Antibody semorinemab reduces tau pathology in a transgenic mouse model and engages tau in patients with Alzheimer's disease.

Tau has become an attractive alternative target for passive immunotherapy efforts for Alzheimer's disease (AD). The anatomical distribution and extent of tau pathology correlate with disease course and severity better than other disease markers to date. We describe here the generation, preclinical characterization, and phase 1 clinical characterization of semorinemab, a humanized anti-tau monoclonal antibody with an immunoglobulin G4 (igG4) isotype backbone. Semorinemab binds all six human tau isoforms and protects neurons against tau oligomer neurotoxicity in cocultures of neurons and microglia. In addition, when administered intraperitoneally once weekly for 13 weeks, murine versions of semorinemab reduced the accumulation of tau pathology in a transgenic mouse model of tauopathy, independent of antibody effector function status. Semorinemab also showed clear evidence of target engagement in vivo, with increases in systemic tau concentrations observed in tau transgenic mice, nonhuman primates, and humans. Higher concentrations of systemic tau were observed after dosing in AD participants compared to healthy control participants. No concerning safety signals were observed in the phase 1 clinical trial at single doses up to 16,800 mg and multiple doses totaling 33,600 mg in a month.

Extended Characterization and Impact of Visible Fatty Acid Particles - A Case Study With a mAb Product.

In recent years, there has been increased scrutiny on the presence and formation of product-related particles in biopharmaceutical formulations. These types of particles, originating from the degradation of the active pharmaceutical ingredient or the excipients, can be challenging to identify and characterize due to their fragility. Additionally, the mechanisms of their formation as well as the impact of their presence on drug product safety can be complicated to elucidate. In this work, a case study is presented in which multiple batches of one formulated monoclonal antibody (mAb-A) were analyzed at different batch ages to better understand the formation of visible particles resulting from degradation of the surfactant polysorbate 20. The particle identity was determined by Raman spectroscopy as free fatty acid (FFA) and the particle composition over time was monitored by mass spectrometry. Further experimental work includes the counts and morphologies of subvisible particles by flow imaging microscopy. Finally, we evaluated the consequences of saline and human plasma exposure to the visible particles to better understand their fate upon dilution and/or administration which is routinely performed in the clinical setting. The experiments performed in this work can be used to support risk assessments of visible product-related particles.

Characterization of Polysorbate Ester Fractions and Implications in Protein Drug Product Stability.

Polysorbates (PS) are commonly used surfactants in biopharmaceutical protein formulations. However, they are susceptible to a variety of degradation pathways, including chemical hydrolysis, oxidation, and enzymatic hydrolysis. Polysorbates are also heterogeneous mixtures, and it has been observed that the patterns of degradation can be strikingly different between the different pathways. Polysorbates (PS20 and PS80) were fractionated, and the fractions were characterized for their physicochemical properties, such as surface tension, micelle size, critical micelle concentration (CMC), and agitation protection for a monoclonal antibody (mAb). This report seeks to use this information to inform how these properties might change in polysorbates as they degrade in biopharmaceutical formulations. The physicochemical properties examined shed light on some of the differences between PS types and the different chemical components of polysorbates. Differences in physicochemical properties for fractionated polysorbates could help inform biopharmaceutical formulations that use PS surfactants. Importantly, they show that subspecies of PS20 are far more distinct from each other than those of PS80. Fractions of PS20 showed highly different critical micelle concentrations and effects on equilibrium surface tension. These differences, and possibly other untested parameters, led to vastly different protective effects for a model mAb under agitation stress. Additionally, the propensity of various PS fractions to form micelles can impact both polysorbate quantitation measurements, some of which rely on micellization, and the effective solubility of hydrophobic compounds (e.g., fatty acids) in the surfactant solution.

A Rapid High-Sensitivity Reversed-Phase Ultra High Performance Liquid Chromatography Mass Spectrometry Method for Assessing Polysorbate 20 Degradation in Protein Therapeutics.

