Monoclonal Antibodies Follow Distinct Aggregation Pathways During Production-Relevant Acidic Incubation and Neutralization.

PURPOSE: Aggregation aspects of therapeutic monoclonal antibodies (mAbs) are of common concern to the pharmaceutical industry. Low pH treatment is applied during affinity purification and to inactivate endogenous retroviruses, directing interest to the mechanisms of acid-induced antibody aggregation. METHODS: We characterized the oligomerization kinetics at pH 3.3, as well as the reversibility upon neutralization, of three model mAbs with identical variable regions, representative of IgG1, IgG2 and IgG4 respectively. We applied size-exclusion high performance liquid chromatography and orthogonal analytical methods, including small-angle X-ray scattering and dynamic light scattering and supplemented the experimental data with crystal structure-based spatial aggregation propensity (SAP) calculations. RESULTS: We revealed distinct solution behaviors between the three mAb models: At acidic pH IgG1 retained monomeric, whereas IgG2 and IgG4 exhibited two-phase oligomerization processes. After neutralization, IgG2 oligomers partially reverted to the monomeric state, while on the contrary, IgG4 oligomers tended to aggregate. Subclass-specific aggregation-prone motifs on the Fc fragments were identified, which may lead to two distinct pathways of reversible and irreversible aggregation, respectively. CONCLUSIONS: We conclude that subtle variations in mAb sequence greatly affect responses towards low-pH incubation and subsequent neutralization, and demonstrate how orthogonal biophysical methods distinguish between reversible and irreversible mAb aggregation pathways at early stages of acidic treatment.

Impacts of polyethylene glycol (PEG) dispersity on protein adsorption, pharmacokinetics, and biodistribution of PEGylated gold nanoparticles.

PEGylated gold nanoparticles (PEG-AuNPs) are widely used in drug delivery, imaging and diagnostics, therapeutics, and biosensing. However, the effect of PEG dispersity on the molecular weight (M W) distribution of PEG grafted onto AuNP surfaces has been rarely reported. This study investigates the effect of PEG dispersity on the M W distribution of PEG grafted onto AuNP surfaces and its subsequent impact on protein adsorption and pharmacokinetics, by modifying AuNPs with monodisperse PEG methyl ether thiols (mPEG n -HS, n = 36, 45) and traditional polydisperse mPEG2k-SH (M W = 1900). Polydisperse PEG-AuNPs favor the enrichment of lower M W PEG fractions on their surface due to the steric hindrance effect, which leads to increased protein adsorption. In contrast, monodisperse PEG-AuNPs have a uniform length of PEG outlayer, exhibiting markedly lower yet constant protein adsorption. Pharmacokinetics analysis in tumor-bearing mice demonstrated that monodisperse PEG-AuNPs possess a significantly prolonged blood circulation half-life and enhanced tumor accumulation compared with their polydisperse counterpart. These findings underscore the critical, yet often underestimated, impacts of PEG dispersity on the in vitro and in vivo behavior of PEG-AuNPs, highlighting the role of monodisperse PEG in enhancing therapeutic nanoparticle performance.

Impact of Stress on the Immunogenic Potential of Adalimumab.

