Daan J.A. Crommelin

Structure-immunogenicity relationships of therapeutic proteins

As more recombinant human proteins become available on the market, the incidence of immunogenicity problems is rising. The antibodies formed against a therapeutic protein can result in serious clinical effects, such as loss of efficacy and neutralization of the endogenous protein with essential biological functions. Here we review the literature on the relations between the immunogenicity of the therapeutic proteins and their structural properties. The mechanisms by which protein therapeutics can induce antibodies as well as the models used to study immunogenicity are discussed. Examples of how the chemical structure (including amino acid sequence, glycosylation, and pegylation) can influence the incidence and level of antibody formation are given. Moreover, it is shown that physical degradation (especially aggregation) of the proteins as well as chemical decomposition (e.g., oxidation) may enhance the immune response. To what extent the presence of degradation products in protein formulations influences their immunogenicity still needs further investigation. Immunization of transgenic animals, tolerant for the human protein, with well-defined, artificially prepared degradation products of therapeutic proteins may shed more light on the structure-immunogenicity relationships of recombinant human proteins.

Overlooking Subvisible Particles in Therapeutic Protein Products: Gaps That May Compromise Product Quality

Therapeutic protein products provide unique and effective treatments for numerous human diseases and medical conditions. In many cases, these treatments are used chronically to slow disease progression, reduce morbidity and/or to replace essential proteins that are not produced endogenously in patients. Therefore, any factor that reduces or eliminates the effectiveness of the treatment can lead to patient suffering and even death. One means by which efficacy of therapeutic proteins can be compromised is by an immune response, resulting in antibody-mediated neutralization of the protein’s activity or alterations in bioavailability.1,2 For example, in the case of treatment of hemophilia A, neutralizing antibodies to Factor VIII can cause life-threatening bleeding episodes, resulting in significant morbidity and necessitating treatment with a prolonged course of a tolerance-inducing therapy to reverse immunity.3,4 In other cases, drug-induced antibodies to a therapeutic version of an endogenous protein can cross-react with and neutralize the patient’s endogenous protein. If the endogenous protein serves a non-redundant biological function, such an immune response can have devastating results. For example, pure red cell aplasia can result from neutralizing antibodies to epoetin alpha. 1,2 It is well established that protein aggregates in therapeutic protein products can enhance immunogenicity2, and such an effect is therefore an important risk factor to consider when assessing product quality. The purpose of this commentary is to accomplish the following: provide brief summaries on the factors affecting protein aggregation and the key aspects of protein aggregates that are associated with immunogenicity; emphasize the current scientific gaps in understanding and analytical limitations for quantitation of species of large protein aggregates that are referred to as subvisible particles, with specific consideration of those particles 0.1–10 μm in size; offer a rationale for why these gaps may compromise the safety and/or efficacy of a product; provide scientifically sound, risked based recommendations/conclusions for assessment and control of such aggregate species. Causes of Protein Aggregation Proteins usually aggregate from partially unfolded molecules, which can be part of the native state ensemble of molecules.5 Even though product formulations are developed to maximize and maintain the fraction of the protein molecules present in the native state, significant amounts of aggregates can form, especially over pharmaceutically-relevant time scales and under stress conditions. For example, exposure to interfaces (e.g., air-liquid and solid-liquid), light, temperature fluctuations or minor impurities can induce aggregation. Such exposure can occur during processing steps, as well as in the final product container during storage, shipment and handling. Furthermore, protein particles (visible and subvisible) can be generated from protein alone or from heterogeneous nucleation on foreign micro- and nanoparticles that are shed, for example, from filling pumps or product container/closures.6–8 The levels and sizes of protein particles present in a given product can be changed by many factors relevant to commercial production of therapeutic proteins. Such factors include a change in the type of filling pump during scale-up to commercial manufacturing, changes in formulation or container/closure, and even unintentional changes in the manufacturing process such as alterations in filling pump mechanical parameters or other unforeseen factors.8,9 Thus, unless appropriate quality controls are in place for subvisible particles, a product that was safe and effective in clinical trials may unexpectedly cause adverse events in patients after commercialization. Effects of Aggregate Characteristics on Immunogenicity From work on fundamental aspects of immunology and vaccine development, it is known that large protein assemblies with repetitive arrays of antigens, in which the protein molecules have native conformation, are usually the most potent at inducing immune responses.2,10,11 Furthermore, efforts to develop more effective vaccines have shown that adsorbing antigenic proteins to nano- or microparticles comprised of other materials (e.g., colloidal aluminum salts or polystyrene) can greatly increase immunogenicity.12,13 Applying these lessons to therapeutic protein products, it has been argued that large aggregates containing protein molecules with native-like conformation pose the greatest risk of causing adverse immune responses in patients.2 Thus, for example, particles of therapeutic proteins formed by adsorption of protein molecules onto foreign micro- and nanoparticles might be particularly prone to cause immunogenicity. These particles contain numerous protein molecules, and in the two examples published to date, the adsorbed protein molecules were shown to retain their native conformations.6,8 Unfortunately, lacking are published studies that comprehensively investigate the range of parameters that could influence immunogenicity of aggregates. Because each protein may differ in aggregate formation and consequences, factors that need to be investigated include but are not limited to type, amount and size of aggregates, as well as protein conformation in aggregates, on a case by case basis. Of course, other factors, particularly pertaining to patient status and treatment protocol, are also critical in determining the propensity to generate immune responses. These include immune competence of the patients, route of administration, and dosing frequency and duration. Given the consequences of aggregate-induced immunogenicity in patients, it is important to understand these issues and to reduce the risk to product quality for every therapeutic protein product. Because the exact characteristics and levels of protein aggregates that lead to an enhanced immune response are unclear and may differ among proteins, it is not possible to predict, a priori, the in vivo effects of different sizes, types or quantities of aggregates for therapeutic protein products. In such situations, careful analysis of the relationship between clinical performance and the presence of protein aggregates in relevant clinical trial material may help in the design of suitable control strategies that ensure product quality. However, the validity and utility such correlations are only optimized when the full spectrum of protein aggregate species are thoroughly characterized by multiple and orthogonal techniques.

