Retrospective assessment of clonal origin of cell lines.

Cell lines used for the manufacture of recombinant proteins are expected to arise from a single cell as a control strategy to limit variability and ensure consistent protein production. Health authorities require a minimum of two rounds of limiting dilution cloning or its equivalent to meet the requirement of single cell origin. However, many legacy cell lines may not have been generated with process meeting this criteria potentially impeding the path to commercialization. A general monoclonality assessment strategy was developed based on using the site of plasmid integration for a cell's identity. By comparing the identities of subclones from a master cell bank (MCB) to each other and that of the MCB, a probability of monoclonality was established. Two technologies were used for cell identity, Southern blot and a PCR assay based on plasmid-genome junction sequences identified by splinkerette PCR. Southern blot analysis revealed that subclones may have banding patterns that differ from each other and yet indicate monoclonal origin. Splinkerette PCR identifies cellular sequence flanking the point(s) of plasmid integration. The two assays together provide complimentary data for cell identity that enables proper monoclonality assessment and establishes that the three legacy cell lines investigated are all of clonal origin.

Metabolomic and quality data for early and late passages of an antibody-producing industrial CHO cell line.

In this article, we provide four data sets for an industrial Chinese Hamster Ovary (CHO) cell line producing antibodies during a 14-day bioreactor run. This cell line was selected for further evaluation because of its significant titer loss as the cells were passaged over time. Four conditions that differed in cell bank ages were run for this dataset. Specifically, cells were passaged to passage 12, 21, 25, and 37 and then used in this experiment. Once the run commenced the following datasets were gathered: 1). Glycosylation data for each reactor 2). Size Exclusion Chromatography (SEC) data for the antibodies produced which allowed for the identification of high and low molecular weight species in the samples (N-Glycan and SEC data was taken on day 14 only). 3/4). Metabolites levels measured using Nuclear Magnetic Resonance (NMR) and liquid chromatography-mass spectroscopy (LC-MS) for all reactors over the time course of days 1, 4, 6, 8, 12, and 14. We also provide a graph of the glutamine levels for cells of different ages as an example of the utility of the data. These metabolomics data provide relative amounts for 36 metabolites (NMR) and 109 metabolites (LC-MS) over the 14-day time course. These data were collected in connection with a co-submitted paper [1].

Implementation of Plate Imaging for Demonstration of Monoclonality in Biologics Manufacturing Development.

Monoclonality of mammalian cell lines used for production of biologics is a regulatory expectation and one of the attributes assessed as part of a larger process to ensure consistent quality of the biologic. Historically, monoclonality has been demonstrated through statistics generated from limiting dilution cloning or through verified flow cytometry methods. A variety of new technologies are now on the market with the potential to offer more efficient and robust approaches to generating and documenting a clonal cell line.Here we present an industry perspective on approaches for the application of imaging and integration of that information into a regulatory submission to support a monoclonality claim. These approaches represent the views of a consortium of companies within the BioPhorum Development Group and include case studies utilising imaging technology that apply scientifically sound approaches and efforts in demonstrating monoclonality. By highlighting both the utility of these alternative approaches and the advantages they bring over the traditional methods, as well as their adoption by industry leaders, we hope to encourage acceptance of their use within the biologics cell line development space and provide guidance for regulatory submission using these alternative approaches.LAY ABSTRACT: In the manufacture of biologics produced in mammalian cells, one recommendation by regulatory agencies to help ensure product consistency, safety, and efficacy is to produce the material from a monoclonal cell line derived from a single, progenitor cell. The process by which monoclonality is assured can be supplemented with single-well plate images of the progenitor cell. Here we highlight the utility of that imaging technology, describe approaches to verify the validity of those images, and discuss how to analyze that information to support a biologic filing application. This approach serves as an industry perspective to increased regulatory interest within the scope of monoclonality for mammalian cell culture-derived biologics.

Codon-Directed Determination of the Biological Causes of Sequence Variants in Therapeutic Proteins.