Monoclonal antibodies against tumor necrosis factor-alpha (TNFα) are widely used for treatment of inflammatory diseases. However, despite the inhibitory effect this class of drugs has on the immune system, anti-drug antibodies are often formed with continuous use. Particles formed during stress conditions, which can be used to simulate storage and handling conditions of commercial antibodies, have previously been associated with the formation of anti-drug antibodies. This study investigates the relationship between particles, oligomerization, folding and chemical degradation on the in vitro cytokine response toward the TNFα inhibitor adalimumab. Adalimumab aggregates generated using stir and heat stress were fractionated into distinct sub-populations, and their structure and immunogenic potential were evaluated. A chemically degraded sample of adalimumab was included to compare particle composition with the milder accelerated heat and stir stressed conditions. Particles from stressed adalimumab samples induced elevated cytokine levels and CD4+ T cell proliferation in vitro compared to non-stressed samples. Samples enriched with both submicron and subvisible particles of adalimumab induced the strongest cytokine release and the strongest CD4+ T cell proliferation despite maintaining some TNFα inhibitory functionality. Samples that were stressed and subsequently purified of subvisible and submicron particles did not elicit a significantly higher cytokine response or show increased CD4+ T cell proliferation compared to a non-stressed sample. Oxidation-induced chemical modifications in adalimumab, mainly in Met, His, Trp, and Tyr, were not found to be sufficient in absence of particle formation to induce increased CD4+ T cell proliferation or cytokine release despite less decreased TNFα inhibitory activity of adalimumab. These observations provide further evidence that particles do indeed potentiate the immunogenic potential of adalimumab.

Endoplasmic reticulum stress mediates cortical neuron apoptosis after experimental subarachnoid hemorrhage in rats.

OBJECTIVE: Neuronal apoptosis plays an important pathological process in early brain injury (EBI) after subarachnoid hemorrhage (SAH). This pathological process leads to a poor neurological prognosis for patients. This study aimed to investigate whether endoplasmic reticulum (ER) stress mediates cortical neuron apoptosis in EBI after SAH. METHODS: Eighty-four male Sprague-Dawley rats were randomly assigned to different groups as follows: the control and the 3, 6, 12, 24, 48, and 72 h groups after SAH. The SAH model was established by injecting 0.3 mL of nonheparinized blood into the prechiasmatic cistern. Hematoxylin-eosin staining, Garcia scoring, Western blotting, transmission electron microscopy, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining were performed. RESULTS: SAH reduced the neurological scores and reached a trough at 24 h after the SAH. The GRP78 expression was significantly upregulated at 6 h after the SAH, peaked at 24 h after the SAH, and then decreased. By comparison, the CHOP, caspase-12, ASK1, and p-c-Jun N-terminal kinase expressions were significantly upregulated at 12 h after the SAH and peaked at 24 h after the SAH. The most serious swelling of the rough ER was observed at 24 h after the SAH and remained notably swollen at 72 h after the SAH. The number of TUNEL-positive cells substantially increased significantly at 12 h after the SAH, and the neuronal apoptosis decreased ratio after reaching a peak at 24 h after the SAH. The apoptosis ratio at 72 h after the SAH was still significantly different from the ratio in the control group. CONCLUSION: Our study clearly demonstrated that ER stress mediates cortical neuron apoptosis after experimental subarachnoid hemorrhage in rats.

Molecular cloning, expression, purification and crystallographic analysis of PRRSV 3CL protease.

3CL protease, a viral chymotrypsin-like proteolytic enzyme, plays a pivotal role in the transcription and replication machinery of many RNA viruses, including porcine reproductive and respiratory syndrome virus (PRRSV). In this study, the full-length 3CL protease from PRRSV was cloned and overexpressed in Escherichia coli. Crystallization experiments yielded crystals that diffracted to 2.1 A resolution and belong to space group C2, with unit-cell parameters a = 112.31, b = 48.34, c = 42.88 A, beta = 109.83 degrees . The Matthews coefficient and the solvent content were calculated to be 2.49 A(3) Da(-1) and 50.61%, respectively, for one molecule in the asymmetric unit.

A Comprehensive Evaluation of Nanoparticle Tracking Analysis (NanoSight) for Characterization of Proteinaceous Submicron Particles.

