IgG Aggregates Research Articles

Immunoglobulin G and Complement as Major Players in the Neurodegeneration of Multiple Sclerosis.

Multiple Sclerosis (MS) is an inflammatory, demyelinating, and neurodegenerative disease of the central nervous system (CNS) and is termed as one of the most common causes of neurological disability in young adults. Axonal loss and neuronal cell damage are the primary causes of disease progression and disability. Yet, little is known about the mechanism of neurodegeneration in the disease, a limitation that impairs the development of more effective treatments for progressive MS. MS is characterized by the presence of oligoclonal bands and raised levels of immunoglobulins in the CNS. The role of complement in the demyelinating process has been detected in both experimental animal models of MS and within the CNS of affected MS patients. Furthermore, both IgG antibodies and complement activation can be detected in the demyelinating plaques and cortical gray matter lesions. We propose here that both immunoglobulins and complement play an active role in the neurodegenerative process of MS. We hypothesize that the increased CNS IgG antibodies form IgG aggregates and bind complement C1q with high affinity, activating the classical complement pathway. This results in neuronal cell damage, which leads to neurodegeneration and demyelination in MS.

Predicting Secondary Progressive Multiple Sclerosis Using Blood IgG Aggregates (S42.005)

Predicting Secondary Progressive Multiple Sclerosis Using Blood IgG Aggregates (S42.005)

Plasma IgG aggregates as biomarkers for multiple sclerosis

We recently reported that multiple sclerosis (MS) plasma contains IgG aggregates and induces complement-dependent neuronal cytotoxicity (Zhou et al., 2023). Using ELISA, we report herein that plasma IgG levels in the aggregates can be used as biomarkers for MS. We enriched the IgG aggregates from samples of two cohorts (190 MS and 160 controls) by collecting flow-through after plasma binding to Protein A followed by detection of IgG subclass. We show that there are significantly higher levels of IgG1, IgG3, and total IgG antibodies in MS IgG aggregates, with an AUC >90%; higher levels of IgG1 distinguish secondary progressive MS from relapsing-remitting MS (AUC = 91%). Significantly, we provided the biological rationale for MS plasma IgG biomarkers by demonstrating the strong correlation between IgG antibodies and IgG aggregate-induced neuronal cytotoxicity. These non-invasive, simple IgG-based blood ELISA assays can be adapted into clinical practice for diagnosing MS and SPMS and monitoring treatment responses.

A distinct variant of the SzM protein of Streptococcus equi subsp. zooepidemicus recruits C1q independent of IgG binding and inhibits activation of the classical complement pathway

ABSTRACT Streptococcus equi subsp. zooepidemicus (SEZ) is a major equine pathogen that causes pneumonia, abortion, and polyarthritis. It can also cause invasive infections in humans. SEZ expresses the M-like protein SzM, which recruits host proteins such as fibrinogen to the bacterial surface. Equine SEZ strain C2, which binds only comparably low amounts of human fibrinogen in comparison to human SEZ strain C33, was previously shown to proliferate in equine and human blood. As the expression of SzM_C2 was necessary for survival in blood, this study investigated the working hypothesis that SzM_C2 inhibits complement activation through a mechanism other than fibrinogen and non-immune immunoglobulin binding. Loss-of-function experiments showed that SEZ C2, but not C33, binds C1q via SzM in IgG-free human plasma. Furthermore, SzM C2 expression is necessary for recruiting purified human or equine C1q to the bacterial surface. Flow cytometry analysis demonstrated that SzM expression in SEZ C2 is crucial for the significant reduction of C3b labelling in human plasma. Addition of human plasma to immobilized rSzM_C2 and immobilized aggregated IgG led to binding of C1q, but only the latter activated the complement system, as shown by the detection of C4 deposition. Complement activation induced by aggregated IgG was significantly reduced if human plasma was pre-incubated with rSzM_C2. Furthermore, rSzM_C2, but not rSzM_C33, inhibited the activation of the classical complement pathway in human plasma, as determined in an erythrocyte lysis experiment. In conclusion, the immunoglobulin-independent binding of C1q to SzM_C2 is associated with complement inhibition.

