Refolding and characterization of two G protein-coupled receptors purified from E. coli inclusion bodies.

Aiming at streamlining GPCR production from E. coli inclusion bodies for structural analysis, we present a generic approach to assess and optimize refolding yield through thermostability analysis. Since commonly used hydrophobic dyes cannot be applied as probes for membrane protein unfolding, we adapted a technique based on reacting cysteins exposed upon thermal denaturation with fluorescent 7-Diethylamino-3-(4-maleimidophenyl)-4-methylcoumarin (CPM). Successful expression, purification and refolding is shown for two G protein-coupled receptors (GPCR), the sphingosine-1-phosphate receptor S1P1, and the orphan receptor GPR3. Refolded receptors were subjected to lipidic cubic phase crystallization screening.

Structure of a Therapeutic Full-Length Anti-NPRA IgG4 Antibody: Dissecting Conformational Diversity.

We report the x-ray crystal structure of intact, full-length human immunoglobulin (IgG4) at 1.8 Å resolution. The data for IgG4 (S228P), an antibody targeting the natriuretic peptide receptor A, show a previously unrecognized type of Fab-Fc orientation with a distorted λ-shape in which one Fab-arm is oriented toward the Fc portion. Detailed structural analysis by x-ray crystallography and molecular simulations suggest that this is one of several conformations coexisting in a dynamic equilibrium state. These results were confirmed by small angle x-ray scattering in solution. Furthermore, electron microscopy supported these findings by preserving molecule classes of different conformations. This study fosters our understanding of IgG4 in particular and our appreciation of antibody flexibility in general. Moreover, we give insights into potential biological implications, specifically for the interaction of human anti-natriuretic peptide receptor A IgG4 with the neonatal Fc receptor, Fcγ receptors, and complement-activating C1q by considering conformational flexibility.

Towards understanding polyol additive effects on the pH shift-induced aggregation of a monoclonal antibody using high throughput screening and quantitative structure-activity modeling.

pH shift-induced aggregation is frequently observed in downstream processing of monoclonal antibodies and has been shown to depend on solvent composition. To quantify the stabilizing effect of polyol additives against aggregation, we determined aggregation rate constants in the presence of a set of 14 compounds. Rate constants were then correlated with molecular descriptors in a quantitative structure activity relationship (QSAR) approach. The molecular size, volume, the charge, number of hydrogen acceptors, the stereochemistry and hydrophobicity of the compounds were identified as important descriptors. Generally larger compounds with a balanced surface polarity tend to inhibit aggregation better while hydrophobicity plays an important role at the nucleation phase, with hydrophobic compounds being more potent at inhibiting aggregation.

High-throughput analysis of sub-visible mAb aggregate particles using automated fluorescence microscopy imaging.

Aggregation of therapeutic proteins is a major concern as aggregates lower the yield and can impact the efficacy of the drug as well as the patient's safety. It can occur in all production stages; thus, it is essential to perform a detailed analysis for protein aggregates. Several methods such as size exclusion high-performance liquid chromatography (SE-HPLC), light scattering, turbidity, light obscuration, and microscopy-based approaches are used to analyze aggregates. None of these methods allows determination of all types of higher molecular weight (HMW) species due to a limited size range. Furthermore, quantification and specification of different HMW species are often not possible. Moreover, automation is a perspective challenge coming up with automated robotic laboratory systems. Hence, there is a need for a fast, high-throughput-compatible method, which can detect a broad size range and enable quantification and classification. We describe a novel approach for the detection of aggregates in the size range 1 to 1000 µm combining fluorescent dyes for protein aggregate labelling and automated fluorescence microscope imaging (aFMI). After appropriate selection of the dye and method optimization, our method enabled us to detect various types of HMW species of monoclonal antibodies (mAbs). Using 10 µmol L-1 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonate (Bis-ANS) in combination with aFMI allowed the analysis of mAb aggregates induced by different stresses occurring during downstream processing, storage, and administration. Validation of our results was performed by SE-HPLC, UV-Vis spectroscopy, and dynamic light scattering. With this new approach, we could not only reliably detect different HMW species but also quantify and classify them in an automated approach. Our method achieves high-throughput requirements and the selection of various fluorescent dyes enables a broad range of applications.

