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PubMedDate not supplied

Modulation of cell cycle for enhancement of antibody productivity in perfusion culture of NS0 cells.

A prolonged period of high productivity at high cell density is desirable for industrial production of biopharmaceuticals. Previous efforts have shown that cessation of cell proliferation in low cell density culture results in increased productivity. We report here further results on multigenic manipulation of cell cycle and apoptosis to enhance productivity at high cell density. The NS0 6A1/4-9F myeloma cell line, which constitutively expresses a chimeric IgG4 antibody and inducibly expresses the p21(CIP1) cyclin-dependent kinase inhibitor has been further engineered to constitutively overexpress the Y28 mutant Bcl-2 anti-apoptotic protein. The effects of overexpression of p21(CIP1) and Bcl-2 on cell proliferation, cell viability, and antibody production has been investigated in batch and continuous perfusion cultures. In both cultures the p21(CIP1) protein arrested cell proliferation, confirming the previous results in low-density culture of 4-fold increase in antibody production, whereas mutant Bcl-2 expression has not resulted in any significant improvement in cell viability of arrested cells. This study demonstrates that it is possible to enhance the productivity of relatively high-density continuous mammalian cell cultures by arresting the cell cycle in G1 phase.

Animals↗

PubMedDate not supplied

Cell growth arrest by nucleotides, nucleosides and bases as a tool for improved production of recombinant proteins.

Arresting cell growth and thus decreasing cell division potentially lessens the chance for genetic drift in the cell population; this would be of utmost importance for the consistent production of biopharmaceuticals during long periods. The drawback of the addition of well-known synchronizing agents, such as chemotherapeutics, is that they cause a disproportionate accumulation of cellular constituents, leading to cell death. The use of compounds that are naturally synthesized by the cell, as is the case of nucleotides, nucleosides, and bases (Nt/Ns/B), is shown in this work to be a promising tool. The addition of purines and pyrimidines was tested using a CHO cell line producing the secreted form of the human placental alkaline phosphatase enzyme (SEAP). From the chemical alternatives tested, AMP was the most promising compound for protein production improvement; it reduced cell growth and maintained the culture with high cell viability for long periods, while increasing SEAP specific productivity 3-fold. The use of CHO and BHK mammalian cells producing Factor VII and the use of a insect cell line (Sf9) showed that the effect of AMP addition seems to be independent of the r-protein and cell line. With the addition of AMP, accumulation of cells at the S phase was accompanied by an increase of the protein specific productivity. Addition of known synchronizing drugs (aphidicolin and doxorubicin) and application of environmental cell growth arrest strategies (depletion of nutrients and byproduct accumulation) showed also to effectively arrest CHO cell growth. A careful look onto cell cycle distribution in the different scenarios created, shows whether it is important to consider r-protein expression dependency upon cell cycle in process optimization and operation strategies.

Alkaline Phosphatase↗

PubMedDate not supplied

Comparison of fluidized bed and ultrasonic cell-retention systems for high cell density mammalian cell culture.

Economically viable biopharmaceutical production is to a high degree dependent on high product yields and stable fermentation systems that are easy to handle. In the current study we have compared two different fermentation systems for the production of recombinant protein from CHO cells. Both systems are fully scaleable and can be used for industrial high cell density bioprocesses. As a model cell line we have used a recombinant CHO cell line producing the enzyme arylsulfatase B (ASB). CHO cells were cultivated as adherent cell culture attached on Cytoline macroporous microcarrier (Amersham Biosciences, Sweden) using a Cytopilot Mini fluidized bed bioreactor (FBR, Vogelbusch-Amersham Biosciences, Austria) and as suspension culture using a stirred tank bioreactor equipped with a BioSep ultrasonic resonator based cell separation device (Applikon, The Netherlands). Both systems are equally well-suited for stable, long-term high cell density perfusion cell culture and provide industrial scalability and high yields. For products such as the recombinant ASB, high perfusion rates and therefore short product bioreactor residence times may be of additional benefit.

