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PubMedDate not supplied

Perspectives of in vitro dissolution tests in establishing in vivo/in vitro correlations.

Perspectives of in vitro dissolution tests in establishing an in vivo/in vitro correlation are considered at three different levels. The first involves the selection of a drug product, or suitable batch(es), for the correlation study. This requires evaluation of the biopharmaceutical properties of the proposed drug from a profile compiled from a set of dissolution tests under different experimental conditions. These characteristics are needed for properly designing the in vivo study and are also referred to when only a poor correlation is obtained. The second is to define the specific in vitro test system either prospectively, by assessing plausibility of in vitro results, or retrospectively, after interpretation of in vivo data. This distinct test system has to be validated both physically and analytically. The third aspect of dissolution testing for correlations is to provide sound data and derived parameters in consideration of statistical aspects (variability) and of a sufficient number of suitable sampling intervals. In vitro models proposed for the special study of the simulation of food effects on bioavailability are discussed but concluded to be as yet not satisfactory for general application.

Animals↗

PubMed1998

Apoptosis and bioprocess technology.

Optimisation of the production of biopharmaceuticals in animal cell lines has become a key area of research. The identification of apoptosis as the major mechanism of cell death during such processes has raised the importance of studies of cell death when implementing culture optimisation strategies. In this article we present an overview of the studies which have demonstrated the induction of apoptosis during the cultivation of industrially important animal cell lines. We also discuss studies which have shown that deprivation of factors such as amino acids, glucose, serum and oxygen are potent inducers of apoptosis in industrial cultures. The suppression of apoptosis under these conditions has been demonstrated by a number of recent reports, and we describe ways in which this knowledge may be applied in the development of novel solutions to some of the technical problems associated with the development of successful large scale culture process. The article concludes with a discussion of future directions for apoptosis research in bioprocess technology.

Animals↗

PubMed2005

[New guidelines for the assessment of bioavailability and bioequivalence].

Bioavailability and bioequivalence studies are essential in the clinical development of medicinal products and the optimization of pharmaceutical forms. Bioavailability means the rate and extent to which the active substance or active moiety is absorbed from a pharmaceutical form and becomes available at the site of action. In practice, drug concentration-time courses are measured in the systemic circulation, and the area under the curve (AUC) as well as the observed maximum concentration (C(max)) are determined. Products are considered bioequivalent if their bioavailabilities after administration of the same molar doses are similar to such a degree that their effects, with respect to both efficacy and safety, will be essentially the same and thus, there are no relevant differences in terms of AUC and C(max). In 2002 a revised version of the 'Note for Guidance on the Investigation of Bioavailability and Bioequivalence' came into effect (CPMP/EWP/QWP/1401/98). Relevant changes in comparison to the previous version are: request for GLP-compliant bioanalytical measurements; for long half-life drugs a truncated AUC is acceptable; acceptance criteria for bioequivalence assessment and requirements for a waiver of bioequivalence studies were further specified. In this context the Biopharmaceutics Classification System (BCS) seems appropriate to decide whether in special cases of rapidly dissolving solid oral dosage forms a biowaiver may be granted or not. Products not considered critical in this matter are medicinal products for which the formulation does not affect the rate and extent of absorption, i. e. bioavailability, of the active moiety. Highly soluble (and highly permeable) drugs (BCS class I) are such candidates. Comprehensive state-of-the-art guidance on the design, conduct and analysis of bioavailability and bioequivalence studies is given in the current European guideline.

Administration, Oral↗

PubMed2002

Direct analysis of nine pharmaceuticals in culture media by use of cartridge separation with electrospray mass spectrometric detection.

A 2-cm cartridge has been used for separation before electrospray mass spectrometric analysis of pharmaceutical compounds in cell culture media, alleviating the need for sample extraction and desalting procedures. Nine representative pharmaceuticals listed in the biopharmaceutical classification system (BCS) were chosen as the candidate compounds and Hank's balanced salt solution with Hepes buffer (HBSS-Hepes buffer) was used as the cell-culture medium in an effort to study permeability of chemicals through cell monolayers. Effects of several conditions, e.g. pH and buffer concentration in the mobile phase, flow rate, and temperature on separation efficiency were examined. The nine pharmaceuticals were separated within 2 min by use of a 2-cm C(8) cartridge. Relative standard deviations (RSD) from repeated analysis within the same day or over five days were 0.03-0.2% for retention times and 0.6-5.3% for peak areas; antipyrine was used as internal standard. Calibration curves based on peak-area measurements were linear over the range 0.1-20 micro mol L(-1). The HBSS-Hepes buffer did not interfere with separation and detection; identical separation and peak intensity were obtained when the samples were separately prepared in distilled water or in the culture medium.

