Lene Jorgensen

Protein adsorption at charged surfaces: the role of electrostatic interactions and interfacial charge regulation.

The understanding of protein adsorption at charged surfaces is important for a wide range of scientific disciplines including surface engineering, separation sciences and pharmaceutical sciences. Compared to chemical entities having a permanent charge, the adsorption of small ampholytes and proteins is more complicated as the pH near a charged surface can be significantly different from the value in bulk solution. In this work, we have developed a phenomenological adsorption model which takes into account the combined role of interfacial ion distribution, interfacial charge regulation of amino acids in the proximity of the surface, electroneutrality, and mass balance. The model is straightforward to apply to a given set of experimental conditions as most model parameters are obtained from bulk properties and therefore easy to estimate or are directly measurable. The model provides a detailed understanding of the importance of surface charge on adsorption and in particular of how changes in surface charge, concentration, and surface area may affect adsorption behavior. The model is successfully used to explain the experimental adsorption behavior of the two model proteins lysozyme and α-lactalbumin. It is demonstrated that it is possible to predict the pH and surface charge dependent adsorption behavior from experimental or theoretical estimates of a preferred orientation of a protein at a solid charged interface.

Recent trends in stabilising peptides and proteins in pharmaceutical formulation – considerations in the choice of excipients

In the area of peptide and protein pharmaceuticals, both the physical and chemical stability of biopharmaceuticals are critical and need to be optimised when formulating a drug product, in order to optimise the outcome after processing and storage. This review focuses on the effects on the stability from various types of excipient and the choices that have to be made during formulation of drug products containing peptides or proteins. It is illustrated, through examples, how the choice of one excipient over another can affect the stability of a protein drug formulation, along with other problems linked to this choice. The excipients used in pharmaceutical preparations are limited and from an academic point of view there is a clear requirement for new excipients.

The importance of interfaces in protein drug delivery – why is protein adsorption of interest in pharmaceutical formulations?

Introduction: In the area of peptide and protein drug products, interfaces are present as part of the basic liquid formulation, when freeze-dried formulations are reconstituted and when particulate delivery systems are prepared. Proteins are known to interact with these interfaces, and the effects seen are often irreversible adsorption and structural changes. Areas covered: This review focuses on the ways in which peptides and proteins interact with surfaces and interfaces, and the effect these interactions have on the stability and safety of the active protein in pharmaceutical formulations. It illustrates, through examples, what can be determined by an adsorption study, and what can change when either the protein or the interfaces are modified. Last but not least, it addresses the value of these studies. The reader will gain an update on the basics of protein adsorption, with a focus on pharmaceutically relevant interfaces and recent advances in the field. Expert opinion: Protein adsorption is widely studied; however, a more unified approach is still needed, especially on the adsorption of pharmaceutically relevant proteins, modified proteins and surfaces.

Secondary structure alterations in insulin and growth hormone water-in-oil emulsions

Water-in-oil (w/o) emulsions have shown a promising release profile of small drug molecules and proteins. However, the major concerns are the structural stability, the retention of the activity and to avoid unwanted immunological reactions caused by the changes in protein structure. In the present study, the secondary structure of insulin and growth hormone is investigated after manufacture of w/o emulsions, using Fourier transform infrared (FTIR) spectroscopy. Initial investigations indicate an altered distribution in the secondary structure elements, e.g. alpha-helix and beta-sheet, measured by area overlap calculations. The changes are more pronounced for growth hormone than for insulin. The overlapping area is 0.93 +/- 0.01 for the emulsion containing insulin manufactured at 0 degrees C and homogenised for 3 min, the corresponding value for growth hormone is 0.83 +/- 0.01. The droplet size changes from 0.27 +/- 0.04 microm in the blank w/o emulsion to 0.79 +/- 0.13 and 0.66 +/- 0.21 microm when insulin or growth hormone is incorporated into the w/o emulsions, respectively.

Interfacial Complexes between a Protein and Lipophilic Ions at an Oil-Water Interface

The interaction between an intact protein and two lipophilic ions at an oil-water interface has been investigated using cyclic voltammetry, impedance based techniques and a newly developed method in which the biphasic oil-water system is analyzed by biphasic electrospray ionization mass spectrometry (BESI-MS), using a dual-channel electrospray emitter. It is found that the protein forms interfacial complexes with the lipophilic ions and that it specifically requires the presence of the oil-water interface to be formed under the experimental conditions. Furthermore, impedance based techniques and BESI-MS with a common ion to polarize the interface indicated that the Galvani potential difference across the oil-water interface significantly influences the interfacial complexation degree. The ability to investigate protein-ligand complexes formed at polarized liquid-liquid interfaces is thus a new analytical method for assessing potential dependent interfacial complexation using a structure elucidating detection principle.