Recombinant monoclonal antibodies (mAbs) manufactured from immortalized mammalian cell lines are becoming increasingly important as therapies. Ensuring the quality of expressed proteins is critical when developing manufacturing processes. Protein sequence variants (PSVs) are a type of product-related variant in which errors in the protein sequence are present. Detecting PSVs and determining their origins, either by DNA mutation or mRNA mistranslation, is critical. Mutations cannot be remediated without developing new clones, which can be costly and time-consuming. In contrast, mistranslation can usually be mitigated by optimizing cell culture conditions. In this work, we first developed a new method to detect low-abundance PSVs with improved sensitivity. Then, a statistical metric was proposed to determine whether the observed PSVs originate from mutation or mistranslation by characterizing the distribution of PSVs. This method was applied to the evaluation of 50 clones from five mAbs programs, allowing for identification of five mutation and 139 mistranslation PSVs. The presence of even a few mutations demonstrates the necessity of clone screening during process development.

CHO cells knocked out for TSC2 display an improved productivity of antibodies under fed batch conditions.

The kinase mTOR operates in two cellular complexes, mTORC1 and mTORC2. mTORC1 adjusts metabolic activity according to external growth conditions and nutrients availability. When conditions are prosperous, mTOR facilitates protein and lipid biosyntheses and inhibits autophagy, while under metabolic constraints, however, its attenuation induces a catabolic program, energy preservation and autophagy. CHO is a key cell line for manufacturing of biologics owing to its remarkable ability to grow to high densities and maintain protein production and secretion for extended times. While high mTOR activity has been associated with high productivity in CHO cells, its inhibition by rapamycin has also been documented to augment productivity via promotion of viability. Here using CRISPR/Cas9 editing we engineered CHO cells to enforce high mTORC1 activity by knocking-out TSC2, a major mTOR inhibitory protein, or PTEN, a phosphatase that attenuates the PI3K/AKT/mTOR pathway. Only TSC2-deleted cells exhibited a constitutive activation of mTORC1 under fed batch conditions. Cells grew larger in size, synthesized more proteins and displayed an over twofold elevation in their specific productivity. While peak viable cell density was compromised, overall titers increased to an extent dependent upon the parental clone. Our data underscore manipulation of TSC as a strategy to improve performance of CHO cell in bioreactors. Biotechnol. Bioeng. 2016;113: 1942-1952. © 2016 Wiley Periodicals, Inc.

Identification of a suppressor mutation that improves the yields of hexon-modified adenovirus vectors.

We have generated hexon-modified adenovirus serotype 5 (Ad5) vectors that are not neutralized by Ad5-specific neutralizing antibodies in mice. These vectors are attractive for the advancement of vaccine products because of their potential for inducing robust antigen-specific immune responses in people with prior exposure to Ad5. However, hexon-modified Ad5 vectors displayed an approximate 10-fold growth defect in complementing cells, making potential vaccine costs unacceptably high. Replacing hypervariable regions (HVRs) 1, 2, 4, and 5 with the equivalent HVRs from Ad43 was sufficient to avoid Ad5 preexisting immunity and retain full vaccine potential. However, the resulting vector displayed the same growth defect as the hexon-modified vector carrying all 9 HVRs from Ad43. The growth defect is likely due to a defect in capsid assembly, since DNA replication and late protein accumulation were normal in these vectors. We determined that the hexon-modified vectors have a 32°C cold-sensitive phenotype and selected revertants that restored vector productivity. Genome sequencing identified a single base change resulting in a threonine-to-methionine amino acid substitution at the position equivalent to residue 342 of the wild-type protein. This mutation has a suppressor phenotype (SP), since cloning it into our Ad5 vector containing all nine hypervariable regions from Ad43, Ad5.H(43m-43), increased yields over the version without the SP mutation. This growth improvement was also shown for an Ad5-based hexon-modified vector that carried the hexon hypervariable regions of Ad48, indicating that the SP mutation may have broad applicability for improving the productivity of different hexon-modified vectors.

Characterization of human adenovirus 35 and derivation of complex vectors.