Adrenergic beta-Antagonists↗

PubMed2004

Therapeutic insulins and their large-scale manufacture.

Biotechnological innovations over the past 25 years have underpinned the rapid development of a thriving biopharmaceutical sector. Therapeutic insulin remains one of the most commonly used products of pharmaceutical biotechnology and insulin-based products command annual global sales in excess of $4.5 billion. Innovations in its method of production and in particular the advent of engineered insulin analogues provide a fascinating insight into how scientific and technological advances have impacted upon the pharmaceutical biotechnology sector as a whole. Current insulin-based diabetes research is increasingly focused not on the insulin molecule per se, but upon areas such as the development of non-parenteral insulin delivery systems, as well as organ-/cell-based and gene therapy-based approaches to controlling the disease.

Amino Acid Sequence↗

PubMed2006

Characterization of the 16S-23S internal transcribed spacer among 34 higher plants: suitability for interspecific plastid transformation.

Biomanufacturing by chloroplast transgene expression has the potential to produce significant amounts of biopharmaceuticals, endow plants with novel commercial or humanitarian capabilities, enhance phytoremediation methods and harden plants against adverse environments. Plastid bioengineering exploits the phenomenon of homologous recombination to specifically integrate heterologous sequences into the plastid genome. Previous research suggests the plastid genome 16S-23S internal transcribed spacer provides an advantageous integration site for transgene expression. To characterize the suitability of the 16S-23S region for interspecific recombination, we developed primers against conserved plastid sequences and amplified approximately 2.6 kb from 25 plant species. We analyzed the amplicons with nine species from Genbank for homeology, phylogenetic relationships, potential to form chimeric rDNA elements disruptive to translational/replication systems, and the potential number of recombination events for various minimal essential processing segments (MEPS) lengths. Multiple sequence alignment of the 34 species revealed considerable conservation, with identities exceeding 95% among the angiosperms. Substitutions were statistically clustered, generally in noncoding sites, although proposed functional elements such as the OriA region and 3' terminus of the 16S rRNA exhibited unexpected variation. The nonrandom distribution of substitutions undermines the established, statistical method of estimating the number of recombination initiation sites. This finding is further substantiated by comparing statistical estimates of the number of MEPS sites to a direct count at three different MEPS lengths. We frame this in silico analysis in terms of the potential of the 16S-23S region as a target for interspecific transformation, and describe a 'primer-to-plastid' system to rapidly generate species-specific flanking regions for transformation vectors.

Base Sequence↗

PubMed2004

Assessing the viability of a clumpy mnn9 strain of Saccharomyces cerevisiae used in the manufacture of recombinant pharmaceutical proteins.

Demonstration of the viability of cryopreserved cell bank used to make a biopharmaceutical product is an important indicator of the ability to consistently manufacture over a long period of time, and is mandated in regulatory guidances. A mnn9 strain of Saccharomyces cerevisiae, chosen for its inability to hypermannosylate vaccine antigens, has a clumpy growth tendency due to the inactivation of the gene MNN9 (wild-type), complicating the interpretation of conventional viability measurements useful for single cells. Therefore, two growth-based measurements as well as staining by a membrane-impermeable dye were examined for their ability to reflect changes in viability of a clumpy mnn9 (defective) strain. The cell clumps proved to be stable to mixing, and variability of agar-plate-based viable counts (VC) of undisrupted suspensions of this clumpy mnn9 strain was consistent with variability observed for cell banks of a non-clumpy MNN9 strain. Both the VC and the growth times in an oxygen-sensing broth-based microplate assay corresponded well with shake-flask growth times for a set of stressed and unstressed samples, although the correlation was highest between the two broth-based systems. Counts of trypan-blue-stained cells within clumps also increased with time of stress, suggesting that this method could be adapted as a simple index of viability as well.

Biotechnology↗

PubMed2005

Purification and characterization of recombinant truncated human interleukin-11 expressed as fusion protein in Escherichia coli.

Mature human interleukin-11 (HuIL-11) is a cytokine consisting of 178 amino acid residues that results from scission of the N-terminal signal peptide, consisting of 21 amino acid residaues, from the corresponding nascent polypeptide. A DNA fragment encoding a truncated HuIL-11 (trHuIL-11), with an additional 5 amino acid residues removed from the N-terminus, was cloned into vector pGEX-2T between the BamHI site and the EcoRI site. Upon transformation with Escherichia coli BL21, the construct over-produced a glutathione S-transferase (GST)-fused protein in a soluble form after IPTG induction. The fusion protein was initially fractionated with butyl-Sepharose 4 fast flow column and by affinity chromatography using a GSH-Sepharose 4B column. On-site enzymatic release with thrombin gave the target protein at 96% purity as judged by SDS-PAGE and HPLC. Expression of the interleukin as a GST-fused protein thus greatly improved downstream processing. Subsequent biological activity assay suggested that trHuIL-11 had similar activity profile to the naturally produced sample and may be a promising candidate for further development as biopharmaceutical.

