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PubMedDate not supplied

A two-dimensional protein map of Chinese hamster ovary cells.

Chinese hamster ovary (CHO) cells are used extensively for the expression of biopharmaceutical protein products. As part of our effort to better understand CHO cell physiology and protein expression changes caused by modified culture conditions, we have begun to map CHO cell polypeptides. A parental cell line reference map was established using two-dimensional gel electrophoresis with immobilized pH gradients (pH 3-10) in the first dimension and a linear acrylamide gradient (9-18%T) in the second dimension. The map is composed of over 1000 silver-stained protein spots. Protein identification is proceeding using a combination of immunostaining, NH2-terminal sequencing, and mass spectrometric analyses. Among the proteins so far identified are glucose-regulated protein 78 (GRP78), protein disulfide isomerase (PDI), galectin-1, and several heat-shock proteins. The goal is to generate a database which emphasizes those proteins most relevant to the use of CHO cells as a host for recombinant protein expression.

Animals↗

PubMed1989

The United States experience with oral controlled-release morphine (MS Contin tablets). Parts I and II. Review of nine dose titration studies and clinical pharmacology of 15-mg, 30-mg, 60-mg, and 100-mg tablet strengths in normal subjects.

The results of nine US multicenter, sequential crossover, dose titration studies of controlled-release oral morphine (MS Contin 30 mg tablets [MSC], Purdue Frederick, Norwalk, CT) are reviewed in Part I. The studies demonstrated the prolonged analgesic efficacy of the preparation in the treatment of patients with moderate to severe cancer-related pain. Approximately 93% of the patients achieved satisfactory to excellent analgesia on a 12-hour regimen when appropriate dose titration was allowed. The remaining patients were successfully maintained on an 8-hour regimen. The preparation was well-tolerated and comparable in safety to immediate-release oral morphine. In global evaluations, MSC was judged to be significantly (P less than 0.05) more effective, and with significantly (P less than 0.05) fewer side effects than both the prestudy opioid analgesics and 4-hour immediate-release oral morphine. Patients had a broad range of morphine requirements (mean daily MSC dose, 240 mg; range, 60 mg/day to 1800 mg/day); therefore various MSC tablet strengths were developed. Part II presents three studies in which the MSC formulations (15-mg, 60-mg, and 100-mg tablets) were compared to the 30-mg tablet within three randomized, single-dose, two-way crossover, analytically blinded bioavailability protocols, to determine bioequivalence and dose proportionality. The maximum morphine concentration, time of maximum morphine concentration, and area under the plasma morphine versus 12-hour and 24-hour time curve (AUC 0.12; AUC 0.24) were determined in each study. There were no significant differences between the values associated with MSC 1 X 30 mg tablet and 2 X 15 mg tablets (study 1), MSC 2 X 30 mg tablets and 1 X 60 mg tablet (study 2), and MSC 3 X 30 mg tablets and 1 X 100 mg tablet (study 3, values adjusted to dose of 90 mg), except for one marginally significant difference in study 3 (AUC 0.24; P = 0.04) which was not clinically or biopharmaceutically significant. The results showed that MSC 15-mg, 30-mg, 60-mg, and 100-mg dosage strengths are bioequivalent and dose proportional, and, therefore, therapeutically interchangeable. It was concluded that with routine assessment of the patient and adherence to the principles of analgesic dosing, MSC can be successfully used to control cancer-related pain. Furthermore, the availability of various MSC tablet strengths can be expected to facilitate the analgesic management of a patient population with widely differing opioid requirements.

Administration, Oral↗

PubMed2000

Lipids, lipophilic drugs, and oral drug delivery-some emerging concepts.

Lipid-based dose forms, which encompass a wide variety of compositional and functional characteristics, can be advantageously utilized for the formulation of lipophilic drugs. There has been a traditional reluctance to develop lipid-based dose forms due to potential problems of chemical and physical instability, and a paucity of knowledge regarding formulation design algorithms and technology transfer issues. However, there is a current resurgence of interest in lipid-based dose forms due to potential commercial and pharmaceutical benefits, and the industry trend towards the discovery/development of increasingly hydrophobic (and potent) new chemical entities. This mini-review describes some emerging formulation and biopharmaceutic strategies that hold promise for better understanding how to design and evaluate lipid-based dose forms.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1992

Rapid toxicological model for use in assessing ocular drugs.