Polysorbate 20 (PS20), a widely used surfactant in protein therapeutics, has been reported to undergo hydrolytic degradation during product storage, causing the release of free fatty acids. The accumulation of free fatty acids in protein therapeutics was found to result in the formation of particles due to their limited aqueous solubility at 2°C-8°C. Quantitation of free fatty acids originating from PS20 degradation is thus important during bioprocess optimization and stability testing in formulation development to ensure optimum PS20 stability as well as product and process consistency in final drug products. This work reports the development of a simple and robust, high-throughput, reversed-phase ultra high performance liquid chromatography mass spectrometry method for high-sensitivity quantitation of lauric acid and myristic acid by using isotope-labeled fatty acid internal standards. The high sensitivity (<100 ng/mL for lauric acid) and suitable precision (intermediate precision relative standard deviation of 11%) of this method enable accurate detection of lauric acid produced from the degradation of less than 1% of PS20 in a 0.2-mg/mL formulation. Using accelerated thermal stability testing, this method identifies processes that exhibit fast PS20 degradation within only days and consequently allows faster iterative optimization of the process.

Degradation Mechanisms of Polysorbate 20 Differentiated by 18O-labeling and Mass Spectrometry.

PURPOSE: To investigate the mechanisms of polysorbate (PS) degradation with the added objective of differentiating the hydrolysis and oxidation pathways. METHODS: Ultra-performance liquid chromatography mass spectrometry (UPLC-MS) was utilized to characterize all-laurate polysorbate 20 (PS20) and its degradants. 18O stable isotope labeling was implemented to produce 18O-labeled degradation products of all-laurate PS20 in H218O, with subsequent UPLC-MS analysis for location of the cleavage site on the fatty acid-containing side chain of PS20. RESULTS: The analysis reveals that hydrolysis of all-laurate PS20 leads to a breakdown of the ester linkage to liberate free lauric acid, showing a distinct dependence on pH. Using a hydrophilic free radical initiator, 2,2-azobis(2-amidinopropane) dihydrochloride (AAPH) to study the oxidative degradation of all-laurate PS20, we demonstrate that free lauric acid and polyoxyethylene (POE) laurate are two major decomposition products. Measurement of 18O incorporation into free lauric acid indicated that hydrolysis primarily led to 18O incorporation into free lauric acid via "acyl-cleavage" of the fatty acid ester bond. In contrast, AAPH-exposure of all-laurate PS20 produced free lauric acid without 18O-incorporation. CONCLUSIONS: The 18O-labeling technique and unique degradant patterns of all-laurate PS20 described here provide a direct approach to differentiate the types of PS degradation.

Quality by Design Approaches to Formulation Robustness-An Antibody Case Study.

The International Conference on Harmonization Q8 (R2) includes a requirement that "Critical formulation attributes and process parameters are generally identified through an assessment of the extent to which their variation can impact the quality of the drug product," that is, the need to assess the robustness of a formulation. In this article, a quality-by-design-based definition of a "robust formulation" for a biopharmaceutical product is proposed and illustrated with a case study. A multivariate formulation robustness study was performed for a selected formulation of a monoclonal antibody to demonstrate acceptable quality at the target composition as well as at the edges of the allowable composition ranges and fulfillment of the end-of-shelf-life stability requirements of 36 months at the intended storage temperature (2°C-8°C). Extrapolation of 24 months' formulation robustness data to end of shelf life showed that the MAb formulation was robust within the claimed formulation composition ranges. Based on this case study, we propose that a formulation can be claimed as "robust" if all drug substance and drug product critical quality attributes remain within their respective end-of-shelf-life critical quality attribute-acceptance criteria throughout the entire claimed formulation composition range.

Understanding Particle Formation: Solubility of Free Fatty Acids as Polysorbate 20 Degradation Byproducts in Therapeutic Monoclonal Antibody Formulations.

The purpose of this work was to determine the aqueous solubilities at 2-8 °C of the major free fatty acids (FFAs) formed by polysorbate 20 (PS20) degradation and identify possible ways to predict, delay, or mitigate subsequent particle formation in monoclonal antibody (mAb) formulations. The FFA solubility limits at 2-8 °C were determined by titrating known amounts of FFA in monoclonal antibody formulations and identifying the FFA concentration leading to visible and subvisible particle formation. The solubility limits of lauric, myristic, and palmitic acids at 2-8 °C were 17 ± 1 µg/mL, 3 ± 1 µg/mL, and 1.5 ± 0.5 µg/mL in a formulation containing 0.04% (w/v) PS20 at pH 5.4 and >22 µg/mL, 3 ± 1 µg/mL, and 0.75 ± 0.25 µg/mL in a formulation containing 0.02% (w/v) PS20 at pH 6.0. For the first time, a 3D correlation between FFA solubility, PS20 concentration, and pH has been reported providing a rational approach for the formulator to balance these with regard to potential particle formation. The results suggest that the lower solubilities of the longer chain FFAs, generated from degradation of the stearate, palmitate, and myristate fraction of PS20, is the primary cause of seeding and subsequent FFA precipitation rather than the most abundant lauric acid.