Dynamic light scattering analysis of immune complexes in sera of rheumatoid arthritis patients

The size of circulating immune complexes (CICs) in rheumatoid arthritis (RA) could be an emerging criterion in disease diagnosis. This study analyzed size and electrokinetic potential of CICs from RA patients, healthy young adults, and RA patients age-matched controls aiming to establish their unique CIC features. Pooled CIC of 30 RA patients, 30 young adults, and 30 RA group's age-matched controls (middle-aged and oldеr healthy adults), and in vitro IgG aggregates from pooled sera of 300 healthy volunteers were tested using dynamic light scattering (DLS). Size distribution of CIC in healthy young adults exhibited high polydispersity. RA CIC patients and their age-matched control showed distinctly narrower size distributions compared with young adults. In these groups, particles clustered around two well-defined peaks. Particles of peak 1 were 36.1 ± 6.8 nm in RA age-matched control, and 30.8 ± 4.2 nm in RA patients. Particles of peak 2 of the RA age-matched control's CIC was 251.7 ± 41.2 nm, while RA CIC contained larger particles (359.9 ± 50.5 nm). The lower zeta potential of RA CIC, compared to control, indicated a disease-related decrease in colloidal stability. DLS identified RA-specific, but also age-specific distribution of CIC size and opened possibility of becoming a method for CIC size analysis in IC-mediated diseases.

A comprehensive review on preparation of pure immunoglobulins Authors

Immunoglobulins are also known as antibodies. Plasma cells are responsible for the production of immunoglobulins. Beta cells are activated against a pathogenic attack and facilitated the formation of different types of immunoglobulins naturally. These immunoglobulins are also artificially synthesized by non-specific laboratorial techniques include fractionation precipitation, electrophoretic methods, gel filtration chromatography, ion exchange chromatography, hydrophobic chromatography and by the specific immuno- adsorbent methods. Third generation immunoglobulins are effectively used for therapeutic purpose against viral infections intravenously. Second generation immunoglobulins synthesis involved removal of anti-complement contaminants and IgG aggregates, through enzymatic degradation and chemical modification. The harmful effects of intravenous immunoglobulins can be reduced by using ultraviolet light, P-propiolactone, and pepsin at pH 4. Intravenous immunoglobulin drugs lead to anti-inflammatory and immunomodulatory effects in various infections. The mechanism of action of intravenous immunoglobulins is dependent on the binding between the Fc portion of injected IgG and target cell receptors. Radiolabeling is performed by two methods as in vivo, in which radiolabeled antibodies are incorporated into the body to bind with the antibodies and in vitro method, radioactive material is bound with already formed antibodies. This chapter highlighted the artificial methods adopted for production of radiolabeled immunoglobulins holding significant therapeutic and diagnostic applications.

Avidity-Based Detection and Quantification of Igg Aggregates Using a Nanoplasmonic Fiber Optical Biosensor System

Avidity-Based Detection and Quantification of Igg Aggregates Using a Nanoplasmonic Fiber Optical Biosensor System

Sample displacement chromatography of monoclonal antibody charge variants and aggregates.

The rise of biosimilar monoclonal antibodies has renewed the interest in monoclonal antibody (mAb) charge variants composition and separation. The sample displacement chromatography (SDC) has the potential to overcome the low separation efficiency and productivity associated with bind-elute separation of mAb charge variants. SDC in combination with weak cation exchanging macroporous monolithic chromatographic column was successfully implemented for a separation of charge variants and aggregates of monoclonal IgG under overloading conditions. The charge variants composition was at-line monitored by a newly developed, simple and fast analytical method, based on weak cation exchange chromatography. It was proven that basic charge variants acted as displacers of IgG molecules with lower pI, when the loading was performed 1 to 1.5pH unit below the pI of acidic charge variants. The efficiency of the SDC process is flow rate independent due to a convection-based mass transfer on the macroporous monolith. The productivity of the process at optimal conditions is 35mg of purified IgG fraction per milliliters of monolithic support with 75-80% recovery. As such, an SDC approach surpasses the standard bind-elute separation in the productivity for a factor of 3, when performed on the same column. The applicability of the SDC approach was confirmed for porous particle-based column as well, but with 1.5 lower productivity compared to the monoliths.

Image Analysis Algorithm-Based Platform for Determining Micron and Higher Aggregate Size Distribution of Therapeutic IgG Using Brightfield and Fluorescence Microscope Images.