Nonsteroidal antiinflammatory drugs (NSAIDs) were applied to corneas either by in vitro or in vivo methods. The in vitro method involved excising and mounting corneas in a perfusion system at 37 degrees and exposing drug for 2.5 h. The in vivo methods represent either topical administration to the rabbit eye or topical in vivo infusion using a fixed well which permitted a constant concentration (0.05 per cent) to be applied to the eye of anesthetized rabbits for up to 120 min. An overlay grid procedure using scanning electron microscopy (SEM) showed less per cent endothelial damage with in vivo methods than with the in vitro method of administration, but per cent damage depended on which section was viewed. Damage to the epithelium and endothelium were also assessed by quantitative carboxyfluorescein and Janus green staining and uptake procedures, respectively, following drug exposure by the in vivo infusion method. Results for the epithelium indicated that the more lipophilic NSAIDs damaged the epithelial layer to a greater degree than newly synthesized hydrophilic NSAIDs. Damage to the epithelium correlated to the surface activity of the NSAIDs. Qualitative assessment of epithelial and endothelial toxicity can be performed with SEM and transmission electron microscopy (TEM) while vital staining procedures and the SEM grid procedure can be used to quantitatively assess corneal toxicity. The staining methods, however, possess advantages over SEM and TEM procedures in that they are rapid and do not require laborious preparation. As a result of these characteristics, the vital staining procedures could be used as part of a biopharmaceutical screening technique in evaluating new ophthalmic drugs.

Administration, Topical↗

PubMed2005

Drop-the-losers design: normal case.

Drop-the-losers designs are statistical designs which have two stages of a trial separated by a data based decision. In the first stage k experimental treatments and a control are administered. During a transition period, the empirically best experimental treatment is selected for continuation into the second phase, along with the control. At the study's end, inference focuses on the comparison of the selected treatment with the control using both stages' data. Traditional methods used to make inferences based on both stages' data can yield tests with higher than advertised levels of significance and confidence intervals with lower than advertised confidence. For normally distributed data, methods are provided to correct these deficiencies, providing confidence intervals with accurate levels of confidence. Drop-the-losers designs are particularly applicable to biopharmaceutical clinical trials where they can allow Phase II and Phase III clinical trials to be conducted under a single protocol with the use of all available data.

Algorithms↗

PubMed2004

The design of a potent inhibitor of the hepatitis C virus NS3 protease: BILN 2061--from the NMR tube to the clinic.

The virally encoded serine protease NS3/NS4A is essential to the life cycle of the hepatitis C virus (HCV), an important human pathogen causing chronic hepatitis, cirrhosis of the liver, and hepatocellular carcinoma. Until very recently, the design of inhibitors for the HCV NS3 protease was limited to large peptidomimetic compounds with poor pharmacokinetic properties, making drug discovery an extremely challenging endeavor. In our quest for the discovery of a small-molecule lead that could block replication of the hepatitis C virus by binding to the HCV NS3 protease, the critical protein-polypeptide interactions between the virally encoded NS3 serine protease and its polyprotein substrate were investigated. Lead optimization of a substrate-based hexapeptide, guided by structural data, led to the understanding of the molecular dynamics and electronic effects that modulate the affinity of peptidomimetic ligands for the active site of this enzyme. Macrocyclic beta-strand scaffolds were designed that allowed the discovery of potent, highly selective, and orally bioavailable compounds. These molecules were the first HCV NS3 protease inhibitors reported that inhibit replication of HCV subgenomic RNA in a cell-based replicon assay at low nanomolar concentrations. Optimization of their biopharmaceutical properties led to the discovery of the clinical candidate BILN 2061. Oral administration of BILN 2061 to patients infected with the hepatitis C genotype 1 virus resulted in an impressive reduction of viral RNA levels, establishing proof-of-concept for HCV NS3 protease inhibitors as therapeutic agents in humans.