Nanoparticle tracking analysis (NTA) has attracted great interest for application in the field of submicron particle characterization for biopharmaceuticals. It has the virtue of direct sample visualization and particle-by-particle tracking, but the complexity of method development has limited its routine applicability. We systematically evaluated data collection and processing parameters as well as sample handling methods using shake-stressed protein samples. The camera shutter and gain were identified as the key factors influencing NTA results. We also demonstrated that sample filtration was necessary for NTA analysis if there were high numbers of micron particles, whereas the choice of filter membrane was critical for data quality. Sample dilution into corresponding formulation buffer did not affect particle size distributions in our study. Finally, NTA analysis exhibited excellent repeatability in intraday comparison of multiple measurements on the same sample and interday comparison on different batches of samples. Shaking-induced protein aggregation could also be sensitively monitored by NTA. In conclusion, NTA analysis can be used as a robust stability-indicating method for the characterization of proteinaceous submicron particles and thereby complement other analytical methods, provided that consistent sample handling and parametric settings are established for the specific case study.

In-depth analysis of subclass-specific conformational preferences of IgG antibodies.

IgG subclass-specific differences in biological function and in vitro stability are often referred to variations in the conformational flexibility, while this flexibility has rarely been characterized. Here, small-angle X-ray scattering data from IgG1, IgG2 and IgG4 antibodies, which were designed with identical variable regions, were thoroughly analysed by the ensemble optimization method. The extended analysis of the optimized ensembles through shape clustering reveals distinct subclass-specific conformational preferences, which provide new insights for understanding the variations in physical/chemical stability and biological function of therapeutic antibodies. Importantly, the way that specific differences in the linker region correlate with the solution structure of intact antibodies is revealed, thereby visualizing future potential for the rational design of antibodies with designated physicochemical properties and tailored effector functions. In addition, this advanced computational approach is applicable to other flexible multi-domain systems and extends the potential for investigating flexibility in solutions of macromolecules by small-angle X-ray scattering.

Structure of Dimeric and Tetrameric Complexes of the BAR Domain Protein PICK1 Determined by Small-Angle X-Ray Scattering.

PICK1 is a neuronal scaffolding protein containing a PDZ domain and an auto-inhibited BAR domain. BAR domains are membrane-sculpting protein modules generating membrane curvature and promoting membrane fission. Previous data suggest that BAR domains are organized in lattice-like arrangements when stabilizing membranes but little is known about structural organization of BAR domains in solution. Through a small-angle X-ray scattering (SAXS) analysis, we determine the structure of dimeric and tetrameric complexes of PICK1 in solution. SAXS and biochemical data reveal a strong propensity of PICK1 to form higher-order structures, and SAXS analysis suggests an offset, parallel mode of BAR-BAR oligomerization. Furthermore, unlike accessory domains in other BAR domain proteins, the positioning of the PDZ domains is flexible, enabling PICK1 to perform long-range, dynamic scaffolding of membrane-associated proteins. Together with functional data, these structural findings are compatible with a model in which oligomerization governs auto-inhibition of BAR domain function.

Small-angle x-ray scattering screening complements conventional biophysical analysis: comparative structural and biophysical analysis of monoclonal antibodies IgG1, IgG2, and IgG4.

A crucial step in the development of therapeutic monoclonal antibodies is the selection of robust pharmaceutical candidates and screening of efficacious protein formulations to increase the resistance toward physicochemical degradation and aggregation during processing and storage. Here, we introduce small-angle X-ray scattering (SAXS) to characterize antibody solution behavior, which strongly complements conventional biophysical analysis. First, we apply a variety of conventional biophysical techniques for the evaluation of structural, conformational, and colloidal stability and report a systematic comparison between designed humanized IgG1, IgG2, and IgG4 with identical variable regions. Then, the high information content of SAXS data enables sensitive detection of structural differences between three IgG subclasses at neutral pH and rapid formation of dimers of IgG2 and IgG4 at low pH. We reveal subclass-specific variation in intermolecular repulsion already at low and medium protein concentrations, which explains the observed improved stability of IgG1 with respect to aggregation. We show how excipients dramatically influence such repulsive effects, hence demonstrating the potential application of extensive SAXS screening in antibody selection, eventual engineering, and formulation development.

Detection of avian influenza virus subtype H5 using a biosensor based on imaging ellipsometry.

