Alternative methods of terminal sterilization for biologically active macromolecules

The traditional perception within the pharmaceutical industry of the manufacture of injectable drug products is that active pharmaceutical ingredients (API) that are peptides, proteins or biopolymers, such as poly(DL-lactide) (PLA) and poly(DL-lactideco-glycolide) (PLGA), cannot be terminally sterilized. This perception exists largely because terminal sterilization is assumed by many to be only carried out by steam sterilization in a standard autoclave. Thus, it is understood that these API candidates must be manufactured by aseptic techniques. With the current technological advances in the area of protein and peptide sterilization, which has largely come from the food industry and has in recent years been developed for pharmaceutical use, techniques have been developed for the terminal sterilization of thermally sensitive APIs and biopolymers. In this review, the focus will be on the four major types of sterilization that are presented in the literature: (i) gamma-irradiation; (ii) e-Beam; (iii) natural light; and (iv) microwave. Each of these sterilization techniques present advantages and disadvantages for use in large-scale terminal sterilization of bioactive macromolecules.     

Protein crystal growth in microgravity.

Protein crystallography is a powerful method for determining the three-dimensional structures of biological macromolecules. Although new methods, such as two-dimensional NMR, have demonstrated promise for determining the structures of small proteins and nucleic acids, the complete atomic arrangements within large proteins can only be determined at present using crystallographic techniques. Such crystallographic studies have been of major importance for establishing structure/function relationships that are fundamental to understanding how enzymes, nucleic acids, and other macromolecules function in biological systems. More recently, crystallographic studies of proteins have become of considerable practical interest within the pharmaceutical and biotechnology industries, as promising tools in drug design and in protein engineering.     

Microfluidic Approaches for the Characterization of Therapeutic Proteins

In the last decades, the pharmaceutical market has experienced an increase in the number of therapeutic proteins. The high activity and selectivity of these macromolecules is often achieved at the expense of complex structures, which exhibit several biophysical properties that must be carefully controlled and optimized for the successful development of these drugs as well as for guaranteeing their quality and safety. This need has motivated the application of a variety of biophysical techniques to analyze properties of therapeutic proteins and protein solutions including interactions, aggregation, solubility, viscosity, and thermal stability. After briefly summarizing currently available experimental approaches, we highlight the emerging possibilities offered by advances in microfluidic technology for the analysis of therapeutic proteins during manufacturing and formulation.     

Characterization of protein and peptide stability and solubility in non-aqueous solvents

Small molecule parenterals have often been formulated as solutions or suspensions in non-aqueous conditions, however, this technology has not found widespread use in the formulation of macromolecules. Formulation of proteins and peptides has primarily been achieved through aqueous solutions or reconstituted lyophilized cakes. The incorporation of non-aqueous techniques has been limited by the lack of general applicability. For example, prediction of solubility, chemical stability, conformational stability (unfolding/denaturation processes), and activity can be difficult. Therefore, macromolecule non-aqueous preformulation work must be performed on a case by case basis. In addition, only a few solvents are pharmaceutically acceptable. This article reviews the characterization of proteins and peptides in a variety of non-aqueous or co-solvent conditions (both acceptable and unacceptable for pharmaceutical applications), and discusses the applicability of non-aqueous conditions for increasing solubility, stability and activity.     

壳聚糖在生物大分子药物给药中的应用

壳聚糖具有促进生物大分子药物透膜渗透和生物粘附作用。利用壳聚糖形成的自身聚集体以及用壳聚糖制备的微粒制剂、混合胶体和包衣制剂等，均可提高生物大分子药物的生物利用度。     

Effects of high-energy electrons and gamma rays directly on protein molecules.