Carbamates↗

PubMed2002

Inhibiting apoptosis in mammalian cell culture using the caspase inhibitor XIAP and deletion mutants.

Lower yields and poorer quality of biopharmaceutical products result from cell death in bioreactors. Such cell death may occur from necrosis but is more commonly associated with apoptosis. During the process of programmed cell death or apoptosis, caspases become activated and cause a cascade of events that eventually destroy the cell. XIAP is the most potent caspase inhibitor encoded in the mammalian genome. The effectiveness of XIAP and its deletion mutants was examined in two cell lines commonly utilized in commercial bioreactors: Chinese hamster ovary (CHO) and 293 human embryonic kidney (293 HEK) cells. CHO cells undergo apoptosis as a result of various insults, including Sindbis virus infection and serum deprivation. In this study, we demonstrate that 293 HEK cells undergo apoptosis during Sindbis virus infection and exposure to the toxins, etoposide and cisplatin. Two deletion mutants of XIAP were created; one containing three tandem baculovirus iap repeat (BIR) domains and the other containing only the C-terminal RING domain, lacking the BIRs. Viability studies were performed for cells expressing each mutant and the wild-type protein on transiently transfected cells, as stable pools, or as stable clonal cell populations after induction of apoptosis by serum deprivation, Sindbis virus infection, etoposide, and cisplatin treatment. Expression of the wild-type XIAP inhibited apoptosis significantly; however, the XIAP mutant containing the three BIRs provided equivalent or improved levels of apoptosis inhibition in all cases. Expression of the RING domain offered no protection and was pro-apoptotic in transient expression experiments. With the aid of an N-terminal YFP fusion to each protein, distribution within the cell was visualized, and the wild-type and mutants showed differing intracellular accumulation patterns. While the wild-type XIAP protein accumulated primarily in aggregates in the cytosol, the RING mutant was enriched in the nucleus. In contrast, the deletion mutant containing the three BIRs was distributed evenly throughout the cytosol. Thus, protein engineering of the XIAP protein can be used to alter the intracellular distribution pattern and improve the ability of this caspase inhibitor to protect against apoptosis for two mammalian cell lines.

Animals↗

PubMed2002

Protein modification during anti-viral heat-treatment bioprocessing of factor VIII concentrates, factor IX concentrates, and model proteins in the presence of sucrose.

To ensure the optimal safety of plasma derived and new generation recombinant proteins, heat treatment is customarily applied in the manufacturing of such biopharmaceuticals as a means of viral inactivation. In subjecting proteins to anti-viral heat-treatment it is necessary to use high concentrations of thermostabilizing excipients to prevent protein damage, and it is therefore imperative that the correct balance between bioprocessing conditions, maintenance of protein integrity and virus kill is found. In this study we have utilized model proteins (lysozyme, fetuin, and human serum albumin) and plasma-derived therapeutic proteins (factor VIII and factor IX) to investigate the protein modifications that occur during anti-viral heat treatment. Specifically, we investigated the relationship between bioprocessing conditions and the type and extent of protein modification under a variety of industrially relevant wet and lyophilized heat treatments using sucrose as a thermostabilizing agent. Heat treatment led to the formation of disulfide crosslinks and aggregates in proteins containing free cysteine residues. Terminal oligosaccharide sialic acid residues were hydrolyzed from the glycan moieties of glycoproteins during anti-viral heat treatment. Heat treatment promoted sucrose hydrolysis to yield glucose and fructose, leading, in turn, to the glycation of lysine amino groups in those proteins containing di-lysine motifs. During extended hear treatments, 1,2-dicarbonyl type advanced glycation end-products were also formed. Glycation-type modifications were more prevalent in wet heat-treated protein formulations.

Disinfection↗

PubMed2002

p27Kip1-mediated controlled proliferation technology increases constitutive sICAM production in CHO-DUKX adapted for growth in suspension and serum-free media.