Amino Acid Sequence↗

PubMed2006

Modeling suppression of cell death by Bcl-2 over-expression in myeloma NS0 6A1 cells.

A novel population-balance model was employed to evaluate the suppression of cell death in myeloma NS0 6A1 cells metabolically engineered to over-express the apoptotic suppressor Bcl-2. The model is robust in its ability to simulate cell population dynamics in batch suspension culture and in response to thymidine-induced growth inhibition: 89% of simulated cell concentrations are within two standard deviations of experimental data. Kinetic rate constants in model equations suggest that Bcl-2 over-expression extends culture longevity from 6 days to at least 15 days by suppressing the specific rate of early apoptotic cell formation by more than 6-fold and necrotic cell formation by at least 3-fold, despite nearly a 3-fold decrease in initial cell growth rate and no significant change in the specific rate of late apoptotic cell formation. This computational analysis supports a mechanism in which Bcl-2 is a common mediator of early apoptotic and necrotic events occurring at rates that are dependent on cellular factors accumulating over time. The model has current application to the rational design of cell cultures through metabolic engineering for the industrial production of biopharmaceuticals.

Apoptosis↗

PubMed2005

Large-scale transient transfection of mammalian cells: a newly emerging attractive option for recombinant protein production.

Mammalian expression systems have an undisputed long-standing and very successful history for the generation of recombinant proteins, mainly as biopharmaceuticals. However, for use as 'tool proteins' in, e.g. assay development and screening, for structure elucidation and as antigens these expression systems were generally regarded as being cumbersome, tedious and expensive. This bias has largely been overcome with the very recent development of large-scale transient transfection (LST) approaches. Especially the HEK.EBNA expression system described here has contributed significantly to this success. The simplicity and speed of this approach compares well with expression trials using the widely applied Baculovirus/insect cell system. In addition, proteins generated in mammalian cells are usually correctly folded, fully processed and functionally active.

Bioreactors↗

PubMed2005

Novel long-acting crystal formulation of human growth hormone.

PURPOSE: The aim of the study is to solve a significant challenge of extending the half-life of therapeutic proteins using crystalline biopharmaceuticals and without redesigning the molecules. METHODS: Crystals of recombinant human growth hormone were coated with a monomolecular layer of positively charged poly(arginine). The pharmacokinetics and pharmacodynamics of this poly(arginine)-coated human growth hormone crystalline formulation were determined in hypophysectomized rats and monkeys. RESULTS: Here we have demonstrated for the first time that crystals of human growth hormone coated with positively charged poly(arginine) allowed for in vivo pharmacokinetic release profiles of over several days in animal models. The efficacy of this crystalline formulation injected subcutaneously once a week was found to be equivalent to seven daily soluble injections in the standard weight gain assay using the hypophysectomized rat model and in measurement of serum insulin-like growth factor in monkeys. The nonviscous nature of the suspension facilitated easy administration through a fine, 30-gauge needle and should provide for improved patient convenience and compliance. CONCLUSIONS: The approach described here offers an exciting possibility of being broadly applicable to other therapeutic proteins.

Adsorption↗

PubMed2006

Analysis of dissolution data using modified versions of Noyes-Whitney equation and the Weibull function.

PURPOSE: The aim of the study is to develop modified, branched versions of the Noyes-Whitney and the Weibull equations, including explicitly the solubility/dose parameter, for the analysis of dissolution data, which reach the plateau either at infinite or finite time. METHODS: The modified Weibull function is applied to the analysis of experimental and literature dissolution data. To demonstrate the usefulness of the mathematical models, two model drugs are used: one highly soluble, metoprolol, and one relatively insoluble, ibuprofen. RESULTS: The models were fitted successfully to the data performing better compared with their classic versions. The advantages of the use of the models presented are several. They fit better to a large range of datasets, especially for fast dissolution curves that reach complete dissolution at a finite time. Also, the modified Weibull presented can be derived from differential equations, and it has a physical meaning as opposed to the purely empirical character of the original Weibull equation. The exponent of the Weibull equation can be attributed to the heterogeneity of the process and can be explained by fractal kinetics concepts. Also, the solubility/dose ratio is present explicitly as a parameter and allows to obtain estimates of the solubility even when the dissolution data do not reach the solubility level. CONCLUSION: The use of the developed branched equations gives better fittings and specific physical meaning to the dissolution parameters. Also, the findings underline the fact that even in the simplest, first-order case, the speed of the dissolution process depends on the dose, a fact of great importance in biopharmaceutic classification for regulatory purposes.

