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PubMed2003

[Capillary electrophoresis for monitoring stability of pharmaceutical proteins].

Like chemical drugs, pharmaceutical proteins have to be of strictly controlled purity to become biopharmaceuticals. Detection, identification and characterization of impurities and compounds present in purified biopharmaceuticals are of central interest. Simultaneous development of structural biology, biotechnologies and of highly resolutive analytical tools, allow significant progress in this field to be achieved. Thus, capillary electrophoresis can be advantageously used for monitoring protein stability. As illustrated by the examples of potential enzymes for detoxification of organophosphates, prophylaxis and treatment of poisoning by pesticides and nerve agents or for skin decontamination, the present feature article shows that this new methodological approach allows: to determine purity and homogeneity of proteins;to analyze their resistance to denaturing conditions such as heat or high electric fields;to detect the presence of unwanted hidden protein-bound ligands capable of altering functional conformation and stability of enzymes, and moreover susceptible to be released and to induce side effects, such as immunologic response; and to establish the role of parameters controlling the "good compromise" between conformational stability and plasticity for allowing optimal functional efficiency of enzymes. Finally, capillary electrophoresis has proved to be a pertinent tool to validate the conformity of purified enzymes to a status of biopharmaceutical.

Acetylcholinesterase↗
PubMed1996

Current issues in viral assays and viral clearance evaluation.

Viral contaminants are less amenable then chemical contaminants to analytical detection due to the lower sensitivity, specificity and precision of existing assays. In combination with the potential for biological amplification of viral contaminants in cell substrates or product recipients these analytical limitations lead directly to potential risk of virus infection of product recipients. While less than perfect, a broad spectrum of assays is available to characterize cell banks for the presence of adventitious and endogenous agents. Each product lot can be tested by an appropriate subset of assays for selected viruses of concern. Engineering and procedural controls over facilities, equipment and operations are necessary to minimize the possibility of viral contamination. Processes can be designed to include unit operations to remove or inactivate specific viruses of potential concern; the efficacy of these measures can be evaluated. While each of these measures has value, each also has limitations. None is solely adequate by itself to the task of preventing potential exposure to viral contaminants in product recipients. Biopharmaceuticals (purified characterizable macromolecules) are prepared by using a combination of such preventive measures. Specific measures will vary from product to product, depending on the nature and origin of the cell substrate, cell bank characterization results, raw materials used, physicochemical properties of the biopharmaceutical, effectiveness of assays, available facilities, and the agents of concern. Thus, flexibility in approach is necessary, resulting in different combinations of measures for each set of circumstances. So far, this approach has been successful in preventing viral infection in large populations treated to date with cell culture-derived biopharmaceuticals. Current issues for discussion include appropriate assays for adventitious and endogenous agents, appropriateness of current control systems and the scientific basis for standards.

Biological Products↗
PubMedDate not supplied

Appropriate specifications at the IND stage.

Biopharmaceutical product specifications are to be "scientifically sound and appropriate." However, how does a biopharmaceutical company determine acceptable specifications for its product at the early stages of clinical development? Are there really enough test data at the start of the IND trials to "establish" specifications? As an industry, are we doing too many tests at the IND stage (i.e., the "must do every test on someone's published list" syndrome), and then finding that specifications need to be set for each test we run? Are we feeling pressured (either by regulatory agencies or by our own corporate cultures) to set unrealistic and light specifications at the IND stage, and then later regretting having to justify loosening them when we gain more experience with the release testing or stability of our products? A discussion on how to set practical product specifications for biopharmaceutical products at the IND stage is presented.

Chromatography, High Pressure Liquid↗
PubMed2005

IV-IVC considerations in the development of immediate-release oral dosage form.

Predictive scientific principles and methods to assess in vivo performance of pharmaceutical dosage forms based on in vitro studies are important in order to minimize costly animal and human experiments during drug development. Because of issues related to poor solubility and low permeability of newer drug candidates, there has in recent years been a special focus on in vitro-in vivo correlation (IV-IVC) of drug products, particularly those used orally. Various physicochemical, biopharmaceutical, and physiological factors that need to be considered in successful IV-IVC of immediate-release oral dosage forms are reviewed in this article. The physicochemical factors include drug solubility in water and physiologically relevant aqueous media, pK(a) and drug ionization characteristics, salt formation, drug diffusion-layer pH, particle size, polymorphism of drug substance, and so forth. The biopharmaceutical factors that need to be considered include effects of drug ionization, partition coefficient, polar surface area, etc., on drug permeability, and some of the physiological factors are gastrointestinal (GI) content, GI pH, GI transit time, etc. Various in silico, in vitro, and in vivo methods of estimating drug permeability and absorption are discussed. Additionally, how IV-IVC may be applied to immediate-release oral dosage form design are presented.