High-energy electrons and gamma rays ionize molecules at random along their trajectories. In each event, chemical bonds are ruptured, releasing radiolytic products that diffuse away. A solution of macromolecules is mostly water whose principal radiation products are H(+) and OH(-). These can diffuse to and react with macromolecules; this indirect action of radiation is responsible for 99.9% of the damage to proteins. In frozen samples, the ionizations still occur randomly and water is still the principle molecular target, but diffusion of radiation products is limited to only a very small distance. At very low temperatures, essentially all the radiation damage to macromolecules is due to primary ionizations occurring directly in those molecules. Therefore, proteins in frozen solutions are only 10(-3) to 10(-4) as sensitive to radiation as in the liquid state. Every molecule that suffered a direct ionization is destroyed; the only surviving molecules are those that escaped ionization. The survival of frozen proteins after irradiation is a direct measure of the mass of the active structures and independent of the presence of other proteins.     

Three ‘D’s: Design approach,dimensional printing,and drug delivery systems as promising tools in healthcare applications

The development of pharmaceutical drug products is required for the treatment of disease, which has resulted in an increasing number of approvals by regulatory agencies across the globe. To establish a hassle-free manufacturing process, the systematic use of a quality-by-design (QbD) approach combined with process analytical technology (PAT) and printing techniques can revolutionize healthcare applications. Printing technology has been emerged in various dimensions, such as 3D, 4D, and 5D printing, with respect to their production capabilities, durability, and accuracy of pharmaceutical manufacturing, which can efficiently deliver novel patient-centric healthcare products with holistic characteristics. In this review, we provide current trends in pharmaceutical product development using a design approach and high-quality printing techniques.     

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.     

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.     

Opportunities and challenges for oral delivery of hydrophobic versus hydrophilic peptide and protein-like drugs using lipid-based technologies

Peptide and protein-like drugs are macromolecules currently produced in increasing numbers by the pharmaceutical biotechnology industry. The physicochemical properties of these molecules posebarriers to oral administration. Lipid-based drug-delivery systems have the potential to overcome these barriers and may be utilized to formulate safe, stable and efficacious oral medicines. This review outlines the design of such lipid-based technologies. The mechanisms whereby these formulations enhance the absorption of lipophilic versus hydrophilic peptide and protein-like drugs are discussed. In the case of lipophilic compounds, the advantages of lipid-based drug-delivery systems including increased solubilization, decreased intestinal efflux, decreased intracellular metabolism and possible lymphatic transport are well established as is evident from the success of Neoral and other drug products on the market. In contrast, with respect to hydrophilic compounds, the situation is more complex and, while promising formulation approaches have been studied, issues including reproducibility of response, intersubject variability and duration of response require further optimization before commercially viable products are possible.     

Application of supercritical fluid to preparation of powders of high-molecular weight drugs for inhalation

The application of supercritical carbon dioxide to particle design has recently emerged as a promising way to produce powders of macromolecules such as proteins and genes. Recently, an insulin powder for inhalation was approved by authorities in Europe and the USA. Other macromolecules for inhalation therapy will follow. In the 1990s proteins were precipitated with supercritical CO(2) from solutions in an organic solvent such as dimethylsulfoxide, which caused significant unfolding of protein. Since 2000, aqueous solutions of proteins and genes have generally been used with a cosolvent such as ethanol to precipitate in CO(2). Operating conditions such as temperature, pressure, flow rates, and concentration of ingredients affect the particle size and integrity of proteins or genes. By optimizing these conditions, the precipitation of proteins and genes with supercritical CO(2) is a promising way to produce protein and gene particles for inhalation.     

Chemistry for peptide and protein PEGylation

Poly(ethylene glycol) (PEG) is a highly investigated polymer for the covalent modification of biological macromolecules and surfaces for many pharmaceutical and biotechnical applications. In the modification of biological macromolecules, peptides and proteins are of extreme importance. Reasons for PEGylation (i.e. the covalent attachment of PEG) of peptides and proteins are numerous and include shielding of antigenic and immunogenic epitopes, shielding receptor-mediated uptake by the reticuloendothelial system (RES), and preventing recognition and degradation by proteolytic enzymes. PEG conjugation also increases the apparent size of the polypeptide, thus reducing the renal filtration and altering biodistribution. An important aspect of PEGylation is the incorporation of various PEG functional groups that are used to attach the PEG to the peptide or protein. In this paper, we review PEG chemistry and methods of preparation with a particular focus on new (second-generation) PEG derivatives, reversible conjugation and PEG structures.     