Polysorbate 20 Degradation in Biopharmaceutical Formulations: Quantification of Free Fatty Acids, Characterization of Particulates, and Insights into the Degradation Mechanism.

Polysorbate 20 (PS20), a commonly used surfactant in biopharmaceuticals, showed degradation upon long-term (∼18-36 months) storage of two monoclonal antibody (mAb, mAb-A, and mAb-B) drug products at 2-8 °C. The PS20 degradation resulted in the accumulation of free fatty acids (FFA), which ultimately precipitated to form particles upon long-term storage. This study documents the development, qualification, and application of a method for FFA quantification in soluble and insoluble fraction of protein formulation. The method was applied to the quantification of capric acid, lauric acid, myristic acid, palmitic/oleic acid, and stearic acid in placebo as well as active protein formulations on stability. Quantification of FFA in both the soluble and insoluble fraction of mAb-A and mAb-B provided a better mechanistic understanding of PS20 degradation and the dynamics of subsequent fatty acid particle formation. Additionally, the use of this method for monitoring and quantitation of the FFA on real time storage stability appears to aid in identifying batches with higher probability for particulate formation upon extended storage at 5 °C.

Solubility Challenges in High Concentration Monoclonal Antibody Formulations: Relationship with Amino Acid Sequence and Intermolecular Interactions.

The purpose of this work was to elucidate the molecular interactions leading to monoclonal antibody self-association and precipitation and utilize biophysical measurements to predict solubility behavior at high protein concentration. Two monoclonal antibodies (mAb-G and mAb-R) binding to overlapping epitopes were investigated. Precipitation of mAb-G solutions was most prominent at high ionic strength conditions and demonstrated strong dependence on ionic strength, as well as slight dependence on solution pH. At similar conditions no precipitation was observed for mAb-R solutions. Intermolecular interactions (interaction parameter, kD) related well with high concentration solubility behavior of both antibodies. Upon increasing buffer ionic strength, interactions of mAb-R tended to weaken, while those of mAb-G became more attractive. To investigate the role of amino acid sequence on precipitation behavior, mutants were designed by substituting the CDR of mAb-R into the mAb-G framework (GM-1) or deleting two hydrophobic residues in the CDR of mAb-G (GM-2). No precipitation was observed at high ionic strength for either mutant. The molecular interactions of mutants were similar in magnitude to those of mAb-R. The results suggest that presence of hydrophobic groups in the CDR of mAb-G may be responsible for compromising its solubility at high ionic strength conditions since deleting these residues mitigated the solubility issue.

Analytical Ultracentrifugation and Its Role in Development and Research of Therapeutical Proteins.

The historical contributions of analytical ultracentrifugation (AUC) to modern biology and biotechnology drug development and research are discussed. AUC developed by Svedberg was used to show that proteins are actually large defined molecular entities and also provided the first experimental verification for the semiconservative replication model for DNA initially proposed by Watson and Crick. This chapter reviews the use of AUC to investigate molecular weight of recombinant-DNA-produced proteins, complex formation of antibodies, intermolecular interactions in dilute and high concentration protein solution, and their impact on physical properties such as solution viscosity. Recent studies using a "competitive binding" analysis by AUC have been useful in critically evaluating the design and interpretation of surface plasmon resonance measurements and are discussed. The future of this technology is also discussed including prospects for a new higher precision analytical ultracentrifuge.

Comparison of binding characteristics and in vitro activities of three inhibitors of vascular endothelial growth factor A.

