibody Portfolio.

Development of stable cell lines for expression of large-molecule therapeutics represents a significant portion of the time and effort required to advance a molecule to enabling regulatory toxicology studies and clinical evaluation. Our development strategy employs two different approaches for cell line development based on the needs of a particular project: a random integration approach for projects where high-level expression is critical, and a site-specific integration approach for projects in which speed and reduced employee time spend is a necessity. Here we describe both our random integration and site-specific integration platforms and their applications in support of monoclonal antibody development and production. We also compare product quality attributes of monoclonal antibodies produced with a nonclonal cell pool or clonal cell lines derived from the two platforms. Our data suggests that material source (pools vs. clones) does not significantly alter the examined product quality attributes. Our current practice is to leverage this observation with our site-specific integration platform, where material generated from cell pools is used for an early molecular assessment of a given candidate to make informed decisions around development strategy. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1463-1467, 2017.

A high‐yielding CHO transient system: Coexpression of genes encoding EBNA‐1 and GS enhances transient protein expression

An efficient rapid protein expression system is crucial to support early drug development. Transient gene expression is an effective route, and to facilitate the use of the same host cells as for subsequent stable cell line development, we have created a high-yielding Chinese hamster ovary (CHO) transient expression system. Suspension-adapted CHO-K1 host cells were engineered to express the gene encoding Epstein-Barr virus (EBV) nuclear antigen-1 (EBNA-1) with and without the coexpression of the gene for glutamine synthetase (GS). Analysis of the transfectants indicated that coexpression of EBNA-1 and GS enhanced transient expression of a recombinant antibody from a plasmid carrying an OriP DNA element compared to EBNA-1-only transfectants. This was confirmed with the retransfection of an EBNA-1-only cell line with a GS gene. The retransfected cell lines showed an increase in transient expression when compared with that of the EBNA-1-only parent. The transient expression process for the best CHO transient cell line was further developed to enhance protein expression and improve scalability by optimizing the transfection conditions and the cell culture process. This resulted in a scalable CHO transient expression system that is capable of expressing 2 g/L of recombinant proteins such as antibodies. This system can now rapidly provide gram amounts of recombinant antibody to supply preclinical development studies that has comparable product quality to antibody produced from a stably transfected CHO cell line.

Chinese hamster ovary K1 host cell enables stable cell line development for antibody molecules which are difficult to express in DUXB11‐derived dihydrofolate reductase deficient host cell

Therapeutic monoclonal antibodies (mAb) are often produced in Chinese hamster ovary (CHO) cells. Three commonly used CHO host cells for generating stable cell lines to produce therapeutic proteins are dihydrofolate reductase (DHFR) positive CHOK1, DHFR-deficient DG44, and DUXB11-based DHFR deficient CHO. Current Genentech commercial full-length antibody products have all been produced in the DUXB11-derived DHFR-deficient CHO host. However, it has been challenging to develop stable cell lines producing an appreciable amount of antibody proteins in the DUXB11-derived DHFR-deficient CHO host for some antibody molecules and the CHOK1 host has been explored as an alternative approach. In this work, stable cell lines were developed for three antibody molecules in both DUXB11-based and CHOK1 hosts. Results have shown that the best CHOK1 clones produce about 1 g/l for an antibody mAb1 and about 4 g/l for an antibody mAb2 in 14-day fed batch cultures in shake flasks. In contrast, the DUXB11-based host produced ∼0.1 g/l for both antibodies in the same 14-day fed batch shake flask production experiments. For an antibody mAb3, both CHOK1 and DUXB11 host cells can generate stable cell lines with the best clone in each host producing ∼2.5 g/l. Additionally, studies have shown that the CHOK1 host cell has a larger endoplasmic reticulum and higher mitochondrial mass.