Animals↗

PubMedDate not supplied

Effect of freezing rates and excipients on the infectivity of a live viral vaccine during lyophilization.

Lyophilization is the most popular method for achieving improved stability of labile biopharmaceuticals, but a significant fraction of product activity can be lost during processing due to stresses that occur in both the freezing and the drying stages. The effect of the freezing rate on the recovery of herpes simplex virus 2 (HSV-2) infectivity in the presence of varying concentrations of cryoprotectant excipients is reported here. The freezing conditions investigated were shelf cooling (223 K), quenching into slush nitrogen (SN2), and plunging into melting propane cooled in liquid nitrogen (LN2). The corresponding freezing rates were measured, and the ice crystal sizes formed within the samples were determined using scanning electron microscopy (SEM). The viral activity assay demonstrated the highest viral titer recovery for nitrogen cooling in the presence of low (0.25% w/v sucrose) excipient concentration. The loss of viral titer in the sample cooled by melting propane was consistently the highest among those results from the alternative cooling methods. However, this loss could be minimized by lyophilization at lower temperature and higher vacuum conditions. We suggest that this is due to a higher ratio of ice recrystallization for the sample cooled by melting propane during warming to the temperature at which freeze-drying was carried out, as smaller ice crystals readily enlarge during warming. Under the same freezing condition, a higher viral titer recovery was obtained with a formulation containing a higher concentration of sugar excipients. The reason was thought to be twofold. First, sugars stabilize membranes and proteins by hydrogen bonding to the polar residues of the biomolecules, working as a water substitute. Second, the concentrated sugar solution lowers the nucleation temperature of the water inside the virus membrane and prevents large ice crystal formation within both the virus and the external medium.

Animals↗

PubMedDate not supplied

Glycosylation of recombinant antibody therapeutics.

The adaptive immune system has the capacity to produce antibodies with a virtually infinite repertoire of specificities. Recombinant antibodies specific for human targets are established in the clinic as therapeutics and represent a major new class of drug. Therapeutic efficacy depends on the formation of complexes with target molecules and subsequent activation of downstream biologic effector mechanisms that result in elimination of the target. The activation of effector mechanisms is dependent on structural characteristics of the antibody molecule that result from posttranslational modifications, in particular, glycosylation. The production of therapeutic antibody with a consistent human glycoform profile has been and remains a considerable challenge to the biopharmaceutical industry. Recent research has shown that individual glycoforms of antibody may provide optimal efficacy for selected outcomes. Thus a further challenge will be the production of a second generation of antibody therapeutics customized for their clinical indication.

Antibodies↗

PubMedDate not supplied

Production of nonclassical inclusion bodies from which correctly folded protein can be extracted.

Human granulocyte-colony stimulating factor (hG-CSF), an important biopharmaceutical drug used in oncology, is currently produced mainly in Escherichia coli. Expression of human hG-CSF gene in E. coli is very low, and therefore a semisynthetic, codon-optimized hG-CSF gene was designed and subcloned into pET expression plasmids. This led to a yield of over 50% of the total cellular proteins. We designed a new approach to biosynthesis at low temperature, enabling the formation of "nonclassical" inclusion bodies from which correctly folded protein can be readily extracted by nondenaturing solvents, such as mild detergents or low concentrations of polar solvents such as DMSO and nondetergent sulfobetaines. FT-IR analysis confirmed different nature of inclusion bodies with respect to the growth temperature and indicated presence of high amounts of very likely correctly folded reduced hG-CSF in nonclassical inclusion bodies. The yield of correctly folded, functional hG-CSF obtained in this way exceeded 40% of the total hG-CSF produced in the cells and is almost completely extractable under nondenaturing conditions. The absence of the need to include a denaturation/renaturation step in the purification process allows the development of more efficient processes characterized by higher yields and lower costs and involving environment-friendly technologies. The technology presented works successfully at the 50-L scale, producing nonclassical inclusion bodies of the same quality. The approach developed for the production of hG-CSF could be extended to other proteins; thus, a broader potential for industrial exploitation is envisaged.