Diversity selection, screening and quantitative structure-activity relationships of osmolyte-like additive effects on the thermal stability of a monoclonal antibody.

Solvents used for therapeutic proteins in downstream processing and in formulations often contain stabilizing additives that inhibit denaturation and aggregation. Such additives are mostly selected based on their positive effect on thermal stability of the protein, and are often derived from naturally occuring osmolytes. To better understand the structural basis underlying the effect of additives, we selected a diverse library of compounds comprising 79 compounds of the polyol, amino acid and methylamine chemical classes and determined the effect of each compound on thermal stability of a monoclonal antibody as a function of compound concentration. Thermal stabilization of the antibody was influenced by solution pH. Quantitative structure-activity relationships (QSAR) were derived by partial least squares regression for individual compound classes and globally. The global model suggests that ligands with a phenyl ring will decrease the Tm, while highly soluble, polar compounds with at least two hydrogen bond donors will increase the Tm. This approach may be beneficial for further studies on the influence of other solution conditions like ionic strength and buffer species on additive-mediated protein stabilization.

Reversible NaCl-induced aggregation of a monoclonal antibody at low pH: Characterization of aggregates and factors affecting aggregation.

We investigated the influence of pH and sodium chloride concentration on aggregation kinetics of a monoclonal antibody. Aggregation was induced by sodium chloride addition at low pH. Protein conformation before and after salt addition was determined as well as the reversibility of aggregation. Aggregation was monitored at pH values between 2 and 7 with NaCl up to 1.5M by turbidity measurement and size-exclusion chromatography. Particle size distribution was assessed by using size-exclusion chromatography as well as nanoparticle tracking analysis and flow imaging microscopy. Structural changes were monitored by circular dichroism, Fourier transform infrared and fluorescence spectroscopy. Thermal stability was measured by differential scanning fluorimetry. Aggregation propensity was maximal at low pH and high ionic strength. While thermal stability decreased with pH, the secondary structure remained unchanged down to pH 3.5 and up to 1.5M NaCl. Precipitated protein could be largely reverted to monomers by dilution into salt-free buffer. The re-solubilized antibody was indistinguishable in structure, solubility and monodispersity from the unstressed protein. Also, binding to Protein A was steady. Aggregation could be reduced in the presence of trehalose. The results suggest a reversible aggregation mechanism characterized by a limited change in tertiary structure at low pH and a subsequent loss of colloidal stability resulting from electrostatic repulsion once salt is added to the sample. The experimental setup is robust and allows high-throughput quantification of the effect of additives on aggregation kinetics.

Experimental Model System to Study pH Shift-Induced Aggregation of Monoclonal Antibodies Under Controlled Conditions.

PURPOSE: To present a convenient screening method for evaluating additive effects on the renaturation of an acid-exposed monoclonal antibody (mAb). METHODS: The assay involves brief incubation of a mAb at acidic pH and subsequent neutralization in the absence or presence of additive to induce mainly aggregation. An increase in absorbance depicted aggregation. The recorded aggregation data traces were fitted with a nucleation-autocatalytic growth model for the extraction of kinetic parameters. RESULTS: All kinetic data traces were fitted successfully with the selected model and the adjusted R square values were greater than 0.99. Trehalose had strongly stabilizing, proline mildly stabilizing and trimethylamine oxide had destabilizing effects on both the nucleation and growth phase of the reaction. Histidine was strongly stabilizing but was limited by its poor solubility. CONCLUSION: The results demonstrate the suitability of the experimental mAb aggregation system and the nucleation-autocatalytic growth fit in the screening and quantification of additive effects on the renaturation of an acid-exposed mAb respectively. This will aid the investigation and derivation of quantitative structure-activity relationships of additive effects on mAb solubility.

A scale-down cross-flow filtration technology for biopharmaceuticals and the associated theory.

Use of microfiltration (MF) and ultrafiltration (UF) in cross-flow mode has been intensifying in downstream processing for expensive biopharmaceuticals. A scale-down cross-flow module with ring channel was constructed for reducing costs and increasing throughput. Commensurate with its validation, a new scale down (or scale up) theoretical framework has been further developed to 3 operational parities: (1) ratio of initial sample volume to membrane area, (2) shear force adjacent to membrane surface, and (3) initial permeate flux. By keeping identical initial physicochemical properties, we show that these 3 operational parities are equivalent to 2 further time-dependent theoretical parities for flux and transmission respectively. Importantly, transmission sensitively reflects membrane conditions for partially transmissible molecules or particles. Computational fluid dynamics simulation was conducted to confirm nearly identical shear forces for the mini and its reference filters. Permeate fluxes in suspension containing Escherichia coli phage T7, a monoclonal antibody (MAb) or other proteins, and transmission (with phage T7) were measured. For application demonstration, diafiltration and concentration modes were applied to the MAb, and separation mode to a mixture of bovine serum albumin and lysozyme. In conclusion, the developed scale-down filter has been shown to behave identically or similarly to its reference filter.

Structure of the chemokine receptor CXCR1 in phospholipid bilayers.

CXCR1 is one of two high-affinity receptors for the CXC chemokine interleukin-8 (IL-8), a major mediator of immune and inflammatory responses implicated in many disorders, including tumour growth. IL-8, released in response to inflammatory stimuli, binds to the extracellular side of CXCR1. The ligand-activated intracellular signalling pathways result in neutrophil migration to the site of inflammation. CXCR1 is a class A, rhodopsin-like G-protein-coupled receptor (GPCR), the largest class of integral membrane proteins responsible for cellular signal transduction and targeted as drug receptors. Despite its importance, the molecular mechanism of CXCR1 signal transduction is poorly understood owing to the limited structural information available. Recent structural determination of GPCRs has advanced by modifying the receptors with stabilizing mutations, insertion of the protein T4 lysozyme and truncations of their amino acid sequences, as well as addition of stabilizing antibodies and small molecules that facilitate crystallization in cubic phase monoolein mixtures. The intracellular loops of GPCRs are crucial for G-protein interactions, and activation of CXCR1 involves both amino-terminal residues and extracellular loops. Our previous nuclear magnetic resonance studies indicate that IL-8 binding to the N-terminal residues is mediated by the membrane, underscoring the importance of the phospholipid bilayer for physiological activity. Here we report the three-dimensional structure of human CXCR1 determined by NMR spectroscopy. The receptor is in liquid crystalline phospholipid bilayers, without modification of its amino acid sequence and under physiological conditions. Features important for intracellular G-protein activation and signal transduction are revealed. The structure of human CXCR1 in a lipid bilayer should help to facilitate the discovery of new compounds that interact with GPCRs and combat diseases such as breast cancer.

The effect of osmolytes on protein fibrillation.

Osmolytes are small molecules that are exploited by cells as a protective system against stress conditions. They favour compact protein states which makes them stabilize globular proteins in vitro and promote folding. Conversely, this preference for compact states promotes aggregation of unstructured proteins. Here we combine a brief review of the effect of osmolytes on protein fibrillation with a report of the effect of osmolytes on the unstructured peptide hormone glucagon. Our results show that osmolytes either accelerate the fibrillation kinetics or leave them unaffected, with the exception of the osmolyte taurine. Furthermore, the osmolytes that affected the shape of the fibrillation time profile led to fibrils with different structure as revealed by CD. The structural changes induced by Pro, Ser and choline-O-sulfate could be due to specific osmolytes binding to the peptides, stabilizing an otherwise labile fibrillation intermediate.

Optimization of purification and refolding of the human chemokine receptor CXCR1 improves the stability of proteoliposomes for structure determination.

The human chemokine receptor CXCR1 is a G-protein coupled receptor that has been successfully expressed in E. coli as inclusion bodies, and purified and refolded in multi-milligram quantities required for structural studies. Expression in E. coli enables selective and uniform isotopic labeling with (13)C and (15)N for NMR studies. Long-term chemical and conformational stability and oligomeric homogeneity of CXCR1 in phospholipid bilayers are crucial for structural studies under physiological conditions. Here we describe substantial refinements in our previously described purification and reconstitution procedures for CXCR1 in phospholipid bilayers. These refinements have led to the preparation of highly purified, completely monomeric, proteoliposome samples that are stable for months at 35°C while subject to the high power radiofrequency irradiations of solid-state NMR experiments. The principal changes from the previously described methods include: 1) ensure that CXCR1 is pure and homogeneously monomeric within the limits of detection (>98%); 2) monitor and control the pH at all times especially following the addition of TCEP, which serves as a reducing agent but also changes the pH; 3) slowly refold CXCR1 with the complete removal of all traces of SDS using a KCl precipitation/dialysis method; and 4) ensure that the molar ratio between the CXCR1 and the phospholipids does not change during refolding and detergent removal. NMR samples prepared with these protocols yield reproducible results over a period of many months at 35°C. This purification and refolding protocol is likely to be applicable with minimal changes to other GPCRs as well as other membrane proteins.

Towards protein crystallization as a process step in downstream processing of therapeutic antibodies: screening and optimization at microbatch scale.

Crystallization conditions of an intact monoclonal IgG4 (immunoglobulin G, subclass 4) antibody were established in vapor diffusion mode by sparse matrix screening and subsequent optimization. The procedure was transferred to microbatch conditions and a phase diagram was built showing surprisingly low solubility of the antibody at equilibrium. With up-scaling to process scale in mind, purification efficiency of the crystallization step was investigated. Added model protein contaminants were excluded from the crystals to more than 95%. No measurable loss of Fc-binding activity was observed in the crystallized and redissolved antibody. Conditions could be adapted to crystallize the antibody directly from concentrated and diafiltrated cell culture supernatant, showing purification efficiency similar to that of Protein A chromatography. We conclude that crystallization has the potential to be included in downstream processing as a low-cost purification or formulation step.

Production and characterisation of monoclonal antibodies against RAI3 and its expression in human breast cancer.

BACKGROUND: RAI3 is an orphan G-protein coupled receptor (GPCR) that has been associated with malignancy and may play a role in the proliferation of breast cancer cells. Although its exact function in normal and malignant cells remains unclear and evidence supporting its role in oncogenesis is controversial, its abundant expression on the surface of cancer cells would make it an interesting target for the development of antibody-based therapeutics. To investigate the link with cancer and provide more evidence for its role, we carried out a systematic analysis of RAI3 expression in a large set of human breast cancer specimens. METHODS: We expressed recombinant human RAI3 in bacteria and reconstituted the purified protein in liposomes to raise monoclonal antibodies using classical hybridoma techniques. The specific binding activity of the antibodies was confirmed by enzyme-linked immunosorbent assay (ELISA), western blot and immunocytochemistry. We carried out a systematic immunohistochemical analysis of RAI3 expression in human invasive breast carcinomas (n = 147) and normal breast tissues (n = 44) using a tissue microarray. In addition, a cDNA dot blot hybridisation assay was used to investigate a set of matched normal and cancerous breast tissue specimens (n = 50) as well as lymph node metastases (n = 3) for RAI3 mRNA expression. RESULTS: The anti-RAI3 monoclonal antibodies bound to recombinant human RAI3 protein with high specificity and affinity, as shown by ELISA, western blot and ICC. The cDNA dot blot and immunohistochemical experiments showed that both RAI3 mRNA and RAI3 protein were abundantly expressed in human breast carcinoma. However, there was no association between RAI3 protein expression and prognosis based on overall and recurrence-free survival. CONCLUSION: We have generated a novel, highly-specific monoclonal antibody that detects RAI3 in formaldehyde-fixed paraffin-embedded tissue. This is the first study to report a systematic analysis of RAI3 expression in normal and cancerous human breast tissue at both the mRNA and protein levels.

High-resolution NMR spectroscopy of a GPCR in aligned bicelles.

Solid-state NMR spectra with single-site resolution of CXCR1, a G protein-coupled receptor (GPCR), were obtained in magnetically aligned phospholipid bicelles. These results demonstrate that GPCRs in phospholipid bilayers are suitable samples for structure determination by solid-state NMR. The spectra also enable studies of drug-receptor interactions.

In vitro folding of alpha-helical membrane proteins.

For large-scale production, as required in structural biology, membrane proteins can be expressed in an insoluble form as inclusion bodies and be refolded in vitro. This requires refolding conditions where the native form is thermodynamically stable and where nonproductive pathways leading to aggregation are avoided. Examples of successful refolding are reviewed and general guidelines to establish refolding protocols of membrane proteins are presented.