Animals↗
PubMed2006

Xbp1-based engineering of secretory capacity enhances the productivity of Chinese hamster ovary cells.

A variety of successful transcription and translation engineering strategies implemented during the past decade have driven the specific productivity of mammalian cells to an apparent limit. Restricted post-translation competence has since been considered the major bottleneck preventing mammalian cells from fully exploiting their physiologic production capacity in a biopharmaceutical manufacturing scenario. Through ectopic expression of the human transcription factor Xbp1 (X-box-binding-protein 1), evolved to manage plasma cell differentiation and coordinate the unfolded protein response, we have specifically expanded the endoplasmic reticulum and the Golgi of transgenic Chinese hamster ovary (CHO-K1)-derived cell lines with a resulting increase in overall production capacity. Xbp-1-based engineering of secretory bottlenecks was compatible with a variety of different promoter–product gene configurations suggesting that Xbp-1 induces generic production increases in CHO-K1 cell derivatives. Secretion engineering, illustrated here by Xbp1-based reprogramming of the post-translational processing machinery, provides a first insight into mastering a major system bottleneck which impacts biopharmaceutical manufacturing of secreted protein therapeutics.

Animals↗
PubMed2002

Immunogenicity of rDNA-derived pharmaceuticals.

Many biopharmaceuticals have been reported to induce the production of antibodies. Several factors influence immunogenicity, including the methods of production, formulation and storage as well as patient characteristics (e.g. type of disease and genetic background). Because immunogenicity is unpredictable, testing for antibodies should always be part of the evaluation of new biopharmaceuticals, although the biological consequences of these antibodies are rarely severe.

Animals↗
PubMed2002

Milestones in chloroplast genetic engineering: an environmentally friendly era in biotechnology.

Chloroplast genomes defied the laws of Mendelian inheritance at the dawn of plant genetics, and continue to defy the mainstream approach to biotechnology, leading the field in an environmentally friendly direction. Recent success in engineering the chloroplast genome for resistance to herbicides, insects, disease and drought, and for production of biopharmaceuticals, has opened the door to a new era in biotechnology. The successful engineering of tomato chromoplasts for high-level transgene expression in fruits, coupled to hyper-expression of vaccine antigens, and the use of plant-derived antibiotic-free selectable markers, augur well for oral delivery of edible vaccines and biopharmaceuticals that are currently beyond the reach of those who need them most.

Animals↗
PubMed2005

Strategies to improve oral drug bioavailability.

Efforts to improve oral drug bioavailability have grown in parallel with the pharmaceutical industry. As the number and chemical diversity of drugs has increased, new strategies have been required to develop orally active therapeutics. The past two decades have been characterised by an increased understanding of the causes of low bioavailability and a great deal of innovation in oral drug delivery technologies, marked by an unprecedented growth of the drug delivery industry. The advent of biotechnology and consequent proliferation of biopharmaceuticals have brought new challenges to the drug delivery field. In spite of the difficulties associated with developing oral forms of this type of therapeutics, significant progress has been made in the past few years, with some oral proteins, peptides and other macromolecules currently advancing through clinical trials. This article reviews the approaches that have been successfully applied to improve oral drug bioavailability, primarily, prodrug strategies, lead optimisation through medicinal chemistry and formulation design. Specific strategies to improve the oral bioavailability of biopharmaceuticals are also discussed.

Absorption↗
PubMed2004

Regulation of biogenerics: a survey of viewpoints.