The objectives of this study were to evaluate the relative binding and potencies of three inhibitors of vascular endothelial growth factor A (VEGF), used to treat neovascular age-related macular degeneration, and assess their relevance in the context of clinical outcome. Ranibizumab is a 48 kDa antigen binding fragment, which lacks a fragment crystallizable (Fc) region and is rapidly cleared from systemic circulation. Aflibercept, a 110 kDa fusion protein, and bevacizumab, a 150 kDa monoclonal antibody, each contain an Fc region. Binding affinities were determined using Biacore analysis. Competitive binding by sedimentation velocity analytical ultracentrifugation (SV-AUC) was used to support the binding affinities determined by Biacore of ranibizumab and aflibercept to VEGF. A bovine retinal microvascular endothelial cell (BREC) proliferation assay was used to measure potency. Biacore measurements were format dependent, especially for aflibercept, suggesting that biologically relevant, true affinities of recombinant VEGF (rhVEGF) and its inhibitors are yet to be determined. Despite this assay format dependency, ranibizumab appeared to be a very tight VEGF binder in all three formats. The results are also very comparable to those reported previously.1-3 At equivalent molar ratios, ranibizumab was able to displace aflibercept from preformed aflibercept/VEGF complexes in solution as assessed by SV-AUC, whereas aflibercept was not able to significantly displace ranibizumab from preformed ranibizumab/VEGF complexes. Ranibizumab, aflibercept, and bevacizumab showed dose-dependent inhibition of BREC proliferation induced by 6 ng/mL VEGF, with average IC50 values of 0.088 ± 0.032, 0.090 ± 0.009, and 0.500 ± 0.091 nM, respectively. Similar results were obtained with 3 ng/mL VEGF. In summary Biacore studies and SV-AUC solution studies show that aflibercept does not bind with higher affinity than ranibizumab to VEGF as recently reported,4 and both inhibitors appeared to be equipotent with respect to their ability to inhibit VEGF function.

Compatibility and stability of pertuzumab and trastuzumab admixtures in i.v. infusion bags for coadministration.

The physical/chemical stability and potential interactions after diluting two immunoglobulin G1 monoclonal antibodies (mAb), pertuzumab (Perjeta®) and trastuzumab (Herceptin®), in a single intravenous (i.v.) infusion bag containing 0.9% saline (NaCl) solution was evaluated. As commercial products, pertuzumab and trastuzumab are administered through i.v. infusion to patients sequentially, that is, one drug after the other. To increase convenience and minimize the in-clinic time for patients, the compatibility of coadministering pertuzumab (420 and 840 mg) mixed with either 420 or 720 mg trastuzumab, respectively, in a single 250 mL polyolefin or polyvinyl chloride i.v. bag stored for up to 24 h at 5°C or 30°C was determined. The controls (i.e., pertuzumab alone in an i.v. bag, trastuzumab alone in an i.v. bag) and the mAb mixture were assessed using color, appearance, and clarity, concentration and turbidity by ultraviolet spectroscopy, particulate analysis by light obscuration, size-exclusion chromatography, capillary electrophoresis-sodium dodecyl sulfate, analytical ultracentrifugation, and ion-exchange chromatography. Additionally, capillary zone electrophoresis, imaged capillary isoelectric focusing, and potency were utilized to measure the stability of the admixtures containing 1:1 mixtures of pertuzumab/trastuzumab and their respective controls (420 mg pertuzumab alone and 420 mg trastuzumab alone). No observable differences were detected by the above methods in the pertuzumab/trastuzumab mixtures stored up to 24 h at either 5°C or 30°C. The physicochemical methods as listed above were able to detect both molecules as well as the minor variants in the drug mixture, even though some overlap of mAb species were seen in the chromatograms and electropherograms. Furthermore, biophysical analysis also did not show any interactions between the two mAbs or any physical instability under these conditions. Additionally, the drug mixture tested by the pertuzumab-specific inhibition of cell proliferation bioassay showed comparable potency before and after storage. On the basis of these results, pertuzumab and trastuzumab admixture in a single i.v. bag is physically and chemically stable for up to 24 h at 5°C or 30°C and can be used for clinical administration.

Assessment and significance of protein-protein interactions during development of protein biopharmaceuticals.

Early development of protein biotherapeutics using recombinant DNA technology involved progress in the areas of cloning, screening, expression and recovery/purification. As the biotechnology industry matured, resulting in marketed products, a greater emphasis was placed on development of formulations and delivery systems requiring a better understanding of the chemical and physical properties of newly developed protein drugs. Biophysical techniques such as analytical ultracentrifugation, dynamic and static light scattering, and circular dichroism were used to study protein-protein interactions during various stages of development of protein therapeutics. These studies included investigation of protein self-association in many of the early development projects including analysis of highly glycosylated proteins expressed in mammalian CHO cell cultures. Assessment of protein-protein interactions during development of an IgG1 monoclonal antibody that binds to IgE were important in understanding the pharmacokinetics and dosing for this important biotherapeutic used to treat severe allergic IgE-mediated asthma. These studies were extended to the investigation of monoclonal antibody-antigen interactions in human serum using the fluorescent detection system of the analytical ultracentrifuge. Analysis by sedimentation velocity analytical ultracentrifugation was also used to investigate competitive binding to monoclonal antibody targets. Recent development of high concentration protein formulations for subcutaneous administration of therapeutics posed challenges, which resulted in the use of dynamic and static light scattering, and preparative analytical ultracentrifugation to understand the self-association and rheological properties of concentrated monoclonal antibody solutions.