We have engineered dihydrofolate reductase-deficient (dhfr(-)) Chinese hamster ovary (CHO)-DUKX B11 cells adapted for growth in serum-free suspension cultures for unlinked muristerone-inducible expression of the cyclin-dependent kinase inhibitor p27Kip1 and constitutive expression of the soluble intercellular adhesion molecule-1 (sICAM), a potent common cold therapeutic. Conditional overexpression of p27Kip1 resulted in a sustained G1-specific growth arrest of transgenic CHO-DUKX associated with up to fivefold-increased specific sICAM productivity. Herein we exemplify the implementation of controlled proliferation technology in a major biopharmaceutical production cell line that is compatible with key requirements for large-scale production procedures, including constitutive transgene expression and anchorage-independent growth in serum-free media.

Animals↗

PubMed2002

Versatile macrolide-responsive mammalian expression vectors for multiregulated multigene metabolic engineering.

The novel macrolide-inducible and -repressible mammalian gene regulation systems (E.REX) have been cloned into a variety of sophisticated expression configurations including (1) multi-purpose expression vectors, (2) pTRIDENT-based artificial operons, (3) dual-regulated expression strategies for independent control of two different transgenes, (4) autoregulated vectors for one-step installation of adjustable multigene expression, and (5) oncoretroviral and lentiviral plasmids for transduction of macrolide-, streptogramin- and tetracycline-dependent transactivators and production of cell lines supporting independent control of three different transgenes. This vector portfolio represents a construction kit-like toolbox for efficient installation of adjustable gene expression responsive to clinically licensed antibiotics and enables the design of multiregulated multigene metabolic engineering strategies required for biopharmaceutical manufacturing, gene therapy, and tissue engineering.

Animals↗

PubMed2003

Novel CNBP- and La-based translation control systems for mammalian cells.

Throughout the development of Xenopus, production of ribosomal proteins (rp) is regulated at the translational level. Translation control is mediated by a terminal oligopyrimidine element (TOP) present in the 5' untranslated region (UTR) of rp-encoding mRNAs. TOP elements adopt a specific secondary structure that prevents ribosome-binding and translation-initiation of rp-encoding mRNAs. However, binding of CNBP (cellular nucleic acid binding protein) or La proteins to the TOP hairpin structure abolishes the TOP-mediated transcription block and induces rp production. Based on the specific CNBP-TOP/La-TOP interactions we have designed a translation control system (TCS) for conditional as well as adjustable translation of desired transgene mRNAs in mammalian cells. The generic TCS configuration consists of a plasmid encoding CNBP or La under control of the tetracycline-responsive expression system (TET(OFF)) and a target expression vector containing a TOP module between a constitutive P(SV40) promoter and the human model product gene SEAP (human secreted alkaline phosphatase) (P(SV40)-TOP-SEAP-pA). The TCS technology showed excellent SEAP regulation profiles in transgenic Chinese hamster ovary (CHO) cells. Alternatively to CNBP and La, TOP-mediated translation control can also be adjusted by artificial phosphorothioate anti-TOP oligodeoxynucleotides. Confocal laser-scanning microscopy demonstrated cellular uptake of FITC-labeled oligodeoxynucleotides and their localization in perinuclear organelles within 24 hours. Besides their TOP-based translation-controlling capacity, CNBP and La were also shown to increase cap-independent translation from polioviral internal ribosomal entry sites (IRES) and La alone to boost cap-dependent translation initiation. CNBP and La exemplify for the first time the potential of RNA-binding proteins to exert translation control of desired transgenes and to increase heterologous protein production in mammalian cells. We expect both of these assets to advance current gene therapy and biopharmaceutical manufacturing strategies.

Animals↗

PubMed2001

Stability of crystalline proteins.