Adrenergic beta-Antagonists↗

PubMed2006

Particle size analysis in pharmaceutics: principles, methods and applications.

Physicochemical and biopharmaceutical properties of drug substances and dosage forms can be highly affected by the particle size, a critical process parameter in pharmaceutical production. The fundamental issue with particle size analysis is the variety of equivalent particle diameters generated by different methods, which is largely ascribable to the particle shape and particle dispersion mechanism involved. Thus, to enable selection of the most appropriate or optimal sizing technique, cross-correlation between different techniques may be required. This review offers an in-depth discussion on particle size analysis pertaining to specific pharmaceutical applications and regulatory aspects, fundamental principles and terminology, instrumentation types, data presentation and interpretation, in-line and process analytical technology. For illustration purposes, special consideration is given to the analysis of aerosols using time-of-flight and cascade impactor measurements, which is supported by a computational analysis conducted for this review.

Aerosols↗

PubMed2004

Generation and phenotypic analysis of a transgenic line of rabbits secreting active recombinant human erythropoietin in the milk.

Production of recombinant human erythropoietin (rhEPO) for therapeutic purposes relies on its expression in selected clones of transfected mammalian cells. Alternatively, this glycoprotein can be produced by targeted secretion into the body fluid of transgenic mammals. Here, we report on the generation of a transgenic rabbits producing rhEPO in the lactating mammary gland. Transgenic individuals are viable, fertile and transmit the rhEPO gene to the offspring. Northern blot data indicated that the expression of the transgene in the mammary gland is controlled by whey acidic protien (WAP) regulatory sequences during the period of lactation. While the hybridization with total RNA revealed the expression only in the lactating mammary gland, the highly sensitive combinatory approach using RT-PCR/hybridization technique detected a minor ectopic expression. The level of rhEPO secretion in the founder female, measured in the period of lactation, varied in the range of 60-178 and 60-162 mIU/ml in the milk and blood plasma, respectively. Biological activity of the milk rhEPO was confirmed by a standard [3H]-thymidine incorporation test. Thus, we describe the model of a rhEPO-transgenic rabbit, valuable for studies of rhEPO glycosylation and function, which can be useful for the development of transgenic approaches designed for the preparation of recombinant proteins by alternative biopharmaceutical production.

Animals↗

PubMed2006

Dynamic control of oligosaccharide modification in the mammary gland: linking recombinant human erythropoietin functional analysis of transgenic mouse milk-derived hEPO.

We analyzed two transgenic mouse lines that secrete rhEPO in their milk to assess the dynamic control of N-linked oligosaccharides. Since pharmaceutically available epoetin alpha and beta are produced in CHO cells, we compared transgenic mammary gland-derived rhEPO to its CHO cell-derived counterpart. The major glycosyltransferases that determine the N-oligosaccharides patterns of rhEPO include N-acetylglycosaminyltransferase (GnT) and alpha1,3/4 fucosyltransferase (Fuc-TIV), GnT-III, -V and Fuc-TIV expression in the mouse mammary gland is significantly higher than that in Chinese hamster ovary (CHO)-derived cells, where the protein is not detectable. The data suggest that N-linked sugar chain patterns of recombinant glycoproteins, produced by the mammary gland differ, since GnT-III alters the sugar pattern extensively. In our experiments, rhEPO produced by the transgenic mice contains more tetra-acidic oligosaccharide structures than epoetin alpha derived from CHO cells, a rhEPO that is widely used therapeutically. Accordingly, we examined milk-derived rhEPO activity, both in vitro and in vivo. The rhEPO protein purified from the milk of mammary glands upregulates the EPO receptor-mediated expression of the STAT5 gene in MCF-7 cells in a dose-dependent manner, similar to the effects of epoetin alpha. Furthermore, direct injection of rhEPO into the mouse tail vein leads to an increase in the levels of blood components, such as red blood cells and platelets. In light of these findings, we suggest that the mammary glands of transgenic animals provide a sufficient environment to generate rhEPO with post-translational modifications for biopharmaceutical use.

Amino Acid Sequence↗

PubMed2026

A stable and potent buffalo EF1α1 promoter for robust gene expression in mammalian systems.