Blotting, Western↗

PubMedDate not supplied

Bioprocess engineering issues that would be faced in producing a DNA vaccine at up to 100 m3 fermentation scale for an influenza pandemic.

The risk of a pandemic with a virulent form of influenza is acknowledged by the World Health Organization (WHO) and other agencies. Current vaccine production facilities would be unable to meet the global requirement for vaccine. As a possible supplement a DNA vaccine may be appropriate, and bioprocess engineering factors bearing on the use of existing biopharmaceutical and antibiotics plants to produce it are described. This approach addresses the uncertainty of timing of a pandemic that precludes purpose-built facilities. The strengths and weaknesses of alternative downstream processing routes are analyzed, and several gaps in public domain information are addressed. The conclusion is that such processing would be challenging but feasible.

Adsorption↗

PubMedDate not supplied

Production of recombinant RNase Ba and its application in downstream processing of plasmid DNA for pharmaceutical use.

The demand for new strategies in downstream processing of biopharmaceutical plasmid DNA has increased in response to the importance of nucleic acids as active pharmaceutical ingredients (API) in gene therapy and genetic vaccination. Led by the problematic usage of animal-derived proteins for producing reagents of clinical applications, we present an opportunity of removing RNA prior to chromatographic steps by using a recombinant RNase Ba (barnase of Bacillus amyloliquefaciens) as an alternative to bovine RNase A. An expression vector for RNase Ba production was constructed enabling periplasmic localization of the recombinant protein. Cultivation of the RNase-producing clone showed stable activity (3.6 kU mL(-1) during stationary phase) throughout the cultivation process. After purification the RNase activity was tested and compared to that of commercially available RNase A. RNase Ba showed no DNase activity even after prolonged incubation with plasmid DNA. Thus, it is a suitable substitute for bovine RNase A in pharmaceutical purification processes.

Animals↗

PubMedDate not supplied

Synthesis of bisected glycoforms of recombinant IFN-beta by overexpression of beta-1,4-N-acetylglucosaminyltransferase III in Chinese hamster ovary cells.

Genetic engineering of oligosaccharide biosynthesis pathways in mammalian cells makes possible generation of new recombinant glycoproteins of potential importance in the biopharmaceutical industry. Most prior investigations of glycosylation engineering of secreted heterologous glycoproteins involve terminal steps of oligosaccharide biosynthesis. In particular, increasing the frequency of bisected structures within the glycoform distribution has not before been considered. A Chinese hamster ovary (CHO) cell line capable of producing bisected oligosaccharides on glycoproteins was created by overexpression of a recombinant N-acetylglucosaminyltransferase III (GnT-III). Interferon beta (IFN-beta) was chosen as a model and potential therapeutic secreted heterologous protein to demonstrate the effect of recombinant GnT-III-expression on product glycosylation. IFN-beta with bisected oligosaccharides was produced by the GnT-III-engineered CHO cells but not by the unmodified parental cell line.

Animals↗

PubMedDate not supplied

A consensus neural network-based technique for discriminating soluble and poorly soluble compounds.

BCUT [Burden, CAS, and University of Texas] descriptors, defined as eigenvalues of modified connectivity matrices, have traditionally been applied to drug design tasks such as defining receptor relevant subspaces to assist in compound selections. In this paper we present studies of consensus neural networks trained on BCUTs to discriminate compounds with poor aqueous solubility from those with reasonable solubility. This level was set at 0.1 mg/mL on advice from drug formulation and drug discovery scientists. By applying strict criteria to the insolubility predictions, approximately 95% of compounds are classified correctly. For compounds whose predictions have a lower level of confidence, further parameters are examined in order to flag those considered to possess unsuitable biopharmaceutical and physicochemical properties. This approach is not designed to be applied in isolation but is intended to be used as a filter in the selection of screening candidates, compound purchases, and the application of synthetic priorities to combinatorial libraries.