A platform for determining size distribution of micron (1-100μm) and larger (> 100μm) aggregates of therapeutic IgG has been established by using image processing algorithms for brightfield and fluorescence microscope images. The algorithm for brightfield images involved conversion to grayscale followed by pixel-based and size-based thresholding. Morphological operations were then applied and the size distribution of aggregates were extracted. Fluorescence images of the aggregates of mAb tagged by a fluorescent dye were captured using widefield fluorescence microscope, confocal laser scanning microscope, and Cytell Cell Imaging System and the images were processed using a series of denoising steps followed by thresholding and morphological operations. The samples were subjected to different stresses, among which the aggregates were visible in the microscope for sample subjected to bubbling, stirring, and temperature. The images of these aggregates were effectively denoised and the size distribution of aggregates was analyzed using the algorithm. The overall aggregate size distribution obtained by image processing ranged in the micron and higher size range. The size obtained from brightfield image processing was validated using images of liquid chromatography resins. Further, the aggregate size distribution obtained using image processing was compared with experimental techniques such as Mastersizer 2000 and Micro Flow Imaging. It was found that analysis of IgG aggregates using image processing could serve as an orthogonal methodology to the existing approaches.

Process development exploiting competitive adsorption-based displacement effects in monoclonal antibody aggregate removal-A new high-throughput screening procedure for membrane chromatography.

High-throughput screening (HTS) approaches are commonly used to accelerate downstream process development. Although most HTS approaches use batch isothermal data (KP screen) or bind and elute mode as screening procedure, different or new process designs are rarely investigated. In this paper, a mechanistic model case study for the separation of two different two-component solutions was conducted and confirmed prior evidence. With these outcomes, a novel HTS screening procedure was developed including the determination of competitive adsorption-based displacement effects and key parameter identification. The screening procedure employing an overload bind and elute (OBE) mode is presented in a case study dealing with IgG aggregate removal in a typical monoclonal antibody purification step, applying a Sartobind®S membrane adsorber (MA). Based on a MA scale down device, the OBE mode allows the determination of classical process parameters and dynamic effects, such as displacement effects. Competitive adsorption-based displacement effects are visualized by introducing a displacement identifier leading to a displacement process map. Based on this map, the approach is transferred to and confirmed by the OBE recycle experiments with 4.6 and 8.2ml benchtop scsale devices resulting in 45% reduced IgG monomer and 88% increased higher molecular weight species binding capacities.

Novel semi-automated fluorescence microscope imaging algorithm for monitoring IgG aggregates in serum

Analysis of therapeutic IgG aggregates in serum is a potential area of investigation as it can give deeper insights about the function, immunogenic issues and protein interaction associated with the aggregates. To overcome various complexities associated with the existing analytical techniques for analyzing aggregates in serum, a novel florescence microscopy-based image processing approach was developed. The monoclonal antibody (mAb) was tagged with a fluorescent dye, fluorescein isothiocyanate (FITC). Aggregates, generated by stirring, were spiked into serum and images were captured at various time points. After denoising, thresholding by weighted median, 1D Otsu, and 2D Otsu was attempted and a modified 2D Otsu, a new mode of thresholding, was developed. This thresholding method was found to be highly effective in removing noises and retaining analyte sizes. Out of 0–255, the optimized threshold value obtained for the images discussed in modified 2D Otsu was 9 while 2D Otsu’s overestimated values were 38 and 48. Other morphological operations were applied after thresholding and the area, perimeter, circularity, and radii of the aggregates in these images were calculated. The proposed algorithm offers an approach for analysis of aggregates in serum that is simpler to implement and is complementary to existing approaches.

Single-Molecule Ex Situ Atomic Force Microscopy Allows Detection of Individual Antibody-Antigen Interactions on a Semiconductor Chip Surface.

Although in situ atomic force microscopy (AFM) allows single‐molecule detection of antibody–antigen binding, the practical applications of in situ AFM for disease diagnosis are greatly limited, due to its operational complexity and long operational times, including the execution time for the surface chemical/biological treatments in the equipped glass liquid cell. Herein, a method of graphically superimposed alignment that enables ex situ AFM analysis of an immobilized antibody at the same location on a semiconductor chip surface before and after incubation with its antigen is presented. All of the required chemical/biological treatments are executed feasibly using standard laboratory containers, allowing single‐molecule ex situ AFM detection to be conducted with great practicality, flexibility, and versatility. As an example, the analysis of hepatitis B virus X protein (HBx) and its IgG antibody is described. Using ex situ AFM, individual information on the topographical characteristics of the immobilized single and aggregated IgG antibodies on the chip surface is extracted and the data are analyzed statistically. Furthermore, in a statistical manner, the changes in AFM‐measured heights of the individual and aggregated IgG antibodies that occur as a result of changes in conformation upon formation of IgG–HBx complexes are investigated.

Systematic use of synthetic 5'-UTR RNA structures to tune protein translation improves yield and quality of complex proteins in mammalian cell factories.

Predictably regulating protein expression levels to improve recombinant protein production has become an important tool, but is still rarely applied to engineer mammalian cells. We therefore sought to set-up an easy-to-implement toolbox to facilitate fast and reliable regulation of protein expression in mammalian cells by introducing defined RNA hairpins, termed ‘regulation elements (RgE)’, in the 5′-untranslated region (UTR) to impact translation efficiency. RgEs varying in thermodynamic stability, GC-content and position were added to the 5′-UTR of a fluorescent reporter gene. Predictable translation dosage over two orders of magnitude in mammalian cell lines of hamster and human origin was confirmed by flow cytometry. Tuning heavy chain expression of an IgG with the RgEs to various levels eventually resulted in up to 3.5-fold increased titers and fewer IgG aggregates and fragments in CHO cells. Co-expression of a therapeutic Arylsulfatase-A with RgE-tuned levels of the required helper factor SUMF1 demonstrated that the maximum specific sulfatase activity was already attained at lower SUMF1 expression levels, while specific production rates steadily decreased with increasing helper expression. In summary, we show that defined 5′-UTR RNA-structures represent a valid tool to systematically tune protein expression levels in mammalian cells and eventually help to optimize recombinant protein expression.

IgG-aggregates rapidly upregulate FcgRI expression at the surface of human neutrophils in a FcgRII-dependent fashion: A crucial role for FcgRI in the generation of reactive oxygen species.

Autoimmune complexes are an important feature of several autoimmune diseases such as lupus, as they contribute to tissue damage through the activation of immune cells. Neutrophils, key players in lupus, interact with immune complexes through Fc gamma receptors (FcgR). Incubation of neutrophils with aggregated-IgGs caused degranulation and increased the surface expression of FcgRI within minutes in a concentration-dependent fashion. After 30minutes, IgG aggregates (1mg/mL) upregulated FcgRI by 4.95±0.45-fold. FcgRI-positive neutrophils reached 67.24%±6.88% on HA-IgGs stimulated neutrophils, from 3.12%±1.62% in non-stimulated cells, ranking IgG-aggregates among the most potent known agonists. FcgRIIa, and possibly FcgRIIIa, appeared to mediate this upregulation. Also, FcgRI-dependent signaling proved necessary for reactive oxygen species (ROS) production in response to IgG-aggregates. Finally, combinations of bacterial materials with aggregates dramatically boosted ROS production. This work suggests FcgRI as an essential component in the response of human neutrophils to immune complexes leading to the production of ROS, which may help explain how neutrophils contribute to tissue damage associated with immune complex-associated diseases, such as lupus.

Quantitative Differentiation of Protein Aggregates From Other Subvisible Particles in Viscous Mixtures Through Holographic Characterization

We demonstrate the use of holographic video microscopy to detect individual subvisible particles dispersed in biopharmaceutical formulations and to differentiate them based on material characteristics measured from their holograms. The result of holographic analysis is a precise and accurate measurement of the concentrations and size distributions of multiple classes of subvisible contaminants dispersed in the same product simultaneously. We demonstrate this analytical technique through measurements on model systems consisting of human IgG aggregates in the presence of common contaminants such as silicone oil emulsion droplets and fatty acids. Holographic video microscopy also clearly identifies metal particles and air bubbles. Being able to differentiate and characterize the individual components of such heterogeneous dispersions provides a basis for tracking other factors that influence the stability of protein formulations including handling and degradation of surfactant and other excipients.

Impact of IgG Fc-fragments on experimental glomerulonephritis induced by <i>Streptococcus pyogenes</i> strain binding various immunoglobulin classes

The pathogenesis of poststreptococcal glomerulonephritis (PSGN), a major complication of acute infections caused by group A streptococci (GAS) remains unclear. Several theories, based on the role of certain streptococcal virulence factors, as well as immunological mimicry between GAS and renal tissue, have been proposed. Earlier, we reported that many virulent clinical GAS isolates showing confirmed nephritogenic activity were capable of nonimmune Fc binding of monomeric or aggregated IgG. Moreover, a rabbit model of PSGN allowed to obtain findings regarding a crucial role of streptococcal IgG Fc binding proteins belonging to the M family surface proteins, in the onset of PSGN. Rabbits injected with inactivated IgGFcBP-positive streptococci, acquired changes in the renal tissue with deposited IgG and complement C3, as well as signs of immune inflammation characteristic for human PSGN. Also, it was shown that the induction of experimental glomerulonephritis could be inhibited after normal IgG or its purified Fc fragments were inoculated at early stages of the process. The data obtained in rabbits injected with group A streptococcal type M60 also showed pathogenic functions of the IgA Fc-binding proteins of GAS. The aim of the study was to examine inhibiting activity of the purified rabbit IgG Fc fragments on the manifestations of glomerulonephritis induced by S. pyogenes strains capable of binding diverse forms of immunoglobulins such native IgG, immune complexes, and IgA.Materials and methods. GAS strains of emml, emml2 and emm60 genotypes were used to induce PSGN or IgA-nephropa-thy in rabbits. Fc fragments derived from rabbit IgG were obtained by enzymatic digestion and purified by affinity chromatography on a protein G-sepharose FF column. Immunomorphological changes of renal tissue were estimated by morphometric analysis.Results. In the present study, using the rabbit model, we revealed pathological changes of different intensity and localization in the renal tissue. For streptococci of the emm1 and emm12 genotypes, PSGN was characterized by deposition of IgG or IgG-anti-IgG immune complexes within the basal glomerular membrane. Morphological changes were evaluated as a membranous-proliferative glomerulonephritis. Meanwhile, IgA-glomerulonephritis is characterized by deposition of IgA in mesangial cells of glomeruli, leading to the mesangial-proliferative glomerulonephritis or IgA-nephropathy. Intravenously administered purified Fc fragments derived from normal rabbit IgG varied in effects on pathological processes: (i) IgG Fc fragments of fully inhibited development of the PSGN induced by IgG Fc binding strain of emml genotype, (ii) IgG Fc fragments of partially reverted changes caused by the emm12 genotype strain, which was binding only to immune complexes, and (iii) had no effects on pathological changes caused by the emm60 genotype GAS strain, which was binding only IgA.Conclusion. The data obtained point and emergence of differences in mechanisms of renal lesions development at glomerulonephritis, depending on the emm genotype of GAS strain. In addition, it also confirmed GAS-derived involvement for various IgFc-receptor proteins in the pathology. Further studies on potential prophylactic or curative effects of IgG Fc fragments in PSGN should therefore be of interest. The findings might suggest a new therapeutic approach for non-suppurative poststreptococcal diseases.

Insight into Heterogeneous Distribution of Protein Aggregates at the Surface Layer Using Attenuated Total Reflection-Fourier Transform Infrared Spectroscopic Imaging.

Monoclonal antibodies (mAbs) have been used as therapeutics for the last few decades. It is necessary to investigate the stability of these mAbs under stress conditions and to elucidate aggregation mechanisms as a means of developing approaches which minimize the problem. Attenuated total reflection (ATR)-FTIR spectroscopic imaging allows probing of a sample at a depth of penetration of around 0.5-5 μm, which makes it suitable for the study of aggregated proteins when accumulated as a layer close to the surface of the ZnSe internal reflection element (IRE). Here, macro ATR-FTIR spectroscopic imaging, along with a variable angle of incidence accessory, have been used to differentiate between the secondary structure of proteins in bulk solution and those that have precipitated onto or near the ZnSe IRE surface. IgG spectra obtained from protein samples in individual wells have been averaged, extracted, and preprocessed, and the Amide I bands of the protein samples were compared and further analyzed to reveal protein distribution at the ZnSe IRE surface. These findings show depth profiling of IgG aggregates at the ZnSe IRE surface (0.5-5 μm) and do not follow a trend of decreasing protein presence with an increasing angle of incidence or increasing depth of penetration, suggesting an irregular distribution of aggregates in the z-direction.

Endocytosis of IgG, Desmoglein 1, and Plakoglobin in Pemphigus Foliaceus Patient Skin.