Weak interactions govern the viscosity of concentrated antibody solutions: high-throughput analysis using the diffusion interaction parameter.

Weak protein-protein interactions are thought to modulate the viscoelastic properties of concentrated antibody solutions. Predicting the viscoelastic behavior of concentrated antibodies from their dilute solution behavior is of significant interest and remains a challenge. Here, we show that the diffusion interaction parameter (k(D)), a component of the osmotic second virial coefficient (B(2)) that is amenable to high-throughput measurement in dilute solutions, correlates well with the viscosity of concentrated monoclonal antibody (mAb) solutions. We measured the k(D) of 29 different mAbs (IgG(1) and IgG(4)) in four different solvent conditions (low and high ion normality) and found a linear dependence between k(D) and the exponential coefficient that describes the viscosity concentration profiles (|R| ≥ 0.9). Through experimentally measured effective charge measurements, under low ion normality where the electroviscous effect can dominate, we show that the mAb solution viscosity is poorly correlated with the mAb net charge (|R| ≤ 0.6). With this large data set, our results provide compelling evidence in support of weak intermolecular interactions, in contrast to the notion that the electroviscous effect is important in governing the viscoelastic behavior of concentrated mAb solutions. Our approach is particularly applicable as a screening tool for selecting mAbs with desirable viscosity properties early during lead candidate selection.

Characterization of particles in protein solutions: reaching the limits of current technologies.

Recent publications have emphasized the lack of characterization methods available for protein particles in a size range comprised between 0.1 and 10 µm and the potential risk of immunogenicity associated with such particles. In the present paper, we have investigated the performance of light obscuration, flow microscopy, and Coulter counter instruments for particle counting and sizing in protein formulations. We focused on particles 2-10 µm in diameter and studied the effect of silicon oil droplets originating from the barrel of pre-filled syringes, as well as the effect of high protein concentrations (up to 150 mg/ml) on the accuracy of particle characterization. Silicon oil was demonstrated to contribute significantly to the particle counts observed in pre-filled syringes. Inconsistent results were observed between different protein concentrations in the range 7.5-150 mg/ml for particles <10 µm studied by optical techniques (light obscuration and flow microscopy). However, the Coulter counter measurements were consistent across the same studied concentration range but required sufficient solution conductivity from the formulation buffer or excipients. Our results show that currently available technologies, while allowing comparisons between samples of a given protein at a fixed concentration, may be unable to measure particle numbers accurately in a variety of protein formulations, e.g., at high concentration in sugar-based formulations.

A therapeutic antibody and its antigen form different complexes in serum than in phosphate-buffered saline: a study by analytical ultracentrifugation.

During the development of protein therapeutics, characterization of the active pharmaceutical ingredient is performed extensively to ensure the stability, safety, and efficacy of the drug. Little is known, however, about the characteristics of protein drugs circulating in the blood. The recent availability of a fluorescence detection system (FDS) in analytical ultracentrifugation (AUC) instruments enables the characterization of fluorescently labeled proteins in biological fluids. AUC provides information about protein size, shape, self-association, and binding while avoiding many limitations associated with size exclusion chromatography. Furthermore, with the specificity and sensitivity of FDS, measurements can be performed at physiological concentrations directly in serum. In the current study, we used omalizumab, an anti-immunoglobulin E (IgE) monoclonal antibody, to demonstrate the potential of using AUC-FDS for the study of a monoclonal antibody and its complexes directly in human serum. Omalizumab properties were essentially unaltered after labeling with the fluorescent dye Alexa Fluor 488. In addition, omalizumab and IgE formed different complexes in serum than in phosphate-buffered saline in terms of both size and affinity.

New methods allowing the detection of protein aggregates: a case study on trastuzumab.