A novel method is reported for the detection of avian influenza virus subtype H5 using a biosensor based on high spatial resolution imaging ellipsometry (IE). Monoclonal antibodies specific to H5 hemagglutinin protein were immobilized on silicon wafers and used to capture virus particles. Resultant changes on the surface of the wafers were visualized directly in gray-scale on an imaging ellipsometry image. This preliminary study has shown that the assay is rapid and specific for the identification of avian influenza virus subtype H5. Compared with lateral-flow immunoassays, this biosensor not only has better sensitivity, but can also simultaneously perform multiplexed tests. These results suggest that this biosensor might be a valuable diagnostic tool for avian influenza virus detection.

Structure and cleavage specificity of the chymotrypsin-like serine protease (3CLSP/nsp4) of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV).

Biogenesis and replication of the porcine reproductive and respiratory syndrome virus (PRRSV) include the crucial step of replicative polyprotein processing by self-encoded proteases. Whole genome bioinformatics analysis suggests that nonstructural protein 4 (nsp4) is a 3C-like serine protease (3CLSP), responsible for most of the nonstructural protein processing. The gene encoding this protease was cloned and expressed in Escherichia coli in order to confirm this prediction. The purified protein was crystallized, and the structure was solved at 1.9 A resolution. In addition, the crystal structure of the Ser118Ala mutant was determined at 2.0 A resolution. The monomeric enzyme folds into three domains, similar to that of the homologous protease of equine arteritis virus, which, like PRRSV, is a member of the family Arteriviridae in the order of Nidovirales. The active site of the PRRSV 3CLSP is located between domains I and II and harbors a canonical catalytic triad comprising Ser118, His39, and Asp64. The structure also shows an atypical oxyanion hole and a partially collapsed S1 specificity pocket. The proteolytic activity of the purified protein was assessed in vitro. Three sites joining nonstructural protein domains in the PRRSV replicative polyprotein are confirmed to be processed by the enzyme. Two of them, the nsp3/nsp4 and nsp11/nsp12 junctions, are shown to be cleaved in trans, while cis cleavage is demonstrated for the nsp4/nsp5 linker. Thus, we provide structural evidence as well as enzymatic proof of the nsp4 protein being a functional 3CLSP. We also show that the enzyme has a strong preference for glutamic acid at the P1 position of the substrate.

Emergence of fatal PRRSV variants: unparalleled outbreaks of atypical PRRS in China and molecular dissection of the unique hallmark.

Porcine reproductive and respiratory syndrome (PRRS) is a severe viral disease in pigs, causing great economic losses worldwide each year. The causative agent of the disease, PRRS virus (PRRSV), is a member of the family Arteriviridae. Here we report our investigation of the unparalleled large-scale outbreaks of an originally unknown, but so-called "high fever" disease in China in 2006 with the essence of PRRS, which spread to more than 10 provinces (autonomous cities or regions) and affected over 2,000,000 pigs with about 400,000 fatal cases. Different from the typical PRRS, numerous adult sows were also infected by the "high fever" disease. This atypical PRRS pandemic was initially identified as a hog cholera-like disease manifesting neurological symptoms (e.g., shivering), high fever (40-42 degrees C), erythematous blanching rash, etc. Autopsies combined with immunological analyses clearly showed that multiple organs were infected by highly pathogenic PRRSVs with severe pathological changes observed. Whole-genome analysis of the isolated viruses revealed that these PRRSV isolates are grouped into Type II and are highly homologous to HB-1, a Chinese strain of PRRSV (96.5% nucleotide identity). More importantly, we observed a unique molecular hallmark in these viral isolates, namely a discontinuous deletion of 30 amino acids in nonstructural protein 2 (NSP2). Taken together, this is the first comprehensive report documenting the 2006 epidemic of atypical PRRS outbreak in China and identifying the 30 amino-acid deletion in NSP2, a novel determining factor for virulence which may be implicated in the high pathogenicity of PRRSV, and will stimulate further study by using the infectious cDNA clone technique.