BACKGROUND AND OBJECTIVES: 'Biogenerics' regulation has brought about a heated debate within the EU as the first biopharmaceuticals are going off patent. This study aims to examine the opportunities and challenges offered by biogenerics by surveying the viewpoints of experts in regulatory agencies and in companies developing novel biopharmaceuticals and biogenerics. METHODS: Oral interviews were conducted in 2002 and 2003 with experts including representatives of the European Generic Medicines Association (EGA) and the European Federation of Pharmaceutical Industries and Associations (EFPIA) in Brussels, three innovator biotech companies, and five other experts in Finland. Additionally, four biogenerics companies and one innovator company abroad answered a structured, written questionnaire. RESULTS AND CONCLUSION: According to this study, biogenerics should be regulated on a case-by-case basis. The interviewees were not unanimous as to whether comparability can be addressed and which are the most challenging areas for proving comparability. Immunogenicity was considered to be a major problem for biogenerics. Therefore, a requirement for an intensified monitoring of the safety profile during post-marketing was thought to be justified in many cases.

Biological Products↗
PubMed1993

Regulatory aspects of modified release dosage forms: special cases of dissolution testing using the flow-through system.

More than 30 years have been devoted to characterizing the biopharmaceutical properties of drug products. The numerous tests developed are generally based on two distinct methodologies--a closed system (beaker method) and an open system (flow-through method). Selected methods were finally standardized and described in such pharmacopoeias as Ph. Eur. 2, USP XXII and Ph. Jap. XII. The most common procedures are the paddle and basket methods. An alternative method--the flow--through method--was also introduced into the pharmacopoeias Ph. Eur. 2 and USP XXII. The advantages of the flow-through method are evident with regard to testing and assessing different types of dosage forms and active ingredients of very slight solubility. Changes of testing fluids (e.g. change to the pH) can be easily performed during the test. Another advantage is seen in the positioning of the specimen. Capsules, even when floating initially, or pellets can be tested using the same equipment and require no additional devices such as sinkers. With slight changes to the cell design, the same apparatus can be used for the testing of powders, or even non-solid dosage forms such as suppositories or soft-gelatine capsules. For long-term testing necessary for extended release products, flow-through apparatus offers a considerable number of advantages for routine work. Irrespective of the methodologies used, dissolution tests should be validated with regard to analytical methods, testing conditions and specifications. The validation of the analytical method should follow EC guidelines. Testing conditions should take into account the choice of the correct apparatus, dissolution media and agitation. The results of in vitro dissolution should be validated by in vivo data in an analogous sense. The practical application of flow-through apparatus is presented on the basis of various examples regarding development and batch control. The assessment of in vitro dissolution of biopharmaceutical characterization of drug substances, enteric-coated tablets, controlled release products, suppositories and implants will be discussed.

Biopharmaceutics↗
PubMed2002

Detection of unwanted protein-bound ligands by capillary zone electrophoresis: the case of hidden ligands that stabilize cholinesterase conformation.

Detection, identification and characterization of compounds present in purified proteins and biopharmaceuticals are of central interest. As well as chemical remedies, proteins of pharmacological interest have to exhibit their nakedness to become therapeutic drugs. Cholinesterases (ChE) are enzymes of major importance for detoxification of poisonous esters. Likewise, ChE are characterized by the high catalytic efficiency of an active site positioned at the bottom of a deep gorge. The gorge can be partially or fully occupied by ligands, i.e., substrates and inhibitors that are currently used in affinity chromatography purification steps. Accordingly, a suitable method allowing to analyse the presence of unwanted ligands and its influence on the functional conformation and stability of these enzymes was essential. We have developed CZE approaches for that purpose. The factors causing discrepancies between data for thermal unfolding of ChE by electrophoretic and by calorimetric methods were investigated. The presence of unwanted hidden ligands bound to purified enzymes was first demonstrated. The incidence of these ligands was discussed. Altogether, our results raised several questions concerning the real conformation of the native state of enzymes. Finally, CZE was proved to be a pertinent tool to validate the conformity of purified enzymes to a status of biopharmaceutical.

Acetylcholinesterase↗
PubMedDate not supplied

Comparative bioavailability of two oral formulations of flurazepam in human subjects.