Perspectives in Anticancer Plant Research

Pharmacognosy, the science of medicinal plants, was born in the nineteenth century from rich traditions of plant remedy use in folk medicine and the historical clinical experience of officinal medicine. Numerous plant species have been reputed by ethnomedicines from around the globe to possess antitumor properties, alleviate symptoms of disease and improve conditions of cancer patients. Systematic study of medicinal plant anticancer properties began in the second half of the twentieth century with the development of stable cell lines. In the 1950th and 1960th, extensive screening of mytotoxic and antiproliferative properties was performed on a multitude of plant compounds in search of agents that could efficiently suppress cell growth. Mitotoxic alkaloids vinblastine and vincristine, and several other plant compounds identified in these studies were introduced into clinical practice for the treatment of haematological and solid tumors. However, the overall outcome of this massive screening was largely disappointing. Only a fraction of ‘anticancer’ plants displayed high antiproliferative activity on tumor cells in culture, while activities of the vast majority of plant metabolites turned out to be low, as compared to those of chemically synthesized drugs.

An efficient method for stable transfection of mouse myogenic C2C12 cell line using a nonviral transfection approach

To describe in detail the use of the T-REx^{TM} System (a tetracycline-regulated mammalian expression system; Invitrogen) for rapid, easy, and effective generation of stable C2C12 cell clones. Tetracycline-inducible stable cell lines are useful tools to study the function of various genes in different types of mammalian cells. However, the generation of stable cell lines is a time-consuming, technically difficult, and expensive process. Materials and methods: Generation of a stable C2C12 cell line was performed by stable transfection of the cells with the pcDNA6/TR plasmid vector, which contains the tetracycline repressor and the blasticidin drug resistance genes. The establishment of the stable cell line and the efficiency of the tetracycline-inducible gene expression system were shown with the transient transfection of reporter plasmid pTO-EGFP and observation of the GFP expression by fluorescence microscopy. Results: We established a stable T-REx^{TM} C2C12 cell line that can be used for muscle research studies by transfecting the gene of interest with a second plasmid. Conclusion: The T-REx^{TM} system is useful in the development of stable cell lines for protein production or regulation of gene expression.

Enhanced CHO Cell-Based Transient Gene Expression with the Epi-CHO Expression System

Transient gene expression systems in mammalian cells continue to grow in popularity due to their capacity to produce significant amounts of recombinant protein in a rapid and scalable manner, without the lengthy time periods and resources required for stable cell line development. Traditionally, production of recombinant monoclonal antibodies for pre-clinical assessment by transient expression in CHO cells has been hampered by low titers. In this report, we demonstrate transient monoclonal antibody titers of 140mg/l with CHO cells using the episomal-based transient expression system, Epi-CHO. Such titers were achieved by implementing an optimized transfection protocol incorporating mild-hypothermia and through screening of a variety of chemically defined and serum-free media for their ability to support elevated and prolonged viable cell densities post-transfection, and in turn, improve recombinant protein yields. Further evidence supporting Epi-CHO's capacity to enhance transgene expression is provided, where we demonstrate higher transgene mRNA and protein levels of two monoclonal antibodies and a destabilized enhanced green fluorescent protein with Epi-CHO compared to cell lines deficient in plasmid DNA replication and/or retention post-transfection. The results demonstrate the Epi-CHO system's capacity for the rapid production of CHO cell-derived recombinant monoclonal antibodies in serum-free conditions.

RD114 envelope proteins provide an effective and versatile approach to pseudotype lentiviral vectors