Incorporation of a synthetic mycobacterial monomycoloyl glycerol analogue stabilizes dimethyldioctadecylammonium liposomes and potentiates their adjuvant effect in vivo

The combination of delivery systems such as cationic liposomes and immunopotentiating molecules is a promising approach for the rational design of vaccine adjuvants. In this study, a synthetic analogue of the mycobacterial lipid monomycoloyl glycerol (MMG), referred to as MMG-1, was synthesized and combined with the cationic surfactant dimethyldioctadecylammonium (DDA). The purpose of the study was to provide a thorough pharmaceutical characterization of the resulting DDA/MMG-1 binary system and to evaluate how incorporation of MMG-1 affected the adjuvant activity of DDA liposomes. Thermal analyses demonstrated that MMG-1 was incorporated into the DDA lipid bilayers, and cryo-transmission electron microscopy (TEM) confirmed that liposomes were formed. The particles had a polydisperse size distribution and an average diameter of approximately 400 nm. Evaluation of the colloidal stability indicated that at least 18 mol% MMG-1 was required to stabilize the DDA liposomes as the average particle size remained constant during storage for 6 months. The improved colloidal stability is most likely caused by increased hydration of the lipid bilayer. This was demonstrated by studying Langmuir-Blodgett monolayers of DDA and MMG-1 which revealed an increased surface pressure in the presence of high concentrations of MMG-1 when the DDA/MMG-1 monolayers were fully compressed, indicating an increased interaction with water due to enhanced hydration of the lipid head groups. Finally, immunization of mice with the tuberculosis fusion antigen Ag85B-ESAT-6 and DDA/MMG-1 liposomes induced a strong cell-mediated immune response characterized by a mixed Th1/Th17 profile and secretion of IgG1 and IgG2c antibodies. The Th1/Th17-biased immunostimulatory effect was increased in an MMG-1 concentration-dependent manner with maximal observed effect at 31 mol% MMG-1. Thus, incorporation of 31 mol% MMG-1 into DDA liposomes results in an adjuvant system with favorable physical as well as immunological properties.

Factors of importance for a successful delivery system for proteins

Protein pharmaceuticals have matured into an important class of drugs, now comprising one in three novel drugs introduced on the market. However, significant gains are still to be made in reducing the costs of production, ensuring proper pharmacokinetics and efficacy, increasing patient compliance and convenience, and reducing side effects such as immunogenicity. This review summarises these issues and provides recent examples of methods to reduce costs, alter pharmacokinetics and increase patient compliance. It also discusses the increasing interest in understanding immunogenicity in order to prevent failure of the protein drug or serious life-threatening side effects due to autoimmunogenicity.

Delivery of dermatan sulfate from polyelectrolyte complex-containing alginate composite microspheres for tissue regeneration.

Dermatan sulfate (DS) is a glycosaminoglycan (GAG) with a great potential as a new therapeutic agent in tissue engineering. The aim of the present study was to investigate the formation of polyelectrolyte complexes (PECs) between chitosan and dermatan sulfate (CS/DS) and delivery of DS from PEC-containing alginate/chitosan/dermatan sulfate (Alg/CS/DS) microspheres for application in tissue regeneration. The CS/DS complexes were initially formed at different conditions including varying CS/DS ratio (positive/negative charge ratio), buffer, and pH. The obtained CS/DS complexes exhibited stronger electrostatic interaction, smaller complex size, and more stable colloidal structure when chitosan was in large excess (CS/DS 3:1) and prepared at pH 3.5 as compared to pH 5 using acetate buffer. The CS/DS complexes were subsequently incorporated into an alginate matrix by spray drying to form Alg/CS/DS composite microspheres with a DS encapsulation efficiency of 90-95%. The excessive CS induced a higher level of sustained DS release into Tris buffer (pH 7.4) from the microspheres formulated at pH 3.5; however, the amount of CS did not have a significant effect on the release from the microspheres formulated at pH 5. Significant cell proliferation was stimulated by the DS released from the microspheres in vitro. The present results provide a promising drug delivery strategy using PECs for sustained release of DS from microspheres intended for site-specific drug delivery and ultimately for use in tissue engineering.

Calcipotriol delivery into the skin with PEGylated liposomes

The D-vitamin analogue calcipotriol is commonly used for topical treatment of psoriasis, but skin penetration is required for calcipotriol to reach its pharmacological target: the keratinocytes in the lower epidermis. Liposomes can enhance the delivery of drugs into the skin, but a major challenge for the development of dosage forms containing liposomes is to maintain the colloidal stability in the formulation. The purpose of this study was to investigate the effect of stabilising liposomes with the lipopolymer poly(ethylene glycol)-distearoylphosphoethanolamine (PEG-DSPE) on the physicochemical properties of the liposomes and the ability to deliver membrane-intercalated calcipotriol into the skin. Inclusion of 0.5, l and 5 mol% PEG-DSPE in the membrane enhanced the colloidal stability of the liposomes without compromising the delivery of calcipotriol from the vehicle into excised pig skin. Calcipotriol-loaded liposomes with 1 mol% PEG-DSPE did even provide for a significantly increased deposition of calcipotriol into the stratum corneum. The size of the liposomes affected the penetration of calcipotriol into the stratum corneum since small unilamellar vesicles enhanced calcipotriol penetration as compared to large multilamellar vesicles, indicating that the liposomes to some extent migrate as intact vesicles into the stratum corneum. However, calcipotriol penetrated the skin better than the lipid component of the liposomes, suggesting that at least a fraction of the drug is released from the liposomes during skin migration. In conclusion, PEGylation is therefore a promising approach for stabilising calcipotriol-containing liposomal dispersions without compromising their favourable skin accumulation properties.

Alternative routes of administration for systemic delivery of protein pharmaceuticals.

Non-invasive and patient-friendly delivery of proteins are important targets for protein formulation development. Traditionally, a lyophilized cake for reconstitution followed by s.c. or i.v. administration has been used. At present, several protein pharmaceuticals delivered by alternative routes of administration are under investigation. The long-term effects of protein delivery by these alternative routes are not known. Nevertheless, these alternative administration routes can in the near future lead to new, successful protein pharmaceuticals on the market.: