Advancing predictions of protein stability in the solid state.

The ß-relaxation associated with the sub-glass transition temperature (Tg,ß) is attributed to fast, localised molecular motions which can occur below the primary glass transition temperature (Tg,α). Consistent with Tg,ß being observed well-below storage temperatures, the ß-relaxation associated motions have been hypothesised to influence protein stability in the solid state and could thus impact the quality of e.g. protein powders for inhalation or reconstitution and injection. Why then do distinct solid state protein formulations with similar aggregation profiles after drying and immediate reconstitution, display different profiles when reconstituted following prolonged storage? Is the value of Tg,ß, associated with the ß-relaxation process of the system, a reliable parameter for characterising the behaviour of proteins in the solid state? Bearing this in mind, in this work we further explore the different relaxation dynamics of glassy solid state monoclonal antibody formulations using terahertz time-domain spectroscopy and dynamical mechanical analysis. By conducting a 52 week stability study on a series of multi-component spray-dried formulations, an approach for characterising and analysing the solid state dynamics and how these relate to protein stability is outlined.

Insights into the architecture and stoichiometry of Escherichia coli PepA*DNA complexes involved in transcriptional control and site-specific DNA recombination by atomic force microscopy.

Multifunctional Aminopeptidase A (PepA) from Escherichia coli is involved in the control of two distinct DNA transaction processes: transcriptional repression of the carAB operon, encoding carbamoyl phosphate synthase and site-specific resolution of ColE1-type plasmid multimers. Both processes require communication at a distance along a DNA molecule and PepA is the major structural component of the nucleoprotein complexes that underlie this communication. Atomic Force Microscopy was used to analyze the architecture of PepA.carAB and PepA.cer site complexes. Contour length measurements, bending angle analyses and volume determinations demonstrate that the carP1 operator is foreshortened by approximately 235 bp through wrapping around one PepA hexamer. The highly deformed part of the operator extends from slightly upstream of the -35 hexamer of the carP1 promoter to just downstream of the IHF-binding site, and comprises the binding sites for the PurR and RutR transcriptional regulators. This extreme remodeling of the carP1 control region provides a straightforward explanation for the strict requirement of PepA in the establishment of pyrimidine and purine-specific repression of carAB transcription. We further provide a direct physical proof that PepA is able to synapse two cer sites in direct repeat in a large interwrapped nucleoprotein complex, likely comprising two PepA hexamers.

Relationship of PEG-induced precipitation with protein-protein interactions and aggregation rates of high concentration mAb formulations at 5 °C.

Native protein-protein interactions can play an important role in determining the tendency of monoclonal antibodies (mAbs) to aggregate under storage conditions. In this context, phase separation of mAb solutions induced by the addition of neutral polymers such as poly(ethylene glycol) (PEG) represents a simple method to assess the tendency of proteins to self-associate in the native state. Here, we investigated their relationships between PEG-induced phase separation, protein-protein interactions and long-term aggregation rate of several formulations of four mAbs at 100 mg/mL and 5 °C over 12 weeks of storage. We observed that the location of the phase boundary correlated well with the osmotic second virial coefficient B22 determined in absence of the polymer, indicating that for our solutions PEG primarily leads to depletion forces between protein molecules, which are additive to protein-protein interactions. However, limited correlation between aggregation rate at 5 °C and phase behavior was observed across different mAbs, pH values and ionic strengths, indicating that colloidal stability is not the only determinant of aggregation even at such low temperature and high protein concentration. Our results contribute to the growing realization that aggregation propensity in the context of antibody developability is a complex feature, which depends on a variety of biophysical properties rather than one single parameter.

Accelerated Aggregation Studies of Monoclonal Antibodies: Considerations for Storage Stability.

Aggregation of mAbs is a crucial concern with respect to their safety and efficacy. Among the various properties of protein aggregates, it is emerging that their size can potentially impact their immunogenicity. Therefore, stability studies of antibody formulations should not only evaluate the rate of monomer loss but also determine the size distribution of the protein aggregates, which in turn depends on the aggregation mechanism. Here, we study the aggregation behavior of different formulations of 2 monoclonal immunoglobulins (IgGs) in the temperature range from 5°C to 50°C over 52 weeks of storage. We show that the aggregation kinetics of both antibodies follow non-Arrhenius behavior and that the aggregation mechanisms change between 40°C and 5°C, leading to different types of aggregates. Specifically, for a given monomer conversion, dimer formation dominates at low temperatures, while larger aggregates are formed at higher temperatures. We further show that the stability ranking of different molecules as well as of different formulations is drastically different at 40°C and 5°C while it correlates better between 30°C and 5°C. Our findings have implications for the level of information provided by accelerated aggregation studies with respect to protein stability under storage conditions.

Formulating monoclonal antibodies as powders for reconstitution at high concentration using spray-drying: Trehalose/amino acid combinations as reconstitution time reducing and stability improving formulations.

To increase their stability, therapeutic (or monoclonal) antibodies (mAbs) are often formulated as solids by using a variety of drying techniques, e.g. freeze-drying, spray-drying, or spray freeze-drying. The addition of excipients is required to preserve stability of the protein during the drying process and subsequent storage of the resulting solid form. The addition of low molecular weight excipients, such as amino acids, to sugar based spray- and freeze-dried formulations has been suggested to improve the storage stability of proteins in the dried state. In this study sugars (sucrose, trehalose), amino acids (Gly, Ala, Pro, Ser, Val, Leu, Ile, Gln, His, Lys, Arg, Phe, Trp) and combinations thereof were investigated for their stabilizing effect during spray-drying and subsequent storage and for their reconstitution time reducing effect. Two IgG4 mAbs were used as model antibodies. From an initial screening study, basic and small neutral amino acids, in combination with a sugar, such as sucrose or trehalose, showed reconstitution time reducing and stabilizing properties. Arg in particular displayed excellent reconstitution and stability enhancing properties. Moreover, Arg was the only amino acid providing stabilizing properties comparable to sucrose or trehalose. Previous work by the authors described a statistically substantiated comparison between the three basic amino acids in a sugar containing formulation, albeit limited to a single concentration level [5]. Therefore, a follow-up design of experiments (DoE) study was performed to determine the optimum trehalose/amino acid content required for an optimal protein stability and reconstitution time and to compare the effects of two basic amino acids, Lys and Arg, to those of two neutral amino acids, Gly and Pro. The conducted DoE covered a wide range of trehalose (30-120 mM) and amino acid (50-150 mM) concentrations. The concentration of trehalose was found to be the main contributor to a reduction in reconstitution time and an increase in stability. Here we show that the addition of amino acids such as Gly, Pro, and Lys does not improve stability nor does it reduce the reconstitution time. Of the tested amino acids, only Arg showed a marked reduction in reconstitution time and improvement in stability compared to a trehalose. Moreover, the properties displayed by Arg could justify its application as the main stabilizer in spray-dried mAb formulations, eliminating the need for a sugar matrix altogether. But the weight ratio of stabilizer to protein was found the factor exerting the strongest overall influence on the formulation's reconstitution time and stability. More specifically, sufficient physical stability and an acceptable reconstitution time could be obtained with a protein to stabilizer weight ratio of at least 1:1.

Feasibility of electrospraying fully aqueous bovine serum albumin solutions.

Electrospraying or electrohydrodynamic atomisation, i.e. the formation of tiny droplets from a jet of conductive liquid under the influence of an electric field, has been gaining in popularity as a particle engineering technique in recent years. In addition to general benefits for particle engineering, e.g. the ability to generate nanometre sized particles with a very narrow size distribution, electrospraying also possesses a number of characteristics, like its applicability at ambient conditions, which could make it especially interesting for formulating therapeutic proteins. However, as fully aqueous solutions of proteins tend to have relatively high electrical conductivities and surface tensions, obtaining a stable Taylor cone-jet mode for these solutions is inherently challenging. This is why in the majority of studies reporting the successful electrospraying of proteins, either emulsions, aqueous suspensions or a mixture of water and one or more organic solvents were used instead of fully aqueous solutions. Therefore, an ab initio electrospraying formulation development study was conducted, using only fully aqueous feed solutions containing protein stabilising excipients commonly used in spray- and freeze-drying of therapeutic proteins. The study included bovine serum albumin (BSA) as a model protein and consisted out of two parts: (1) a one parameter at a time screening study, designed to improve the understanding of how various formulation components influence relevant physicochemical properties and the electrospraying process and (2) two subsequent mixture design of experiments (DoE) studies, designed to aid in the statistical description and prediction of the influence of different protein-excipient combinations on the electrospraying process. Additionally, the influence of physicochemical properties relevant to the electrospraying process, i.e. the volumetric mass density, electrical conductivity, kinematic viscosity and surface tension, was assessed for all feed solutions included in the study.

Crystal structure of a cold-adapted class C beta-lactamase.