Lentiviral vectors derived from the HIV-1 genome offer great promise for gene therapy due to their ability to transduce non-dividing cells and sustain long-term expression of transgenes. The majority of current lentiviral vectors are pseudotyped with the vesicular stomatitis viral envelope (VSV-G). VSV-G equips lentiviral vectors with a broad host cell tropism and increased stability. Increased particle stability enables viral supernatants to be concentrated by high-speed centrifugation to enhance their infectivity. Despite its efficacy, VSV-G is cytotoxic - a feature that prohibits the development of stable cell lines that constitutively express this envelope. Therefore, non-toxic envelope proteins are being investigated. RD114 is an attractive alternative because it also provides increased particle stability and its receptor is widely expressed on hematopoietic stem cells (HSCs). In this study, the packaging efficiency of three envelope proteins, RD114, RDpro and VSV-G, were evaluated with two lentiviral vectors (TRIP GFP and HPV-402). RDpro is an RD114-HIV chimera designed to pseudotype lentiviral vectors more efficiently. In transient systems, VSV-G generated titers of 10(8) and 10(7) viral particles/mL for TRIP GFP and HPV-402. RDpro possessed titers of 10(7) and 10(6), while RD114 titers were one log lower for each vector. Despite having relatively lower titers, RD114 proteins are less toxic; this was demonstrated in the extension of transient transfection reactions from 48 to 96 h. VSV-G transfections are generally limited to 48 h. In regard to gene therapy applications, we show that RDpro supernatants efficiently transduce peripheral blood HSCs. The versatility of RD114 envelopes was again demonstrated by using a 'mixed' expression system; composed of stably expressed RD114 envelope proteins to pseudotype lentiviral vectors generated in trans (titer range 10(3)-10(5)). Our data show that RD114 envelope proteins are effective and versatile constructs that could prove to be essential components of therapeutic lentiviral gene transfer systems.

A rapid screening method for cell lines producing singly-tagged recombinant proteins using the “TARGET tag” system

Recombinant production of extracellular glycoproteins in stable mammalian cell lines is an ideal technique for obtaining a large quantity of high-quality proteins. In most cases, however, current methodologies do not allow for sufficiently rapid cell line development and protein purification. Here, we describe a 21-residue peptide tag (designated as TARGET tag) and its use for rapid stable cell line development and purification. The ability of the anti-tag antibody P20.1 to withstand repetitive regeneration cycles has enabled the development of a sensitive surface plasmon resonance-based screening format that requires only 20 microl of cell culture supernatants. Direct and semi-quantitative screening at the 96-well culture scale eliminated the need for a second screening, re-cloning, or sorting, thereby minimizing culture pre-production time. Using this system, "high producer" cell lines were established in less than a month, and milligram quantities of target proteins could be purified with a standardized protocol.

Mechanisms of unintended amino acid sequence changes in recombinant monoclonal antibodies expressed in Chinese Hamster Ovary (CHO) cells

An amino acid sequence variant is defined as an unintended amino acid sequence change and contributes to product heterogeneity. Recombinant monoclonal antibodies (MAbs) are primarily expressed from Chinese Hamster Ovary (CHO) cells using stably transfected production cell lines. Selections and amplifications with reagents such as methotrexate (MTX) are often required to achieve high producing stable cell lines. Since MTX is often used to generate high producing cell lines, we investigated the genomic mutation rates of the hypoxanthine-guanine phosphoribosyltransferase (HGPRT or HPRT) gene using a 6-thioguanine (6-TG) assay under various concentrations of MTX selection in CHO cells. Our results show that the 6-TG resistance increased as the MTX concentration increased during stable cell line development. We also investigated low levels of sequence variants observed in two stable cell lines expressing different MAbs. Our data show that the replacement of serine at position 167 by arginine (S167R) in the light chain of antibody A (MAb-A) was due to a genomic nucleotide sequence change whereas the replacement of serine at position 63 by asparagine (S63N) in the heavy chain of antibody B (MAb-B) was likely due to translational misincorporation. This mistranslation is codon specific since S63N mistranslation is not detectable when the S63 AGC codon is changed to a TCC or TCT codon. Our results demonstrate that both a genomic nucleotide change and translational misincorporation can lead to low levels of sequence variants and mistranslation of serine to asparagine can be eliminated by substituting the TCC or TCT codon for the S63 AGC codon without impacting antibody productivity.