BACKGROUND: Replication-deficient recombinant adenoviral vectors based on human serotype 35 (Ad35) are desirable due to the relatively low prevalence of neutralizing antibodies in the human population. The structure of the viral genome and life cycle of Ad35 differs from the better characterized Ad5 and these differences require differences in the strategies for the generation of vectors for gene delivery. RESULTS: Sequences essential for E1 and E4 function were identified and removed and the effects of the deletions on viral gene transcription were determined. In addition, the non-essential E3 region was deleted from rAd35 vectors and a sequence was found that did not have an effect on viability but reduced viral fitness. The packaging capacity of rAd35 was dependent on pIX and vectors were generated with stable genome sizes of up to 104% of the wild type genome size. These data were used to make an E1-, E3-, E4-deleted rAd35 vector. This rAd35 vector with multiple gene deletions has the advantages of multiple blocks to viral replication (i.e., E1 and E4 deletions) and a transgene packaging capacity of 7.6 Kb, comparable to rAd5 vectors. CONCLUSIONS: The results reported here allow the generation of larger capacity rAd35 vectors and will guide the derivation of adenovirus vectors from other serotypes.

Repeat administration of proteins to the eye with a single intraocular injection of an adenovirus vector.

Delivery of therapeutic proteins, such as antiangiogenic proteins, to the eye is a demonstrated method for the control of age-related macular degeneration (AMD). However, one of the key limitations is the requirement for frequent and repeated intraocular injections. In this article, we demonstrate that repeated protein production in the eye can be stimulated from the cytomegalovirus (CMV) promoter without repeat intraocular injections using a small molecule, all-trans retinoic acid (ATRA). ATRA by systemic delivery can stimulate protein production multiple times in the eye. Administration of ATRA resulted in stimulation of gene expression to relevant levels that block abnormal blood vessel growth in an experimental animal model for AMD. These data support the principles of this technological discovery to therapeutic applications for chronic ocular diseases.

Rescue and production of vaccine and therapeutic adenovirus vectors expressing inhibitory transgenes.

Expression of certain transgenes from an adenovirus vector can be deleterious to its own replication. This can result in the inhibition of virus rescue, reduced viral yields, or, in the worst case, make it impossible to construct a vector expressing the inhibiting transgene product. A gene regulation system based on the tet operon was used to allow the rescue and efficient growth of adenovectors that express transgenes to high levels. A key advantage to this system is that repression of transgene expression is mediated by the packaging cell line, thus, expression of regulatory products from the adenovector are not required. This provides a simple, broadly applicable system wherein transgene repression is constitutive during vector rescue and growth and there is no effect on adenovector-mediated expression of gene products in transduced cells. Several high-level expression vectors based on first- and second-generation adenovectors were rescued and produced to high titer that otherwise could not be grown. Yields of adenovectors expressing inhibitory transgene products were increased, and the overgrowth of cultures by adenovectors with nonfunctional expression cassettes was prevented. The gene regulation system is a significant advancement for the development of adenovirus vectors for vaccine and other gene transfer applications.

Repeated administration of adenovector in the eye results in efficient gene delivery.

PURPOSE: To determine whether repeat administration of an adenovector (Ad) into the eye results in efficient gene delivery and to test whether transgenes can be expressed from an adenovector expression system in the presence of preexisting, neutralizing anti-Ad antibodies. METHODS: To assess the efficiency of repeated gene delivery of an adenovector expression system, C57Bl/6 mice received one, two, or three injections (intravitreal [IVT] or periocular [PO]) of AdNull.11D (empty cassette) at 2-week intervals, followed by a single AdLuciferase (AdL.11D) IVT or PO injection. Mice were killed approximately 24 hours after AdL.11D injection and the eyes were enucleated and stored until assayed. Serum samples were also analyzed to determine whether repeated IVT or PO injections lead to induction of neutralizing antibodies directed against an adenovector delivery system. To determine whether preexisting neutralizing anti-Ad antibodies would block transgene expression, mice were preimmunized with one, two, or three intramuscular (IM) injection(s) of AdNull.11D (1 x 10(9) particle units [pu]). Fourteen days later, when systemic anti-Ad antibody titers were expected to exist, mice were given a single AdL.11D injection (IVT or PO) and killed, and the eyes and serum collected. RESULTS: These studies show that multiple injections at 2-week intervals with adenovectors (IM, IVT, or PO) did not prevent transgene expression in the eye. Moreover, measurement of neutralizing anti-Ad antibody titers revealed that measurable anti-Ad antibody titers in mice did not ablate transgene expression. CONCLUSIONS: These studies suggest that transgene expression after repeated adenovector administration into the eye is feasible and repeated injections, whether given IVT or PO, do not lead to an immediate increase in neutralizing anti-Ad antibody titers. Moreover, preimmunization of mice by systemic exposure to adenovector, does not block transgene expression in the eye. These studies indicate that repeat administration of adenovectors (IVT and PO) into the eye can be considered in designing future clinical trials and that the pre-existence of neutralizing anti-Ad antibodies probably does not mitigate activity.