Journal Article↗

PubMedDate not supplied

Metabotropic glutamate receptor-mediated currents at the climbing fiber to Purkinje cell synapse.

Different forms of synaptic plasticity in the cerebellum expressed at the synapses onto Purkinje cells (PCs) are mediated by membrane metabotropic glutamate receptors (mGluRs). There are three main mGluR groups with a total of 8 subtypes. Although mGluRs are also found at the climbing fiber (CF) to PC synapses, the distribution and biological activity of their types is not well-known. Using whole cell patch-clamp recordings from PCs in rat cerebellar slices with inhibitors of ionotropic receptors and glutamate uptake blockers we demonstrate a complex pharmacology of currents obtained by CF stimulation. The mGluR1 specific antagonist CPCCOEt in a group of cells suppressed this response, but in a similar number of other cells it induced a potentiating effect. It was found that a switch between these two biopharmaceutical effects might occur with age.

Aging↗

PubMed2005

Occurrence of (+)- and (-)-gossypol in wild species of cotton and in Gossypium hirsutum Var. marie-galante (Watt) Hutchinson.

Gossypol occurs as a mixture of enantiomers in cottonseed. These enantiomers exhibit different biological activities. The (-)-enantiomer is toxic to animals, but it has potential medicinal uses. Therefore, cottonseed with >95% (-)-gossypol could have biopharmaceutical applications. The (+)-enantiomer shows little, if any, toxicity to nonruminant animals. Thus, cottonseed with >95% (+)-gossypol could be more readily utilized as a feed for nonruminants. The (+)- to (-)-gossypol ratio in commercial Upland (Gossypium hirsutum) cottonseed is usually about 3:2, whereas that in commercial Pima cottonseed (Gossypium barbadense) is approximately 2:3. Herein are reported the (+)- to (-)-gossypol ratios in the seed from 28 wild species of cotton (194 accessions), 94 accessions of G. hirsutum var. marie-galante, and 3 domesticated species (11 accessions). It was found that some or all of the accessions of Gossypium darwinii, Gossypium sturtianum, Gossypium areysianum, Gossypium longicalyx, Gossypium harknessii, and Gossypium costulatum produce an excess of (-)-gossypol but none >65%. At least one accession of Gossypium anomalum, Gossypium mustelinum, Gossypium gossypioides, and Gossypium capitis-viridis contained >94% (+)-gossypol. One of the 94 accessions of G. hirsutum var. marie-galante (i.e., no. 2469) contained 97% (+)-gossypol.

Animal Feed↗

PubMed2002

Structure-activity profiles of complex biantennary glycans with core fucosylation and with/without additional alpha 2,3/alpha 2,6 sialylation: synthesis of neoglycoproteins and their properties in lectin assays, cell binding, and organ uptake.

The consideration of oligosaccharides and glycoconjugates as biopharmaceuticals is an emerging topic in drug design. Chemoenzymatic synthesis of N-glycans was performed to examine the influence of N-glycan core fucosylation on lectin-binding properties and biodistribution. As a first step in a systematic comparison of N-glycans, the core fucose moiety was chemically introduced into a complex-type biantennary heptasaccharide azide. After deprotection and attachment of a spacer, the terminal sections of the N-glycan were elongated enzymatically. Conversion of the amino group in the spacer to an isothiocyanate gave derivatives allowing convenient ligand attachment to bovine serum albumin (BSA). The resulting neoglycoproteins contained an average of 2.9-4.6 chains per carrier molecule. Relative to unsubstituted biantennary complex-type N-glycans, the core fucosylation appears to favor the extended orientation of the alpha 1,6-arm. This was deduced from an up to 5-fold alteration of affinity for lectins in solid-phase assays. Marked differences were also found for cell surface binding of cultured tumor cells, for staining of tumor cells in lung sections, and in organ distribution. In vivo, the alpha 2,6-sialylated neoglycoproteins showed a reduced serum half-life in mice relative to the alpha 2,3-sialylated isomer and the non-fucosylated congeners. These results support the notion that changing the shape of a glycan provides a promising strategy to optimize the affinity of protein-carbohydrate interactions. Overall, our study underscores the importance of chemoenzymatic synthesis to define the effect of chain orientation on the ligand properties of N-glycans.