The systemic availability of an investigational oral formulation of flurazepam was compared to that of a commercially available product whose therapeutic efficacy has been well established by usage. The experiment was designed to dissociate formula on factors from all other sources of variation including differences between subjects, sexes, sequences of administration, experimental periods, as well as sex by sequence, sex by period, and sex by formulation interactions. Systemic availability was assessed by conventional pharmacokinetic techniques. Pharmacokinetic interpretation and statistical analysis of plasma concentrations of flurazepam and its major blood metabolites namely N-1-hydroxyethylfurazepam and N-1-desalkylflurazepam as a function of time and of systemic availability indicators revealed a nearly identical biopharmaceutical behaviour for the two preparations. A significant difference could be seen in the plasma levels of N-1-desalkylflurazepam between male and female subjects. The results collectively indicate a very similar biopharmaceutical performance of the two oral formulations of flurazepam.

Adult↗
PubMed2006

Heterologous protein production capacity of mammalian cells cultivated as monolayers and microtissues.

A precise understanding of processes managing heterologous protein production in vitro and in vivo is essential for the manufacture of sophisticated biopharmaceuticals as well as for future gene therapy and tissue engineering initiatives. Capitalizing on the gravity-enforced self-assembly of monodispersed cells into coherent (multicellular) microtissues we studied heterologous protein production of microtissues and monolayers derived from cell lines and primary cells engineered/transduced for (i) constitutive, (ii) proliferation-controlled, (iii) macrolide-, or (iv) gas-inducible expression of the human placental secreted alkaline phosphatase (SEAP) and of the Bacillus stearothermophilus-derived secreted alpha-amylase (SAMY). Specific productivity of cells assembled in microtissues was up to 20-fold higher than isogenic monolayer cultures. Diffusion across microtissues could be further increased by HUVEC-mediated vascularization. As well as higher specific protein productivities, microtissues were also more efficient than monolayer cultures in assembling transgenic lentiviral particles. Our results showed that mammalian cells embedded in a tissue-like three-dimensional (3D) microenvironment exhibit increased production capacity. This observation should be considered for gene therapy and tissue engineering scenarios as well as for biopharmaceutical manufacturing.

Alkaline Phosphatase↗
PubMed2005

Engineered vascular-targeting antibody-interferon-gamma fusion protein for cancer therapy.

A number of cytokines are either approved drugs or are in advanced clinical trials, yet these biopharmaceuticals do not typically localize efficiently in solid tumors and manifest their therapeutic potential at the expense of severe side effects. The targeted delivery of cytokines to solid tumors is a promising avenue for increasing the therapeutic index of these biopharmaceuticals. We engineered a fusion protein between scFv(L19), a human antibody fragment specific to the EDB domain of fibronectin, and a cysteine-free mutant of murine interferon-gamma. The resulting fusion protein was capable of targeting new blood vessels in solid tumors, and the targeting efficiency was strikingly increased in tumor-bearing knockout mice lacking the interferon-gamma receptor. ScFv(L19)-interferon-gamma displayed a strong antitumor effect in both subcutaneous and metastatic murine F9 teratocarcinomas, but was not efficacious as single agent when used to treat C51 and CT26 tumors. The potency of this fusion protein could be substantially enhanced by combination with doxorubicin and other immunocytokines. These findings are of clinical relevance, as the EDB domain is a marker of angiogenesis, with identical sequence in mouse and man, which is abundantly expressed in a variety of aggressive solid tumors but is undetectable in most normal tissues.

Adenocarcinoma↗
PubMedDate not supplied

Dual-regulated expression technology: a new era in the adjustment of heterologous gene expression in mammalian cells.

BACKGROUND: On the basis of the compatible streptogramin- and tetracycline-responsive expression systems, a series of dual-regulated expression systems have been established for use in sophisticated biopharmaceutical manufacturing, advanced gene therapy, and tissue engineering. METHODS: Dual-regulated expression concepts enable streptogramin- and tetracycline-responsive control of two different (sets of) transgenes (multi-regulated multigene metabolic engineering), dual-autoregulated expression configurations for one-step chromosomal integration of two antibiotic-adjustable expression units, and artificial regulatory cascades for multi-level regulation of transgenes and optimized integration of molecular interventions into mammalian regulatory networks. RESULTS: This report describes the construction and testing of a family of dual-regulated expression vectors which are compatible with the pTRIDENT vector construction kit, and, in some cases, adapted for retroviral expression technology enabling straightforward transduction of difficult-to-transfect cell lines such as primary cells and stem cells. CONCLUSIONS: Dual-regulated expression technology will probably become of prime interest for a variety of therapeutic applications, including biopharmaceutical manufacturing, gene therapy, and tissue engineering.