Adenovirus vector library: an approach to the discovery of gene and protein function.

A method was developed to generate a complex cDNA expression library within an adenovirus type 5 (Ad5)-based vector backbone, termed AdLibrary. Construction of the AdLibrary entailed the conversion of an Ad5 genome-containing cosmid to infectious virus particles. The Ad5 genome was modified by replacing the E1A and E1B genes with a Rous sarcoma virus-driven expression cassette. Conversion was accomplished by liberating the viral genome by restriction enzyme digestion and transfection in HEK 293 cells, which support the growth of E1A/E1B-deficient virus. A test AdLibrary demonstrated the possibility of converting and identifying a marker gene present at a frequency of 1/10(5) in the cosmid library. To demonstrate the utility of this technology, an AdLibrary was used to isolate a viral gene by its biological function. Virus growth was selected for with an AdLibrary on A549 cells, which do not complement for E1A/E1B function. The AdLibrary was generated with cDNAs derived from HeLa cells productively infected with Ad5. A cDNA corresponding to Ad5 E1A 13S was selected and isolated from the AdLibrary using this strategy. Since multiple genes are assayed simultaneously, this technology should expedite the discovery of genes affecting defined biological activities. This AdLibrary approach provides an opportunity to exploit the efficient gene delivery capabilities of adenovirus vectors for the rapid discovery of gene and protein function.

Regression of ocular neovascularization in response to increased expression of pigment epithelium-derived factor.

PURPOSE: Several pharmacologic treatments have been shown to reduce ocular neovascularization when administered before the onset of angiogenic stimuli, but none have been shown to cause regression of already established ocular neovascularization. In this study, the authors tested the effect of adenoviral vectored pigment epithelium-derived factor (PEDF) gene transfer on established neovascularization in transgenic mice with expression of vascular endothelial growth factor (VEGF) in photoreceptors (rho/VEGF mice) and in a model of choroidal neovascularization. METHODS: Two weeks after the onset of VEGF transgene expression in rho/VEGF mice or 2 weeks after laser-induced rupture of Bruch's membrane in wild-type mice, subgroups of mice were killed, and the baseline amount of neovascularization was measured by image analysis. The remainder of the mice received an intravitreous or subretinal injection of adenoviral vector containing a PEDF expression construct (AdPEDF.11) or control vector (AdNull.11). RESULTS: Seven days after injection in rho/VEGF mice or 10 days after injection in the choroidal neovascularization model, the amount of neovascularization in AdPEDF.11-injected eyes was significantly less than the baseline level, indicating that regression of neovascularization had occurred. There was TUNEL staining within choroidal neovascular lesions in eyes injected with AdPEDF.11. Eyes given a subretinal injection of AdNull.11 had TUNEL-positive cells in the retina, but none within areas of choroidal neovascularization. CONCLUSIONS: These data indicate that increased expression of PEDF causes regression of ocular neovascularization by promoting apoptosis of cells within neovascular lesions and possibly represents a new treatment paradigm for patients with established ocular neovascularization.

Intraocular adenoviral vector-mediated gene transfer in proliferative retinopathies.