Animals↗

PubMed2005

Design and synthesis of the first generation of novel potent, selective, and in vivo active (benzothiazol-2-yl)acetonitrile inhibitors of the c-Jun N-terminal kinase.

Several lines of evidence support the hypothesis that c-Jun N-terminal kinase (JNKs) plays a critical role in a wide range of diseases including cell death (apoptosis)-related disorders (neurodegenerative diseases, brain, heart, and renal ischemia, epilepsy) and inflammatory disorders (multiple sclerosis, rheumatoid arthritis, inflammatory bowel diseases). Screening of our internal compound collection for inhibitors of JNK3 led to the identification of (benzothiazol-2-yl)acetonitrile derivatives as potent and selective JNK1, -2, -3 inhibitors. Starting from initial hit 1 (AS007149), the chemistry and initial structure-activity relationship (SAR) of this novel and unique kinase inhibitor template were explored. Investigation of the SAR rapidly revealed that the benzothiazol-2-ylacetonitrile pyrimidine core was crucial to retain a good level of potency on rat JNK3. Therefore, compound 6 was further optimized by exploring a number of distal combinations in place of the chlorine atom. This led to the observation that the presence of an aromatic group, two carbons away from the aminopyrimidine moiety and bearing substituents conferring hydrogen bond acceptor (HBA) properties, could improve the potency. Further improvements to the biological and biopharmaceutical profile of the most promising compounds were performed, resulting in the discovery of compound 59 (AS601245). The in vitro and in vivo anti-inflammatory potential of this new JNK inhibitor was investigated and found to demonstrate efficacy per oral route in an experimental model of rheumatoid arthritis (RA).

Acetonitriles↗

PubMed1996

Evaluation of selected chemotypes in coupled cellular and molecular target-based screens identifies novel HIV-1 zinc finger inhibitors.

Conservation of the Cys-Xaa2-Cys-Xaa4-His-Xaa4-Cys retroviral zinc finger sequences and their absolute requirement in both the early and late phases of retroviral replication make these chemically reactive structures prime antiviral targets. We recently reported that select 2,2'-dithiobisbenzamides (DIBAs) chemically modify the zinc finger Cys residues, resulting in release of zinc from the fingers and inhibition of HIV replication. In the current study we surveyed 21 categories of disulfide-based compounds from the chemical repository of the National Cancer Institute for their capacity to act as retroviral zinc finger inhibitors. Aromatic disulfides that exerted anti-HIV activity tended to cluster in the substituted aminobenzene, benzoate, and benzenesulfonamide disulfide subclasses. Only one thiuram derivative exerted moderate anti-HIV activity, while a number of nonaromatic thiosulfones and miscellaneous disulfide congeners were moderately antiviral. Two compounds (NSC 20625 and NSC 4493) demonstrated anti-cultures. The two compounds chemically modified the p7NC zinc fingers in two separate in vitro assays, and interatomic surface molecular modeling docked the compounds efficiently but differentially into the zinc finger domains. The combined efforts of rational drug selection, cell-based screening, and molecular target-based screening led to the identification of zinc finger inhibitors that can now be optimized by medicinal chemistry for the development of biopharmaceutically useful anti-HIV agents.

Anti-HIV Agents↗

PubMed1998

Correlation of human jejunal permeability (in vivo) of drugs with experimentally and theoretically derived parameters. A multivariate data analysis approach.