Chemistry for peptide and protein PEGylation

Poly(ethylene glycol) (PEG) is a highly investigated polymer for the covalent modification of biological macromolecules and surfaces for many pharmaceutical and biotechnical applications. In the modification of biological macromolecules, peptides and proteins are of extreme importance. Reasons for PEGylation (i.e. the covalent attachment of PEG) of peptides and proteins are numerous and include shielding of antigenic and immunogenic epitopes, shielding receptor-mediated uptake by the reticuloendothelial system (RES), and preventing recognition and degradation by proteolytic enzymes. PEG conjugation also increases the apparent size of the polypeptide, thus reducing the renal filtration and altering biodistribution. An important aspect of PEGylation is the incorporation of various PEG functional groups that are used to attach the PEG to the peptide or protein. In this paper, we review PEG chemistry and methods of preparation with a particular focus on new (second-generation) PEG derivatives, reversible conjugation and PEG structures.     

Biotechnology and genetic engineering in the new drug development. Part I. DNA technology and recombinant proteins

Pharmaceutical biotechnology has a long tradition and is rooted in the last century, first exemplified by penicillin and streptomycin as low molecular weight biosynthetic compounds. Today, pharmaceutical biotechnology still has its fundamentals in fermentation and bioprocessing, but the paradigmatic change affected by biotechnology and pharmaceutical sciences has led to an updated definition. The biotechnology revolution redrew the research, development, production and even marketing processes of drugs. Powerful new instruments and biotechnology related scientific disciplines (genomics, proteomics) make it possible to examine and exploit the behavior of proteins and molecules.Recombinant DNA(rDNA) technologies (genetic, protein, and metabolic engineering) allow the production of a wide range of peptides, proteins, and biochemicals from naturally nonproducing cells. This technology, now approximately 25 years old, is becoming one of the most important technologies developed in the 20^th century.Pharmaceutical products and industrial enzymes were the first biotech products on the world market made by means of rDNA. Despite important advances regarding rDNA applications in mammalian cells, yeasts still represent attractive hosts for the production of heterologous proteins. In this review we describe these processes.     

Rapid methods for the microbiological surveillance of pharmaceuticals

The use of rapid microbiological methods in pharmaceutical laboratories has improved the quality control analysis of water, products, raw materials, and enhanced the antimicrobial effectiveness testing of pharmaceutical finished products. Rapid release of samples has resulted in the optimization of manufacturing, product testing, and release allowing high throughput and simultaneous analysis of pharmaceutical formulations. ATP Bioluminescence, Impedance, Direct Viable Counts, and Flow Cytometry determine the total microbial content in a given pharmaceutical sample while PCR and Immunoassays detect the presence or absence of specific microbial species. Rapid methods provide reliable and cost effective analysis for the microbiological evaluation of pharmaceutical environments. The dramatic reduction in detection times and analysis, e.g., from 30 hours to 90 minutes, by using rapid methods will ultimately lead the pharmaceutical industry closer to real time monitoring of processes and samples.     

Thermoanalytical techniques for the investigation of the freeze drying process and freeze-dried products

A key challenge facing the pharmaceutical industry is the production of biotechnological drug products such as proteins in a stable form. Freeze-drying is preferred for manufacturing such products because of the low temperatures used. However, the protein may still degrade during the process necessitating the inclusion of a protectant. This review describes the range of thermal analysis techniques that have been used to investigate the properties of formulations to be freeze dried and the resultant products. This approach has allowed insight into the key parameters required for design of formulations and processes that will generate the best possible products.     