By using two model proteins, glucose oxidase and lipase, we demonstrate that dry crystalline formulations are significantly more stable than their amorphous counterparts. The results of Fourier-transform infrared spectroscopy indicate that crystalline proteins better maintain their native conformation in accelerated stability studies. The lower tendency of crystalline proteins to aggregate is confirmed by size-exclusion chromatography. The data suggest that protein crystallization may significantly improve some aspects of protein handling, and change the way biopharmaceuticals are produced, formulated, and delivered.

Chromatography, Gel↗

PubMed2003

Scalable inoculation strategies for microcarrier-based animal cell bioprocesses.

Scalability is a major demand for high-yield, stable bioprocess systems in animal cell culture-based biopharmaceutical production. Increased yields can be achieved through high-density cell culture, such as in the combination of microcarrier and fluidized bed bioreactor technology. To minimize inocula volume in industrial applications of fluidized bed fermentation systems, it is crucial to increase the bed volume in the reactor during the fermentation process. We tested scale-up strategy for the production of recombinant human arylsulfatase B (ASB) enzyme used in enzyme replacement therapy in patients afflicted with mucopolysaccharidosis type VI (MPS VI). This enzyme was derived from Chinese hamster ovary (CHO) cells cultivated as adherent cell culture on Cytoline macroporous microcarriers (Amersham Biosciences, Uppsala, Sweden) using a Cytopilot Mini fluidized bed bioreactor (FBR; Amersham Biosciences, Vogelbusch, Austria). Both 1:2 expansion (herein referred to as the addition of fresh, not-yet-colonized microcarriers) and 1:6 expansion of the carrier bed were performed successfully; the cells restarted to proliferate for colonizing these newly added carriers; and the stability of the culture was not negatively affected.

Animals↗

PubMed2001

Novel pristinamycin-responsive expression systems for plant cells.

Novel gene regulation systems were designed for plant cells responsive to the streptogramin antibiotic pristinamycin. The pristinamycin-repressible plant gene regulation concept (PIPpOFF) is based on a transcriptional activator (PIT) which consists of the Pip protein, the repressor of the pristinamycin resistance operon of Streptomyces coelicolor, fused to the VP16 transactivation domain of the Herpes simplex virus. PIT mediates pristinamycin-repressible activation of a synthetic plant promoter (P(pPIR)) in tobacco cells consisting of a nine Pip-binding site-containing artificial operator (PIR3) placed upstream of a TATA-box derived from the cauliflower mosaic virus 35S promoter (P(CaMV35S)). Pristinamycin interferes with induction by negatively regulating the DNA-binding capacity of the Pip moiety of PIT. A second, streptogramin-inducible plant gene regulation system (PIPpON) was constructed by combining Pip expression with a plant-specific pristinamycin-inducible promoter (P(pPIRON)). P(pPIRON) consists of a PIR3 module cloned downstream of the strong constitutive plant promoter P(CaMV35S). As in the native Streptomyces configuration, Pip binds to its cognate sequence within P(pPIRON) in the absence of regulating antibiotic and silences the chimeric plant promoter. Upon addition of pristinamycin, Pip is released from the PIR3 operator and full P(CaMV35S)-driven expression of desired plant genes is induced. The PIPpOFF and PIPpON systems performed well in Nicotiana tabacum suspension cultures and promise to provide an attractive extension of existing plant gene regulation technology for basic plant research or biopharmaceutical manufacturing using plant tissue culture.

Amino Acid Sequence↗

PubMed2001

Novel surface tagging technology for selection of complex proliferation-controlled mammalian cell phenotypes.

Regulated overexpression of the cyclin dependent kinase inhibitor p27 enables biphasic production processes which consist of a nonproducing expansion phase followed by an extended proliferation-arrested production phase. During the growth-arrested production phase proliferation-competent mutants emerge as a consequence of genetic drift and strong counterselection. Here, we evaluate the use of cell surface markers for ex vivo selection of growth-arrested phenotypes by magnetic or FACS-mediated cell sorting. Multigene metabolic engineering resulted in a Chinese hamster ovary- (CHO) derived cell line CHO-SS101(5), which expresses the model product protein SEAP (secreted alkaline phosphatase), the human cyclindependent kinase inhibitor p27, and a membrane-anchored multidomain surface marker Hook in a tricistronic tetracycline-repressible manner. In the absence of tetracycline in the cell culture medium, p27 mediated a G1-phase-specific cell-cycle arrest of CHO-SS101(5) and resulted in a fivefold increase in SEAP production compared to proliferation-competent control cells. Concomitant expression of Hook enabled FACS- or magnetic-based selection of CHO-SS101(5) cells from various mixed populations. Surface selection of engineered cells will likely become important for biopharmaceutical manufacturing and for in vivo maintenance of treated cells in gene therapy and tissue engineering.