Effect of Size and Serum Proteins on Transfection Efficiency of Poly ((2-dimethylamino)ethyl Methacrylate)-Plasmid Nanoparticles

AbstractPurpose. The aim of this study was to gain insight into the relation between the physical characteristics of particles formed by a plasmid and a synthetic cationic polymer (poly(2-dimethylamino)ethyl methacrylate, PDMAEMA) and their transfection efficiency. Methods. The PDMAEMA-plasmid particles were characterized by dynamic light scattering (size) and electrophoretic mobility measurements (charge). The transfection efficiency was evaluated in cell culture (COS-7 cells) using a pCMV-lacZ plasmid coding for β-galactosidase as a reporter gene. Results. It was shown that the optimal transfection efficiency was found at a PDMAEMA-plasmid ratio of 3 (w/w), yielding stable and rather homogeneous particles (diameter 0.15 µm) with a narrow size distribution and a slightly positive charge. Particles prepared at lower weight ratios, showed a reduced transfection efficiency and were unstable in time as demonstrated by DLS measurements. Like other cationic polymers, PDMAEMA is slightly cytotoxic. This activity was partially masked by complexing the polymer with DNA. Interestingly, the transfection efficiency of the particles was not affected by the presence of serum proteins. Conclusions. PDMAEMA is an interesting vector for the design of in vivo and ex vivo gene transfection systems.

Artificial viruses: a nanotechnological approach to gene delivery

Nanotechnology is a rapidly expanding multidisciplinary field in which highly sophisticated nanoscale devices are constructed from atoms, molecules or (macro)molecular assemblies. In the field of gene medicine, systems for delivering nucleic acids are being developed that incorporate virus-like functions in a single nanoparticle. Although their development is still in its infancy, it is expected that such artificial viruses will have a great impact on the advancements of gene therapeutics.

Lymphatic uptake and biodistribution of liposomes after subcutaneous injection.: II. Influence of liposomal size, lipid composition and lipid dose

The present paper reports on the results of a systematic study on liposome variables potentially affecting lymphatic disposition and biodistribution of liposomes after sc injection. Liposomal size was found to be the most important factor influencing lymphatic uptake and lymph node localization of sc administered liposomes. Lymphatic uptake from the s.c. injection site of small liposomes (about 0.04 microm) was relatively high (76% of the injected dose (%ID)) as compared to large, non-sized liposomes, which remained almost completely at the site of injection. Small liposomes were less efficiently retained by regional lymph nodes than larger liposomes. Liposomal lipid composition did not influence lymphatic uptake with one exception: Lymphatic uptake was decreased in case of neutral liposomes composed of (DPPC). Lymph node localization was substantially enhanced by inclusion of phosphatidylserine (PS) into the liposomal bilayers. Saturation of lymphatic uptake and lymph node localization did not occur over a large liposomal lipid dose range, illustrating the efficient performance of lymph nodes in capturing s.c. administered particles.

