Patrick Garidel

Strategies for the assessment of protein aggregates in pharmaceutical biotech product development.

ABSTRACTWithin the European Immunogenicity Platform (EIP) (http://www.e-i-p.eu), the Protein Characterization Subcommittee (EIP-PCS) has been established to discuss and exchange experience of protein characterization in relation to unwanted immunogenicity. In this commentary, we, as representatives of EIP-PCS, review the current state of methods for analysis of protein aggregates. Moreover, we elaborate on why these methods should be used during product development and make recommendations to the biotech community with regard to strategies for their application during the development of protein therapeutics.

Single Particle Characterization of Iron-induced Pore-forming α-Synuclein Oligomers

Aggregation of α-synuclein is a key event in several neurodegenerative diseases, including Parkinson disease. Recent findings suggest that oligomers represent the principal toxic aggregate species. Using confocal single-molecule fluorescence techniques, such as scanning for intensely fluorescent targets (SIFT) and atomic force microscopy, we monitored α-synuclein oligomer formation at the single particle level. Organic solvents were used to trigger aggregation, which resulted in small oligomers (“intermediate I”). Under these conditions, Fe3+ at low micromolar concentrations dramatically increased aggregation and induced formation of larger oligomers (“intermediate II”). Both oligomer species were on-pathway to amyloid fibrils and could seed amyloid formation. Notably, only Fe3+-induced oligomers were SDS-resistant and could form ion-permeable pores in a planar lipid bilayer, which were inhibited by the oligomer-specific A11 antibody. Moreover, baicalein and N′-benzylidene-benzohydrazide derivatives inhibited oligomer formation. Baicalein also inhibited α-synuclein-dependent toxicity in neuronal cells. Our results may provide a potential disease mechanism regarding the role of ferric iron and of toxic oligomer species in Parkinson diseases. Moreover, scanning for intensely fluorescent targets allows high throughput screening for aggregation inhibitors and may provide new approaches for drug development and therapy.

Interaction of quorum signals with outer membrane lipids: insights into prokaryotic membrane vesicle formation

Bacteria have evolved elaborate communication strategies to co‐ordinate their group activities, a process termed quorum sensing (QS). Pseudomonas aeruginosa is an opportunistic pathogen that utilizes QS for diverse activities, including disease pathogenesis. P. aeruginosa has evolved a novel communication system in which the signal molecule 2‐heptyl‐3‐hydroxy‐4‐quinolone (Pseudomonas Quinolone Signal, PQS) is trafficked between cells via membrane vesicles (MVs). Not only is PQS packaged into MVs, it is required for MV formation. Although MVs are involved in important biological processes aside from signalling, the molecular mechanism of MV formation is unknown. To provide insight into the molecular mechanism of MV formation, we examined the interaction of PQS with bacterial lipids. Here, we show that PQS interacts strongly with the acyl chains and 4′‐phosphate of bacterial lipopolysaccharide (LPS). Using PQS derivatives, we demonstrate that the alkyl side‐chain and third position hydroxyl of PQS are critical for these interactions. Finally, we show that PQS stimulated purified LPS to form liposome‐like structures. These studies provide molecular insight into P. aeruginosa MV formation and demonstrate that quorum signals serve important non‐signalling functions.

A Fourier transform infrared spectroscopic study of the interaction of alkaline earth cations with the negatively charged phospholipid 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol

The interaction of aqueous phospholipid dispersions of negatively charged 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol, sodium salt (DMPG) with the divalent cations Mg(2+), Ca(2+) and Sr(2+) at equimolar ratios in 100 mM NaCl at pH 7 was investigated by Fourier transform infrared spectroscopy. The binding of the three cations induces a crystalline-like gel phase with highly ordered and rigid all-trans acyl chains. These features are observed after storage below room temperature for 24 h. When the gel phase is heated after prolonged incubation at low temperature phase transitions into the liquid crystalline phase are observed at 58 degrees C for the DMPG:Sr(2+), 65 degrees C for the DMPG:Mg(2+), and 80 degrees C for the DMPG:Ca(2+) complex. By subsequent cooling from temperatures above T(m) these complexes retain the features of a liquid crystalline phase with disordered acyl chains until a metastable gel phase is formed at temperatures between 38 and 32 degrees C. This phase is characterized by predominantly all-trans acyl chains, arranged in a loosely packed hexagonal or distorted hexagonal subcell lattice. Reheating the DMPG:Sr(2+) samples after a storage time of 2 h at 4 degrees C results in the transition of the metastable gel to the liquid crystalline phase at 35 degrees C. This phase transition into the liquid crystalline state at 35 degrees C is also observed for the Mg(2+) complex. However, for DMPG:Mg(2+) at higher temperatures, a partial recrystallization of the acyl chains occurs and the high temperature phase transition at 65 degrees C is also detected. In contrast, DMPG:Ca(2+) exhibits only the phase transition at 80 degrees C from the crystalline gel into the fluid state upon reheating. Below 20 degrees C, the rate of conversion from the metastable gel to a thermodynamically stable, crystalline-like gel phase decreases in the order Ca(2+)&z. Gt;Mg(2+)>Sr(2+). This conversion into the crystalline gel phase is accompanied by a complete dehydration of the phosphate groups in DMPG:Mg(2+) and by a reorientation of the polar lipid head groups in DMPG:Ca(2+) and in DMPG:Sr(2+). The primary binding sites of the cations are the PO(2)(-) groups of the phosphodiester moiety. Our infrared spectroscopic results suggest a deep penetration of the divalent cations into the polar head group region of DMPG bilayers, whereby the ester carbonyl groups, located in the interfacial region of the bilayers, are indirectly affected by strong hydrogen bonding of immobilized water molecules. In the liquid crystalline phase, the interaction of all three cations with DMPG is weak, but still observable in the infrared spectra of the DMPG:Ca(2+) complex by a slight ordering effect induced in the acyl chains, when compared to pure DMPG liposomes.

Stabilization of IgG1 in spray-dried powders for inhalation.

The protein stabilizing capabilities of spray-dried IgG1/mannitol formulations were evaluated. The storage stability was tested at different residual moisture levels prepared by vacuum-drying or equilibration prior to storage. Vacuum-drying at 32 degrees C/0.1mbar for 24h reduced the moisture level below 1%, constituting an optimal basis for improved storage stability. The crystalline IgG1/mannitol powders with a weight ratio of 20/80 up to 40/60 failed to prevent the antibody aggregation as assessed by size exclusion chromatography during storage. Ratios of 60/40 up to 80/20 IgG1/mannitol provided superior stability of the antibody and the powders could be produced with high yields. The lower the residual moisture, the better was the stabilizing capability. An amount of 20% mannitol provided the best stabilization. Storage stability of 60/40, 70/30, and 80/20 IgG1/mannitol formulations over one year was adequate at 2-8 degrees C and 25 degrees C. Closed storage (sealed in vials) at 40 degrees C/75% RH and open storage at 25 degrees C/60% RH revealed that the stability still required optimization. The lower the protein content, the better was the powder flowability. The aerodynamic properties of powders spray-dried with 10% solids content were inadequate, as the particle size ranged between 5.1 and 7.2 microm and the fine particle fraction accounted for only 4-11%. Reduction of the solids content to 2.5% did improve the aerodynamic properties as the mass mean aerodynamic diameter was reduced to 3.6 microm and the fine particle fraction was increased to about 14%. The reduction of the solids content did not influence the storage stability significantly. Also spray-drying at higher temperatures had no significant impact on the storage stability, despite a higher tendency to form amorphous systems. In order to improve the storage stability and to maintain the good flowability of 70/30 IgG1/mannitol powder or to keep the storage stability but to improve the flowability of the 80/20 IgG1/mannitol powder, mannitol was partially substituted by a second excipient such as trehalose, sucrose, glycine, lactose, lactosucrose, or dextran 1. Differences in the stabilizing capability were noticeable upon closed storage at 40 degrees C/75% RH and open powder storage. Protein stabilization was improved by the addition of glycine but trehalose and sucrose were most effective in preventing aggregation, which can be primarily attributed to the water replacement properties of the sugars. The addition of another excipient, isoleucine had positive effects on both flowability and protein stability.

Systematic investigation of the effect of lyophilizate collapse on pharmaceutically relevant proteins I: Stability after freeze-drying

The objective of this work was to investigate the effect of cake collapse during freeze-drying on the stability of protein lyophilizates containing a monoclonal IgG(1)-antibody or a second pharmaceutically relevant protein, referred to as PA01. In addition, L-lactic dehydrogenase was investigated because of its well-documented sensitivity towards freeze-drying stresses. Collapse was induced by two different means. First, by varying the ratio of the crystalline bulking agent mannitol to the amorphous stabilizer sucrose, different extents of collapsed cakes were generated. Second, formulations were freeze-dried using an aggressive collapse-cycle and a conventional freeze-drying protocol and collapsed and noncollapsed cakes of identical formulation were produced. Lyophilizates were analyzed using a comprehensive set of analytical techniques to monitor protein stability in terms of formation of soluble and insoluble aggregates, the biological activity and the conformational stability. The stability of excipients, namely the glass transition temperature, crystallinity, reconstitution behavior, and the residual moisture content was analyzed as well. In addition, the extent of collapse was quantified using the decrease of the specific surface area (SSA). Collapsed cakes had comparable residual moisture levels to noncollapsed lyophilizates. Reconstitution times were not increased. Protein stability was not relevantly different between collapsed and noncollapsed cakes.

The membrane-activity of Ibuprofen, Diclofenac, and Naproxen: A physico-chemical study with lecithin phospholipids

Nonsteroidal anti-inflammatory drugs (NSAIDs) represent non-specific inhibitors of the cycloxygenase pathway of inflammation, and therefore an understanding of the interaction process of the drugs with membrane phospholipids is of high relevance. We have studied the interaction of the NSAIDs with phospholipid membranes made from dimyristoylphosphatidylcholine (DMPC) by applying Fourier-transform infrared spectroscopy (FTIR), Förster resonance energy transfer spectroscopy (FRET), differential scanning calorimetry (DSC) and isothermal titration calorimetry (ITC). FTIR data obtained via attenuated total reflectance (ATR) show that the interaction between DMPC and NSAIDs is limited to a strong interaction of the drugs with the phosphate region of the lipid head group. The FTIR transmission data furthermore are indicative of a strong effect of the drugs on the hydrocarbon chains inducing a reduction of the chain-chain interactions, i.e., a fluidization effect. Parallel to this, from the DSC data beside the decrease of T(m) a reduction of the peak height of the melting endotherm connected with its broadening is observed, but leaving the overall phase transition enthalpy constant. Additionally, phase separation is observed, inducing the formation of a NSAID-rich and a NSAID-poor phase. This is especially pronounced for Diclofenac. Despite the strong influence of the drugs on the acyl chain moiety, FRET data do not reveal any evidence for drug incorporation into the lipid matrix, and ITC measurements performed do not exhibit any heat production due to drug binding. This implies that the interaction process is governed by only entropic reactions at the lipid/water interface.

Membranolytic activity of bile salts: influence of biological membrane properties and composition.

The two main steps of the membranolytic activity of detergents: 1) the partitioning of detergent molecules in the membrane and 2) the solubilisation of the membrane are systematically investigated. The interactions of two bile salt molecules, sodium cholate (NaC) and sodium deoxycholate (NaDC) with biological phospholipid model membranes are considered. The membranolytic activity is analysed as a function of the hydrophobicity of the bile salt, ionic strength, temperature, membrane phase properties, membrane surface charge and composition of the acyl chains of the lipids. The results are derived from calorimetric measurements (ITC, isothermal titration calorimetry). A thermodynamic model is described, taking into consideration electrostatic interactions, which is used for the calculation of the partition coefficient as well as to derive the complete thermodynamic parameters describing the interaction of detergents with biological membranes (change in enthalpy, change in free energy, change in entropy etc). The solubilisation properties are described in a so-called vesicle-to-micelle phase transition diagram. The obtained results are supplemented and confirmed by data obtained from other biophysical techniques (DSC differential scanning calorimetry, DLS dynamic light scattering, SANS small angle neutron scattering).