The effective permeability (Peff) in the human jejunum (in vivo) of 22 structurally diverse compounds was correlated with both experimentally determined lipophilicity values and calculated molecular descriptors. The permeability data were previously obtained by using a regional in vivo perfusion system in the proximal jejunum in humans as part of constructing a biopharmaceutical classification system for oral immediate-release products. pKa, log P, and, where relevant, log Pion values were determined using the pH-metric technique. On the basis of these experiments, log D values were calculated at pH 5.5, 6.5, and 7.4. Multivariate data analysis was used to derive models that correlate passive intestinal permeability to physicochemical descriptors. The best model obtained, based on 13 passively transcellularly absorbed compounds, used the variables HBD (number of hydrogen bond donors), PSA (polar surface area), and either log D5.5 or log D6.5 (octanol/water distribution coefficient at pH 5.5 and 6.5, respectively). Statistically good models for prediciting human in vivo Peff values were also obtained by using only HBD and PSA or HBD, PSA, and CLOGP. These models can be used to predict passive intestinal membrane diffusion in humans for compounds that fit within the defined property space. We used one of the models obtained above to predict the log Peff values for an external validation set consisting of 34 compounds. A good correlation with the absorption data of these compounds was found.

Humans↗

PubMed1998

Human intestinal permeability.

This review focuses on permeability measurements in humans, briefly discussing different perfusion techniques, the relevance of human Peff values, and various aspects of in vivo transport mechanisms. In addition, human Peff values are compared with corresponding data from three preclinical transport models. The regional human jejunal perfusion technique has been validated in several important ways. One of the most important findings is that there is a good correlation between the measured human effective permeability values and the extent of absorption of drugs in humans determined by pharmacokinetic studies. Estimations of the absorption half-lives from the measured Peff agree very well with the time to maximal amount of the dose absorbed achieved after an oral dose in humans. We have also shown that it is possible to determine the Peff for carrier-mediated transported compounds and to classify them according to the proposed biopharmaceutical classification system (BCS). Furthermore, human in vivo permeabilities can be predicted using preclinical permeability models, such as in situ perfusion of rat jejunum, the Caco-2 model, and excised intestinal segments in the Ussing chamber. The permeability of passively transported compounds can be predicted with a particularly high degree of accuracy. However, special care must be taken for drugs with a carrier-mediated transport mechanism, and a scaling factor has to be used. Finally, the data obtained in vivo in humans emphasize the need for more clinical studies investigating the effect of physiological in vivo factors and molecular mechanisms influencing the transport of drugs across the intestinal and as well as other membrane barriers. It will also be important to study the effect of antitransport mechanisms (multidrug resistance, MDR), such as efflux by P-glycoprotein(s) and gut wall metabolism, for example CYP 3A4, on bioavailability.

Animals↗

PubMed1999

Hydroxypropyl-beta-cyclodextrin inhibits spray-drying-induced inactivation of beta-galactosidase.

The single-step, fast spray-drying process may represent a valuable alternative to the multistep, time-consuming freeze-drying process in the area of formulation and processing of biopharmaceuticals. In this study, we tested the use of sucrose and hydroxypropyl-beta-cyclodextrin (HP-beta-CD) as stabilizing excipients in the spray-drying of a model protein, beta-galactosidase. The solutions were processed using a Büchi 190 cocurrent Mini Spray Dryer at an outlet temperature of 61 +/- 2 degrees C. The powders were redissolved and analyzed for catalytic activity, aggregation, chemical decomposition, and thermal susceptibility as observed by high-resolution calorimetry. Spray-drying significantly inactivated beta-galactosidase. Spray-drying beta-galactosidase in the presence of sucrose did not prevent inactivation. However, after spray-drying beta-galactosidase in the presence of HP-beta-CD, or HP-beta-CD and sucrose, full catalytic activity was exhibited on reconstitution. Furthermore, the reconstituted product was unchanged in terms of molecular weight, charge, and thermal stability. These findings are consistent with a hypothesis that the change responsible for inactivation of beta-galactosidase was mainly a monomolecular, noncovalent change, i. e., the formation of incorrect structures, that arose from surface denaturation. This study clearly demonstrates that cyclodextrins can be useful stabilizing excipients in the preparation of spray-dried protein pharmaceuticals.

2-Hydroxypropyl-beta-cyclodextrin↗