Animals↗
PubMed2003

General pharmaceutics--the new physical pharmacy.

General Pharmaceutics is proposed as the broad study of the biopharmaceutical and physical chemical properties of each potential drug substance. When the first quality bulk lot is delivered, usually the first GMP bulk lot, an extensive profiling of the potential drug substance should commence. This profile should include solid-state characterization as well as thorough analyses of solubility, stability, and absorption properties of the drug substance that could affect the development of a viable medicine. As a result of these studies, a number of initial specifications could be developed: the preferred polymorphic or crystalline form identified, the preferred particle size to optimize absorption/development, and an initial biopharmaceutics classification with a dose limit to identify those cases in which the formulation can be expected to improve absorption and exposure. The broad topic of General Pharmaceutics is discussed in this Minireview including many advances in technology in this field as well as the rationale behind the proposed initial specifications.

Dosage Forms↗
PubMed2004

Frog intestinal sac: a new in vitro method for the assessment of intestinal permeability.

The aim of this study was to evaluate a new experimental protocol utilizing isolated frog intestinal sacs for the assessment of intestinal drug permeability in humans. Segments of approximately 5.0 cm in length were used for these experiments. The intestinal sacs were filled with a solution of the appropriate drug in frog Ringer (FR) and immersed in a vial containing fresh FR. The transport was monitored for a period ranging from 2 to 5 h by moving the intestinal sac at each time point to a new vial containing fresh medium (Method A). Alternatively, according to Method B, at predetermined times aliquots of receiving mixture were taken up without removing the intestinal sac, and replaced with fresh drug-free FR. In all cases, the samples were analyzed by HPLC. A series of 20 noncongeneric drugs, predominantly absorbed by passive diffusion mechanism, was examined. The results indicate that drugs completely absorbed in humans had Papp values greater than 1 x 10(-6) cm/s, while drugs absorbed <90% had Papp values lower than 1 x 10(-6) cm/s. By plotting Log Papp values against percent human absorption, an approximately sigmoidal relationship was obtained. The frog intestinal sac method was evaluated as a permeability model to classify the 20 studied drugs into the Biopharmaceutics Classification System (BCS). By comparing the predictions made by this new approach with those reported in literature, it can be concluded that a satisfactory biopharmaceutical classification may be based on the Papp values determined by the frog intestinal permeability. Molecular properties relevant to passive intestinal permeability were considered to evaluate the correlation with the frog intestinal permeability rates recorded. Good relationships were observed when hydrogen-bonding parameters were expressed as a function of the Log Papp values. It can be concluded that the in vitro permeability coefficient deduced from isolated frog intestinal experiments can be used for predicting peroral absorption in humans for passively absorbed compounds. Computational methods for prediction of frog intestinal permeability may be applied in a highly simplified manner.

Animals↗
PubMed2001

Evaluation of protein modification during anti-viral heat bioprocessing by electrospray ionization mass spectrometry.

During the preparation of therapeutic plasma and recombinant protein biopharmaceuticals heat-treatment is routinely applied as a means of viral inactivation. However, as most proteins denature and aggregate under heat stress, it is necessary to add thermostabilizing excipients to protein formulations destined for anti-viral heat-treatment in order to prevent protein damage. Anti-viral heat-treatment bioprocessing therefore requires that a balance be found between the bioprocessing conditions, virus kill and protein integrity. In this study we have utilized a simple model protein, beta-lactoglobulin, to investigate the relationship between virucidal heat-treatment conditions (protein formulation and temperature) and the type and extent of protein modification in the liquid state. A variety of industrially relevant heat-treatments were undertaken, using formulations that included sucrose as a thermostabilizing excipient. Using liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) we show here that protein modifications do occur with increasingly harsh heat-treatment. The predominant modification under these conditions was protein glycation by either glucose or fructose derived from hydrolyzed sucrose. Advanced glycation end products and additional unidentified products were also present in beta-lactoglobulin protein samples subjected to extended heat-treatment. These findings have implications for the improvement of anti-viral heat-treatment bioprocesses to ensure the safety and efficacy of protein biopharmaceuticals. CopyrightCopyright 2001 John Wiley & Sons, Ltd.

Blood Proteins↗