PURPOSE: The purpose of this study was to compare levels and patterns of expression of reporter genes achieved with an E1-deleted and partially E3-deleted type 5 adenoviral (Ad) vector after intravitreous or subretinal injections, or after intravitreous injections in mouse eyes with proliferative retinopathies. METHODS: Ad vectors containing reporter gene constructs were injected into the vitreous cavity or subretinal space of wild-type mice or mice with proliferative retinopathies, and quantitative comparisons were made of expression of transgenes. RESULTS: In normal eyes, peak Ad-mediated expression of luciferase, driven by a cytomegalovirus (CMV) promoter, occurred after injection of 10(7) to 10(8) viral particles and was 10 times greater after subretinal injections than after intravitreous injections. Intravitreous injections of Ad containing beta-galactosidase (LacZ) expression constructs (AdLacZ.10) resulted in strong expression of LacZ in epithelial cells of the iris and ciliary body and focal expression in the retina. Subretinal injections of AdLacZ.10 resulted in strong expression in RPE cells. Expression of LacZ after intravitreous injection of AdLacZ.10 was significantly greater in mice with two types of proliferative retinopathy (ischemic retinopathy or transgenic mice with retina-specific expression of platelet-derived growth factor (PDGF)-BB or PDGF-AB) than littermate control animals. Cells within epiretinal membranes and activated Müller cells were preferentially transduced in eyes with proliferative retinopathy. CONCLUSIONS: These data suggest that although higher intraocular expression levels can be achieved after subretinal injection of adenoviral vectors, intravitreous injections provide good transduction of cells lining the vitreous cavity. Compared with normal eyes, eyes with proliferative retinopathy showed increased transduction, which occurred preferentially in cells participating in the disease process. Intravitreous injection of adenoviral vectors containing appropriate expression constructs may provide a good strategy for acute treatment of proliferative retinopathies, such as diabetic retinopathy and proliferative vitreoretinopathy.

Rapid construction of adenoviral vectors by lambda phage genetics.

Continued improvements of adenoviral vectors require the investigation of novel genome configurations. Since adenovirus can be generated directly by transfecting packaging cell lines with viral genomes isolated from plasmid DNA, it is possible to separate genome construction from virus production. In this way failure to generate a virus is not associated with an inability to generate the desired genome. We have developed a novel lambda-based system that allows rapid modification of the viral genome by double homologous recombination in Escherichia coli. The recombination reaction and newly generated genome may reside in a recombination-deficient bacterial host for enhanced plasmid stability. Furthermore, the process is independent of any restriction endonucleases. The strategy relies on four main steps: (i) homologous recombination between an adenovirus cosmid and a donor plasmid (the donor plasmid carries the desired modification[s] and flanking regions of homology to direct its recombination into the viral genome); (ii) in vivo packaging of the recombinant adenoviral cosmids during a productive lambda infection; (iii) transducing a recombination-deficient E. coli lambda lysogen with the generated lysate (the lysogen inhibits the helper phage used to package the recombinant andenoviral cosmid from productively infecting and destroying the host bacteria); (iv) effectively selecting for the desired double-recombinant cosmid. Approximately 10,000 double-recombinant cosmids are recovered per reaction with essentially all of them being the correct double-recombinant molecule. This system was used to generate quickly and efficiently adenoviral genomes deficient in the E1/E3 and E1/E3/E4 regions. The basis of this technology allows any region of the viral genome to be readily modified for investigation of novel configurations.

Encainide for refractory supraventricular tachycardia in children.

Fifteen children, aged 5 days to 19 years (mean 4.7 years), with medically refractory supraventricular tachycardia were given oral encainide. In 10 of 15 children with "incessant" tachycardia (greater than 10% of the day), encainide alone controlled supraventricular tachycardia in 5 children; in combination with other antiarrhythmic agents, it partially controlled supraventricular tachycardia in 4 and was ineffective in 1. In 5 children with accessory atrioventricular connections, encainide eliminated supraventricular tachycardia in 3 and was ineffective in 2. Therapeutic encainide dosages ranged from 60 to 120 mg/m2/day (mean 90) (2.0 to 5.7 mg/kg/day). Encainide caused prolongation of the PR interval by 35%, RP interval by 17%, QRS interval by 44% and corrected QT interval by 10%. In 5 children with depressed left ventricular function administration of encainide, by controlling the arrhythmia, increased echocardiographic left ventricular shortening fraction from a mean of 24% to a mean of 36%. Three patients developed excessive QRS aberrancy, which was associated with wide QRS tachycardia in 2. No adverse reactions were noted in the absence of QRS aberration. Side effects were minor and noted in only 1 of 9 patients continuing to take the drug in 9 months of follow-up. Encainide was effective, or partially effective, in the control of resistant or incessant supraventricular tachycardia in 80% of children treated. Encainide allowed rapid resolution of arrhythmia-induced cardiomyopathy by controlling chronic supraventricular tachycardia.