In this study, the crystal structure of a class C beta-lactamase from a psychrophilic organism, Pseudomonas fluorescens, has been refined to 2.2 A resolution. It is one of the few solved crystal structures of psychrophilic proteins. The structure was compared with those of homologous mesophilic enzymes and of another, modeled, psychrophilic protein. The elucidation of the 3D structure of this enzyme provides additional insights into the features involved in cold adaptation. Structure comparison of the psychrophilic and mesophilic beta-lactamases shows that electrostatics seems to play a major role in low-temperature adaptation, with a lower total number of ionic interactions for cold enzymes. The psychrophilic enzymes are also characterized by a decreased number of hydrogen bonds, a lower content of prolines, and a lower percentage of arginines in comparison with lysines. All these features make the structure more flexible so that the enzyme can behave as an efficient catalyst at low temperatures.

ArgR-dependent repression of arginine and histidine transport genes in Escherichia coli K-12.

In Escherichia coli L-arginine is taken up by three periplasmic binding protein-dependent transport systems that are encoded by two genetic loci: the artPIQM-artJ and argT-hisJQMP gene clusters. The transcription of the artJ, artPIQM and hisJQMP genes and operons is repressed by liganded ArgR, whereas argT, encoding the LAO (lysine, arginine, ornithine) periplasmic binding protein, is insensitive to the repressor. Here we characterize the repressible Esigma70 P artJ, P artP and P hisJ promoters and demonstrate that the cognate operators consist of two 18 bp ARG boxes separated by 3 bp. Determination of the energy landscape of the ArgR-operator contacts by missing contact probing and mutant studies indicated that each box of a pair contributes to complex formation in vitro and to the repressibility in vivo, but to a different extent. The organization of the ARG boxes and promoter elements in the control regions of the uptake genes is distinct from that of the arginine biosynthetic genes. The hisJQMP operon is the first member of the E. coli ArgR regulon, directly repressed by liganded ArgR, where none of the core promoter elements overlaps the ARG boxes. Single round in vitro transcription assays and DNase I footprinting experiments indicate that liganded ArgR inhibits P artJ and P artP promoter activity by steric exclusion of the RNA polymerase. In contrast, ArgR-mediated repression of P hisJ by inhibition of RNA polymerase binding appears to occur through topological changes of the promoter region.

Formulating monoclonal antibodies as powders for reconstitution at high concentration using spray drying: Models and pitfalls.

In anticipation of non-invasive routes capable of delivering adequately high, systemic monoclonal antibody (mAb) concentrations, subcutaneous (SC) injection is arguably the most patient friendly alternative administration route available for this drug class. However, due to the limited volume that can be administered through this route and mAbs' relatively low therapeutic activity, solutions for subcutaneous injection often need to be highly concentrated, making them inherently more prone to potentially detrimental protein (self-) interaction, which is why mAb formulations for SC injection and other highly concentrated mAb solutions are often dried to increase their stability. In this work we investigated spray drying (SD) as a drying technique for formulating mAbs as powders for reconstitution, assessing the influence of SD process parameters, as well as excipients present in the feed solution on both mAb stability and relevant powder characteristics for reconstitution using a model mAb. By employing a design of experiments approach, we were able to provide statistically substantiated evidence for the reconstitution time reducing and stability improving properties of l-arginineHCl, l-histidineHCl, l-lysineHCl and polysorbate 20 when combined with a disaccharide in SD mAb powders for reconstitution. Additionally, the study yielded several statistical models describing process parameter influences on relevant powder and mAb stability characteristics.

Crystallization and preliminary X-ray diffraction analysis of the arginine repressor of the hyperthermophile Thermotoga neapolitana.

The arginine repressor of Thermotoga neapolitana (ArgRTnp) is a member of the family of multifunctional bacterial arginine repressors involved in the regulation of arginine metabolism. This hyperthermophilic repressor shows unique DNA-binding features that distinguish it from its homologues. ArgRTnp exists as a homotrimeric protein that assembles into hexamers at higher protein concentrations and/or in the presence of arginine. ArgRTnp was crystallized with and without its corepressor arginine using the hanging-drop vapour-diffusion method. Crystals of the aporepressor diffracted to a resolution of 2.1 A and belong to the orthorhombic P2(1)2(1)2(1) space group, with unit-cell parameters a = 117.73, b = 134.15, c = 139.31 A. Crystals of the repressor in the presence of its corepressor arginine diffracted to a resolution of 2.4 A and belong to the same space group, with similar unit-cell parameters.

Kinetics of Monoclonal Antibody Aggregation from Dilute toward Concentrated Conditions.