Nonideal mixing and phase separation in phosphatidylcholine-phosphatidic acid mixtures as a function of acyl chain length and pH

The miscibilities of phosphatidic acids (PAs) and phosphatidylcholines (PCs) with different chain lengths (n = 14, 16) at pH 4, pH 7, and pH 12 were examined by differential scanning calorimetry. Simulation of heat capacity curves was performed using a new approach that incorporates changes of cooperativity of the transition in addition to nonideal mixing in the gel and the liquid-crystalline phase as a function of composition. From the simulations of the heat capacity curves, first estimates for the nonideality parameters for nonideal mixing as a function of composition were obtained, and phase diagrams were constructed using temperatures for onset and end of melting, which were corrected for the broadening effect caused by a decrease in cooperativity. In all cases the composition dependence of the nonideality parameters indicated nonsymmetrical mixing behavior. The phase diagrams were therefore further refined by simulations of the coexistence curves using a four-parameter approximation to account for nonideal and nonsymmetrical mixing in the gel and the liquid-crystalline phase. The mixing behavior was studied at three different pH values to investigate how changes in headgroup charge of the PA influences the miscibility. The experiments showed that at pH 7, where the PA component is negatively charged, the nonideality parameters are in most cases negative, indicating that electrostatic effects favor a mixing of the two components. Partial protonation of the PA component at pH 4 leads to strong changes in miscibility; the nonideality parameters for the liquid-crystalline phase are now in most cases positive, indicating clustering of like molecules. The phase diagram for 1,2-dimyristoyl-sn-glycero-3-phosphatidic acid:1,2-dipalmitoyl-sn-glycero-3-phosphorylcholine mixtures at pH 4 indicates that a fluid-fluid immiscibility is likely. The results show that a decrease in ionization of PAs can induce large changes in mixing behavior. This occurs because of a reduction in electrostatic repulsion between PA headgroups and a concomitant increase in attractive hydrogen bonding interactions.

Biophysical Mechanisms of Endotoxin Neutralization by Cationic Amphiphilic Peptides

Bacterial endotoxins (lipopolysaccharides (LPS)) are strong elicitors of the human immune system by interacting with serum and membrane proteins such as lipopolysaccharide-binding protein (LBP) and CD14 with high specificity. At LPS concentrations as low as 0.3 ng/ml, such interactions may lead to severe pathophysiological effects, including sepsis and septic shock. One approach to inhibit an uncontrolled inflammatory reaction is the use of appropriate polycationic and amphiphilic antimicrobial peptides, here called synthetic anti-LPS peptides (SALPs). We designed various SALP structures and investigated their ability to inhibit LPS-induced cytokine secretion in vitro, their protective effect in a mouse model of sepsis, and their cytotoxicity in physiological human cells. Using a variety of biophysical techniques, we investigated selected SALPs with considerable differences in their biological responses to characterize and understand the mechanism of LPS inactivation by SALPs. Our investigations show that neutralization of LPS by peptides is associated with a fluidization of the LPS acyl chains, a strong exothermic Coulomb interaction between the two compounds, and a drastic change of the LPS aggregate type from cubic into multilamellar, with an increase in the aggregate sizes, inhibiting the binding of LBP and other mammalian proteins to the endotoxin. At the same time, peptide binding to phospholipids of human origin (e.g., phosphatidylcholine) does not cause essential structural changes, such as changes in membrane fluidity and bilayer structure. The absence of cytotoxicity is explained by the high specificity of the interaction of the peptides with LPS.