Alkaline Phosphatase↗

PubMed2004

Toward construction of a self-sustained clock-like expression system based on the mammalian circadian clock.

Despite recent advances in circadian biology, detailed understanding of how a biological pacemaker system is assembled, maintained, and regulated continues to be a significant challenge. We have assembled and characterized a first-generation, regulatable, self-sustained clock-like expression system based on key components of the mammalian circadian clock. The molecular setup of the clock-like oscillator was reduced to the core set of positive and negative elements common to all known circadian pacemakers. Sophisticated tetracycline-responsive multi-cistronic expression integrated with forefront lentiviral transduction tools enabled autoregulated reporter transgene expression in a human cell line. We characterized transgene expression kinetics of an artificial oscillator and showed that its expression profiles could be modulated by a serum shock and administration of regulating tetracycline antibiotics. Design of a generic mammalian clock-like expression system will offer novel opportunities to study circadian biology and may provide a unique tool for rhythmic expression of desired transgenes fostering advances in biopharmaceutical manufacturing, gene therapy, and tissue engineering.

ARNTL Transcription Factors↗

PubMed2004

Macrolide- and tetracycline-adjustable siRNA-mediated gene silencing in mammalian cells using polymerase II-dependent promoter derivatives.

RNA interference has emerged as a powerful technology for downregulation of specific genes in cells and animals. We have pioneered macrolide- and tetracycline-adjustable short interfering RNA (siRNA) expression for conditional target gene translation fine-tuning in mammalian/human cell lines based on modified RNA polymerase II promoters. Established macrolide- and tetracycline-dependent transactivators/trans-silencers bound and activated modified target promoters tailored for optimal siRNA expression in response to clinical antibiotics' dosing regimes and modulated desired target genes in Chinese hamster ovary (CHO-K1) and human fibrosarcoma (HT-1080) cells with high precision. Further optimization of adjustable RNA polymerase II-based siRNA-specific promoters as well as their combination with various transmodulators enabled near-perfect regulation configurations in specific cell types. Devoid of major genetic constraints compared to basic RNA polymerase III-based siRNA-specific promoters, we expect RNA polymerase II counterparts to significantly advance siRNA-based molecular interventions in biopharmaceutical manufacturing and gene-function analysis as well as gene therapy and tissue engineering.

Animals↗

PubMed2004

Screening for improved cell performance: selection of subclones with altered production kinetics or improved stability by cell sorting.

One of the major problems in the biotechnology industry is the selection of cell lines well suited for production of biopharmaceutical proteins. Usually, the most important selection criterion is the cell specific production rate. Nevertheless, a good producer cell line should have a number of additional advantageous properties, which allow the cell line to perform well in the type of bioreactor chosen for the process. However, the time and work required to select for high production rates as well as the lack of methods to specifically select for other cellular properties, usually prevents researchers from including such criteria into their screening program. With the Single Cell Secretion Assay it is possible to measure the specific production rates of individual cells by catching secreted product in an artificial matrix applied to the cell surface. Flow cytometric cell sorting then allows selection of rare cells with high production rates, which occur at frequencies as low as 10(-6). By combining this method with culture conditions that bring out a desired cellular property, we were able to isolate subclones with similar production rates, but improved performance from a recombinant Chinese hamster ovary cell line producing a human monoclonal antibody. The two desired cellular properties screened for were a non-growth associated production kinetic and improved stability in the absence of selective pressure.

Animals↗