Gaining understanding on the aggregation behavior of proteins under concentrated conditions is of both fundamental and industrial relevance. Here, we study the aggregation kinetics of a model monoclonal antibody (mAb) under thermal stress over a wide range of protein concentrations in various buffer solutions. We follow experimentally the monomer depletion and the aggregate growth by size exclusion chromatography with inline light scattering. We describe the experimental results in the frame of a kinetic model based on population balance equations, which allows one to discriminate the contributions of the conformational and of the colloidal stabilities to the global aggregation rate. Finally, we propose an expression for the aggregation rate constant, which accounts for solution viscosity, protein-protein interactions, as well as aggregate compactness. All these effects can be quantified by light scattering techniques. It is found that the model describes well the experimental data under dilute conditions. Under concentrated conditions, good model predictions are obtained when the solution pH is far below the isoelectric point (pI) of the mAb. However, peculiar effects arise when the solution pH is increased toward the mAb pI, and possible explanations are discussed.

New experimental approaches for investigating interactions between Pyrococcus furiosus carbamate kinase and carbamoyltransferases, enzymes involved in the channeling of thermolabile carbamoyl phosphate.

A somewhat neglected but essential aspect of the molecular physiology of hyperthermophiles is the protection of thermolabile metabolites and coenzymes. An example is carbamoyl phosphate (CP), a precursor of pyrimidines and arginine, which is an extremely labile and potentially toxic intermediate. The first evidence for a biologically significant interaction between carbamate kinase (CK) and ornithine carbamoyltransferase (OTC) from Pyrococcus furiosus was provided by affinity electrophoresis and co-immunoprecipitation in combination with cross-linking (Massant et al. 2002). Using the yeast two-hybrid system, Hummel-Dreyer chromatography and isothermal titration calorimetry, we obtained additional concrete evidence for an interaction between CK and OTC, the first evidence for an interaction between CK and aspartate carbamoyltransferase (ATC) and an estimate of the binding constant between CK and ATC. The physical interaction between CK and OTC or ATC may prevent thermodenaturation of CP in the aqueous cytoplasmic environment. Here we emphasize the importance of developing experimental approaches to investigate the mechanism of thermal protection of metabolic intermediates by metabolic channeling and the molecular basis of transient protein-protein interactions in the physiology of hyperthermophiles.

Refined structure of Pyrococcus furiosus ornithine carbamoyltransferase at 1.87 A.

Using synchrotron radiation, X-ray data have been collected from Pyrococcus furiosus ornithine carbamoyltransferase (Pfu OTCase) to a maximal resolution of 1.87 A, allowing the refinement of a previous structure at 2.7 A [Villeret et al. (1998), Proc. Natl Acad. Sci. USA, 95, 2801-2806]. Thanks to the high resolution of this refined structure, two sulfate ions and 191 water molecules could be localized directly from the electron-density maps. The identification of these molecules allowed a more rigorous description of the active site and the identification of residues involved in binding carbamoyl phosphate. The improved quality of the model resulted in a better definition of several loops and the various interfaces. The dodecameric protein is composed of four catalytic trimers disposed in a tetrahedral manner. The extreme thermal stability of Pfu OTCase is mainly the result of the strengthening of the intersubunit interactions in a trimer and oligomerization of the trimers into a dodecamer. Interfaces between monomers in a catalytic trimer are characterized by an increase in ion-pair networks compared with mesophilic OTCases. However, the interfaces between catalytic trimers in the dodecameric oligomer are mainly hydrophobic and also involve aromatic-aromatic and cation-pi interactions.

Metabolic channeling of carbamoyl phosphate, a thermolabile intermediate: evidence for physical interaction between carbamate kinase-like carbamoyl-phosphate synthetase and ornithine carbamoyltransferase from the hyperthermophile Pyrococcus furiosus.

Two different approaches provided evidence for a physical interaction between the carbamate kinase-like carbamoyl-phosphate synthetase (CKase) and ornithine carbamoyltransferase (OTCase) from the hyperthermophilic archaeon Pyrococcus furiosus. Affinity electrophoresis indicated that CKase and OTCase associate into a multienzyme cluster. Further evidence for a biologically significant interaction between CKase and OTCase was obtained by co-immunoprecipitation combined with formaldehyde cross-linking experiments. These experiments support the hypothesis that CKase and OTCase form an efficient channeling cluster for carbamoyl phosphate, an extremely thermolabile and potentially toxic metabolic intermediate. Therefore, by physically interacting with each other, CKase and OTCase prevent the thermodenaturation of carbamoyl phosphate in the aqueous cytoplasmic environment.