Photochemical internalization: a new tool for drug delivery

The utilisation of macromolecules in the therapy of cancer and other diseases is becoming increasingly important. Recent advances in molecular biology and biotechnology have made it possible to improve targeting and design of cytotoxic agents, DNA complexes and other macromolecules for clinical applications. In many cases the targets of macromolecular therapeutics are intracellular. However, degradation of macromolecules in endocytic vesicles after uptake by endocytosis is a major intracellular barrier for the therapeutic application of macromolecules having intracellular targets of action. Photochemical internalisation (PCI) is a novel technology for the release of endocytosed macromolecules into the cytosol. The technology is based on the activation by light of photosensitizers located in endocytic vesicles to induce the release of macromolecules from the endocytic vesicles. Thereby, endocytosed molecules can be released to reach their target of action before being degraded in lysosomes. PCI has been shown to stimulate intracellular delivery of a large variety of macromolecules and other molecules that do not readily penetrate the plasma membrane, including type I ribosome-inactivating proteins (RIPs), DNA delivered as gene-encoding plasmids or by means of adenovirus or adeno-associated virus, peptide nucleic acids (PNAs) and chemotherapeutic agents such as bleomycin and in some cases doxorubicin. PCI of PNA may be of particular importance due to the low therapeutic efficacy of PNA in the absence of an efficient delivery technology and the 10-100-fold increased efficacy in combination with PCI. The efficacy and specificity of PCI of macromolecular therapeutics has been improved by combining the macromolecules with targeting moieties, such as the epidermal growth factor. In general, PCI can induce efficient light-directed delivery of macromolecules into the cytosol, indicating that it may have a variety of useful applications for site-specific drug delivery as for example in gene therapy, vaccination and cancer treatment.     

Therapeutic synthetic polymers: a game of Russian roulette?

Synthetic polymer-based drug-delivery systems have been applied in drug delivery for the past 50 years. So why are there so few examples of these macromolecules being used successfully in the clinic? It is our view that many products are failing because of a neglect of the fundamental science surrounding the architectural control of the molecules present, their behaviour following in vivo administration and host response. Adverse events following parenteral administration of approved synthetic polymer-based systems have resulted in unpredictable and fatal responses in a significant number of individuals. Acceptance of the importance of immunotoxicological factors in response to the presence of these macromolecules must be addressed if emergent technologies, such as polymer-based gene-delivery systems, are going to succeed.     

Permeation enhancers in the transmucosal delivery of macromolecules

The present article presents a compilation of information regarding various chemical permeation enhancers useful for transmucosal delivery of macromolecules. In the recent past, biotechnology has provided a great number of macromolecules for treatment of various disorders. With the rise in importance of macromolecules, especially proteins and peptides, an enormous volume of research on various novel routes of drug delivery has been carried out. Inspite of its giving the highest and fastest bioavailability, the parenteral route is not a preferred option, due to its inconvenience and the noncompliance of patients. Mucosal surfaces are the most common and convenient routes for delivering drugs to the body. However, macromolecular drugs such as peptides and proteins are unable to overcome the mucosal barriers and/or are degraded before reaching the blood stream. Transmucosal drug delivery with various bioavailability enhancers is receiving increasing attention as a possible alternative to parenteral delivery of macromolecules. Among the various bioavailability enhancers, chemical permeation enhancers have been most studied. Permeation enhancers reversibly modulate the permeability of the barrier layer in favor of drug absorption. Newer permeation enhancers like zonula occludin toxin, poly-L-arginine, chitosan derivatives etc have shown a significant increase in drug absorption through transmucosal routes without serious damage to the barrier layer. In particular delivery of macromolecules via the nasal and pulmonary routesusing newer permeation enhancers has emerged as a possible alternative to the parenteral delivery ofmacromolecules.     

Nondestructive detection of glass vial inner surface morphology with differential interference contrast microscopy

Glass particles generated by glass dissolution and delamination of the glass container for pharmaceutical products have become a major issue in the pharmaceutical industry. The observation of glass particles in certain injectable drugs, including several protein therapeutics, has recently resulted in a number of product recalls. Glass vial surface properties have been suggested to play a critical role in glass dissolution and delamination. Surface characterization of glass container, therefore, is important to evaluate the quality of the glass container. In this work, we demonstrate that differential interference contrast (DIC) microscopy is a powerful, effective, and convenient technique to examine the inner surface morphology of glass vials nondestructively. DIC microscopy does not require the cutting of the glass vial for scanning the inner surface and has sufficient spatial resolution to reveal glass pitting, phase separation, delamination scars, and other defects. Typical surface morphology of pharmaceutical glass vials with different alkalinity are compared and discussed.