2-(dimethylamino)ethyl methacrylate based (co)polymers as gene transfer agents

Poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA) is a water-soluble cationic polymer, which is able to bind to DNA by electrostatic interactions. At a polymer/plasmid ratio above 2 (w/w) positively charged complexes were formed with a size around 0.2 microm. The transfection efficiency of polymer/plasmid complexes was evaluated in cell culture (COS-7 and OVCAR-3 cells) using a pCMV-lacZ plasmid, encoding for beta-galactosidase, as a reporter gene. The optimal transfection efficiency was found at a PDMAEMA/plasmid ratio of 3-5 (w/w). Under these conditions 3-6% of the cells were actually transfected. Like other cationic polymers, PDMAEMA is slightly cytotoxic. This activity was partially masked by complexing the polymer with DNA. A pronounced effect of the molecular weight of the polymer on the transfection efficiency was observed. An increasing molecular weight resulted in an increasing number of transfected cells. Dynamic light scattering experiments showed that high molecular weight polymers (Mw>300 kDa) were able to condense DNA effectively (particle size 0.15-0.20 microm). In contrast, when plasmid was incubated with low molecular weight PDMAEMA, large complexes were formed (size 0.5-1.0 microm). Copolymers of DMAEMA with methyl methacrylate (MMA), ethoxytriethylene glycol methacrylate (triEGMA) or N-vinyl-pyrrolidone (NVP) also acted as transfection agents. A copolymer with 20 mol % of MMA showed a reduced transfection efficiency and a substantial increased cytotoxicity compared with a homopolymer of the same molecular weight. A copolymer with triEGMA (48 mol %) showed both a reduced transfection efficiency and a reduced cytotoxicity, whereas a copolymer with NVP (54 mol %) showed an increased transfection efficiency and a decreased cytotoxicity as compared to a DMAEMA homopolymer.

Liposomal and Lipid Formulations of Amphotericin B: Clinical Pharmacokinetics

SummaryAmphotericin B remains a very important drug for the treatment of fungal infections despite its toxicity. Encapsulation of amphotericin B into liposomes appears to reduce the toxic effects and to improve the clinical efficacy, allowing higher dosages to be given. The exact mechanism behind the reduced toxicity is not yet known.Amphotericin B is widely distributed after intravenous administration as the deoxycholate solubilisate. The highest concentrations are found in the liver, spleen and kidney. Protein binding and binding to the tissues is very high. The fate of the drug in the body is not known in detail. Renal and biliary excretion are both low and no metabolites have been identified. The drug is still detectable in the liver, spleen and kidney for as long as 1 year after stopping therapy.The pharmacokinetics of the different liposomal amphotericin B or lipid complexes of amphotericin B, which were recently developed, are quite diverse. A number of these preparations, such as amphotericin B lipid complex (ABLC), ‘AmBisome’ and amphotericin B colloidal dispersion (ABCD) are in clinical development. Their pharmacokinetics depend to a large extent on the composition and particle size of the liposomes or lipid complexes. Relatively large structures such as ABLC are rapidly taken up by the mononuclear phagocyte system, whereas smaller liposomes remain in the circulation for prolonged periods. In all studies only the total amphotericin B (both free and liposome- or lipid-associated) concentrations were determined.There is a need for studies correlating clinical efficacy and tolerability of liposomal amphotericin B with the pharmacokinetic properties of these formulations.

Chemical stability of liposomes: implications for their physical stability

In the first part of this article, chemical and physical stability of aqueous liposome dispersions have been addressed. Chemical stability of phospholipids has been considered in two parts: oxidation and hydrolysis. Major attention has been paid to hydrolysis kinetics of phospholipids as a function of pH, temperature, buffer concentration and ionic strength. Furthermore, the effect of chain length, head group, state of aggregation, addition of cholesterol and presence of charge on the hydrolysis kinetics of phospholipids has been dealt with. In the second part physical stability of chemically degraded liposome dispersions has been evaluated. In the final part quality control assays for liposome dispersions is presented and a HPLC method with a refractive index detector for the analysis of phospholipids from aqueous liposome dispersions is described.

Liposomes: quo vadis?

Abstract Liposomes have matured as a delivery system for therapeutic agents. It has taken two decades to develop the liposome carrier concept to a pharmaceutical product level, but commercial preparations are now available in important disease areas. In this review, the authors provide their perspective on where the field is going and where opportunities can be found for rational improvement of drug therapy with liposomes.

Cellular Uptake of Cationic Polymer-DNA Complexes Via Caveolae Plays a Pivotal Role in Gene Transfection in COS-7 Cells

PurposeKnowledge about the uptake mechanism and subsequent intracellular routing of non-viral gene delivery systems is important for the development of more efficient carriers. In this study we compared two established cationic polymers pDMAEMA and PEI with regard to their transfection efficiency and mechanism of cellular uptake.Materials and MethodsThe effects of several inhibitors of particular cellular uptake routes on the uptake of polyplexes and subsequent gene expression in COS-7 cells were investigated using FACS and transfection. Moreover, cellular localization of fluorescently labeled polyplexes was assessed by spectral fluorescence microscopy.ResultsBoth pDMAEMA- and PEI-complexed DNA showed colocalization with fluorescently-labeled transferrin and cholera toxin after internalization by COS-7 cells, which indicates uptake via the clathrin- and caveolae-dependent pathways. Blocking either routes of uptake with specific inhibitors only resulted in a marginal decrease in polyplex uptake, which may suggest that uptake routes of polyplexes are interchangeable. Despite the marginal effect of inhibitors on polyplex internalization, blocking the caveolae-mediated uptake route resulted in an almost complete loss of polyplex-mediated gene expression, whereas gene expression was not negatively affected by blocking the clathrin-dependent route of uptake.ConclusionsThese results show the importance of caveolae-mediated uptake for successful gene expression and have implications for the rational design of non-viral gene delivery systems.