Pemphigus foliaceus (PF) is one of the two main forms of pemphigus and is characterized by circulating IgG to the desmosomal cadherin desmoglein 1 (DSG1) and by subcorneal blistering of the skin. The pathomechanism of blister formation in PF is unknown. Previously we have shown that PF IgG induces aggregation of DSG1, plakoglobin (PG), and IgG outside of desmosomes, what in immunofluorescence of PF patient skin visualizes as a granular IgG deposition pattern with a limited number of coarse IgG aggregates between cells. Here we have investigated the fate of these aggregates in skin and found that these are cleared by endocytosis. We performed double immunofluorescence staining on snap-frozen skin biopsies of six PF patients for the following molecules: IgG, the desmosomal proteins DSG1 and DSG3, desmocollins 1 and 3, PG, desmoplakin and plakophilin 3, and for the endosomal marker early endosomal antigen 1 and the lysosomal markers cathepsin D and lysosomal-associated membrane protein 1. Endosomes were present in all cells but did not make contact with the aggregates in the basal and suprabasal layers. In the higher layers they moored to the aggregates, often symmetrically from two adjacent cells, and IgG, DSG1, and PG were taken up. Finally these endosomes became localized perinuclear. Endocytosis was only observed in perilesional or lesional skin but not in non-lesional skin. Older immunoelectron microscopic studies have suggested that in PF skin endocytosis of detached desmosomes takes place but we found no other desmosomal proteins to be present in these endosomes. Double staining with cathepsin D and LAMP-1 revealed no overlap with IgG, DSG1, or PG suggesting that lysosomes have no role in the clearing process. Collectively, our results show that endocytosis is part of the pathogenic process in PF but that no detached desmosomes are taken up but instead the deposited IgG is taken up together with DSG1 and PG.

Suppression of Aggregation of Therapeutic Monoclonal Antibodies during Storage by Removal of Aggregation Precursors Using a Specific Adsorbent of Non-Native IgG Conformers.

The quality of preparations of therapeutic IgG molecules, widely used for the treatment of various diseases, should be maintained during storage and administration. Nevertheless, recent studies demonstrate that IgG aggregation is one of the most critical immunogenicity risk factors that compromises safety and efficacy of therapeutic IgG molecules in the clinical setting. During the IgG manufacturing process, 0.22-μm membrane filters are commonly used to remove aggregates. However, particles with a diameter below 0.22 μm (small aggregates) are not removed from the final product. The residual species may grow into large aggregates during the storage period. In the current study, we devised a strategy to suppress IgG aggregate growth by removing aggregation precursors using the artificial protein AF.2A1. This protein efficiently binds the Fc region of non-native IgG conformers generated under chemical and physical stresses. Magnetic beads conjugated with AF.2A1 were used to remove non-native monomers and aggregates from solutions of native IgG and from native IgG solutions spiked with stressed IgG. The time-dependent growth of aggregates after the removal treatment was monitored. The removal of aggregation precursors, i.e., non-native monomers and nanometer aggregates (<100 nm), suppressed the aggregate growth. The presented findings demonstrate that a removal treatment with a specific adsorbent of non-native IgG conformers enables long-term stable storage of therapeutic IgG molecules and will facilitate mitigation of the immunogenicity of IgG preparations.

Power Law Approach as a Convenient Protocol for Improving Peak Shapes and Recovering Areas from Partially Resolved Peaks

Separation techniques have developed rapidly where sub-second chromatography, ultrahigh resolution recycling chromatography, and two-dimensional liquid chromatography have become potent tools for analytical chemists. Despite the popularity of high-efficiency materials and new selectivity columns, peak overlap is still observed because as the number of analytes increases, Poisson statistics predicts a higher probability of peak overlap. This work shows the application of the properties of exponential functions and Gaussian functions for virtual resolution enhancement. A mathematical protocol is derived to recover areas from overlapping signals and overcomes the previously known limitations of power laws of losing area and height information. This method also reduces noise and makes the peaks more symmetrical while maintaining the retention time and selectivity. Furthermore, it does not require a prior knowledge of the total number of components as needed in curve fitting techniques. Complex examples are shown using chiral chromatography for enantiomers, and twin-column recycling HPLC of IgG aggregates and with tailing or fronting peaks. The strengths and weaknesses of the power law protocol for area recovery are discussed with simulated and real examples.