Aggregation compromises the safety and efficacy of therapeutic proteins. According to the manufacturer, the therapeutic immunoglobulin trastuzumab (Herceptin) should be diluted in 0.9% sodium chloride before administration. Dilution in 5% dextrose solutions is prohibited. The reason for the interdiction is not mentioned in the Food and Drug Administration (FDA) documentation, but the European Medicines Agency (EMEA) Summary of Product Characteristics states that dilution of trastuzumab in dextrose solutions results in protein aggregation. In this paper, asymmetrical flow field-flow fractionation (FFF), fluorescence spectroscopy, fluorescence microscopy and transmission electron microscopy (TEM) have been used to characterize trastuzumab samples diluted in 0.9% sodium chloride, a stable infusion solution, as well as in 5% dextrose (a solution prone to aggregation). When trastuzumab samples were injected in the FFF channel using a standard separation method, no difference could be seen between trastuzumab diluted in sodium chloride and trastuzumab diluted in dextrose. However, during FFF measurements made with appropriate protocols, aggregates were detected in 5% dextrose. The parameters enabling the detection of reversible trastuzumab aggregates are described. Aggregates could also be documented by fluorescence microscopy and TEM. Fluorescence spectroscopy data were indicative of conformational changes consistent with increased aggregation and adsorption to surfaces. The analytical methods presented in this study were able to detect and characterize trastuzumab aggregates.

Characterization of protein aggregation: the case of a therapeutic immunoglobulin.

In this paper, a therapeutic immunoglobulin (Antibody A) has been characterized in two solutions: (1) 0.1% acetic acid containing 50 mM magnesium chloride, a solution in which the immunoglobulin is stable, and (2) 10 mM sodium phosphate buffer pH approximately 7. The protein solutions were characterized by microscopy, asymmetrical flow field-flow fractionation (FFF), light scattering, circular dichroism, fluorescence and fluorescence lifetime spectroscopy. The results show that Antibody A dissolved in 0.1% acetic acid containing 50 mM magnesium chloride exists as 88% monomer, 2% low molecular weight aggregates and 10% high molecular weight aggregates (>1 million Dalton). In phosphate buffer, Antibody A formed micrometre-sized aggregates that were best characterized by fluorescence microscopy. The aggregation of Antibody A in phosphate buffer was shown to be concomitant with conformational changes in amino acid residue side chains. The aggregates formed in phosphate buffer were easily disrupted during FFF analysis, indicating that they are formed by weak interactions. The combination of microscopy, asymmetrical flow field-flow fractionation (FFF) and spectroscopy allowed a reliable assessment of protein self association and aggregation.

Detection and characterization of protein aggregates by fluorescence microscopy.

Aggregation may compromise the stability as well as the biological activity of protein drugs. Detection of protein aggregates is needed in the process of protein characterization and during optimization of pharmaceutical formulations. This paper describes a technique, which consists of analysing protein aggregates by fluorescence microscopy after staining with the hydrophobic probe Nile Red. Dilution, filtration or other modifications of the sample are not needed. Assessment of aggregation was possible in highly concentrated protein samples (193 mg/ml). Fluorescence microscopy observations allowed the detection and characterization of protein aggregates not easily detected by spectroscopic techniques. Nile Red was shown to be very sensitive for the detection and analysis of immunoglobulin aggregates. Nile Red, Congo red and Thioflavine T stainings were compared. Nile Red and Thioflavine T fluorescence were colocalized. The diameter of immunoglobulin aggregates was determined, and the number of aggregates was correlated with 90 degrees light scattering measurements. Studies of human calcitonin aggregates brought to light new aspects of the human calcitonin aggregation mechanisms. Thus, Nile Red staining not only allows detection of very low levels of protein aggregates, but also contributes to a better understanding of the complex mechanisms governing protein aggregation.

Where disease pathogenesis meets protein formulation: renal deposition of immunoglobulin aggregates.

Aggregation is one of the important issues encountered during the development of immunoglobulin-based drugs. The aim of the current review is to discuss the causes and consequences of immunoglobulin aggregation as well as the relevance of immunoglobulin aggregation to disease pathogenesis. Extracellular deposition of immunoglobulins, either monoclonal light chains or intact polyclonal antibodies, induces renal failure in various nephropathies. The aggregates can present fibrillar or amorphous structures. In this review, factors known to influence protein aggregation, such as the primary structure of the protein, local environment and glycosylation are assessed, as well as the subsequent altered clearance, fibril formation and toxicity. The role of the protein local environment is emphasized. Even if the local environment causes only minor perturbations in the protein structure, these perturbations might be sufficient to trigger aggregate formation. This fact underlines the importance of choosing appropriate formulations for protein drugs. If the formulation provides a slightly destabilizing environment to the protein, the long-term stability of the drug may be compromised by aggregate formation.