This study reports the first isolation and characterization of the buffalo EF1α1 promoter, demonstrating its strong gene expression activity both in vitro across diverse cultured cell types and in vivo across multiple mouse organs. Although viral promoters, such as cytomegalovirus (CMV) and simian virus (SV40), are widely used for their strong expression in various cell lines in mammalian expression systems and in animal tissues, they are prone to methylation-induced transcriptional silencing and subsequent loss of exogenous gene expression. The most effective alternative to viral promoters is the synthetic hybrid CAG promoter (cytomegalovirus major immediate-early enhancer combined with the chicken beta-actin promoter) or mammalian cellular promoter such as human elongation factor 1 alpha (hEF1α), which drives strong gene expression but lacks consistency and is limited in their in vivo expression potential due to their vulnerability to epigenetic silencing. To overcome these challenges, the bbEF1α1 promoter was cloned and evaluated both in vitro and in vivo. It consistently drives higher levels of exogenous gene expression than CMV in diverse cell lines. Importantly, transgene expression was achieved in various organs of transgenic mice and in muscle tissue following in vivo electroporation. These findings establish the bbEF1α1 promoter as a powerful ubiquitous driver of gene expression, offering high stability with broad applications in gene therapy, biopharmaceutical production, and functional genomics.

Animals↗

PubMed2026

Generation of germline-transmitting transgenic sheep by piggyBac-mediated transgenesis using pronuclear and cytoplasmic gene injection approaches.

Sheep represent an important large-animal model for biomedical research and biopharmaceutical production. Although the piggyBac transposon system offers efficient and stable genomic integration, the optimal gene delivery strategy for ovine embryos remains unclear. This study evaluated piggyBac-mediated transgenesis using pronuclear injection (PNI) in both in vivo- and in vitro-derived embryos and assessed cytoplasmic injection (CTI) as an alternative approach. In vivo-derived embryos were obtained from superovulated Kıvırcık ewes approximately 40 h after gonadotropin-releasing hormone administration, whereas in vitro-derived embryos were produced from slaughterhouse-derived oocytes. All embryos were injected with the hyperactive piggyBac transposase-based pmhyGENIE-3 construct (10 ng/µL). In vivo-derived embryos were transferred immediately after injection, whereas in vitro-derived embryos were cultured for 3 days and screened for EGFP expression prior to transfer. Among 65 in vitro-derived embryos injected by PNI, no transgenic offspring was obtained. In contrast, PNI of 19 in vivo-derived embryos resulted in one transgenic lamb (5.3%). CTI of 12 in vivo-derived embryos similarly produced one transgenic lamb (8.3%). Whole-genome sequencing of the healthy founder male produced by CTI identified a single detectable genomic integration locus on chromosome 10 within a non-coding RNA locus (LOC121820439). Germline transmission was confirmed by in vitro fertilization using sperm from the founder male, with EGFP expression detected in 10.1% (10/99) resulting embryos. These findings provide proof-of-concept evidence for piggyBac-mediated transgenesis in sheep and support the feasibility of cytoplasmic injection as an alternative gene-delivery approach under the conditions tested.

Animals↗

PubMed1998

Sensitivity of isoenzyme analysis for the detection of interspecies cell line cross-contamination.

The analysis of the gel electrophoresis banding patterns and relative migration distances for the individual isoforms of intracellular enzymes, such as lactate dehydrogenase, purine nucleoside phosphorylase, glucose-6-phosphate dehydrogenase, and malate dehydrogenase, is used routinely in the biopharmaceutical industry for confirmation of cell line species of origin. In the present study, the sensitivity of the technique (AuthentiKit, Innovative Chemistry, Marshfield, MA) for determining interspecies cell line cross-contamination was examined. Extracts were prepared from a CHO-K1 line (AA8, Chinese hamster), MRC-5 (human) cells, and L929 (mouse) cells and from several proportional mixtures of the various binary combinations of cells. The isoenzymes were analyzed according to standard procedures for the technique. Contamination of MRC-5 cells with CHO-K1 or with L929 cells was clearly detectable with each enzyme analyzed. Similarly, the contamination of L929 or CHO-K1 cells with MRC-5 cells was readily apparent with each enzyme. On the other hand, contamination of CHO-K1 cells with L929 cells was only detected with lactate dehydrogenase analysis, and contamination of L929 cells with CHO-K1 cells was not detected with any of the four enzymes examined. For the latter case, the analysis of an additional enzyme (peptidase B) was required. The results indicate that interspecies cross-contamination should be detectable with isoenzyme analysis if the contaminating cells represent at least 10% of the total cell population.

Animals↗