Characterization of ADCs by Capillary Electrophoresis.

Capillary electrophoresis (CE) is a highly efficient separation technique that resolves ions based on their electrophoretic mobility in the presence of an applied voltage. It has been broadly applied for characterizing biotherapeutics including ADCs. In this chapter, step-by-step procedures for characterizing ADCs using CE will be described with focus placed on reduced and non-reduced capillary electrophoresis sodium dodecyl sulfate (CE-SDS) for purity determination and imaged capillary isoelectric focusing (iCIEF) for charge heterogeneity analysis.

Comparison of Sebia Free Light Chain Assay With Freelite Assay for the Clinical Management of Diagnosis, Response, and Relapse Assessment in Multiple Myeloma.

BACKGROUND: Serum free light chain (FLC) measurement has become an important marker for the management of multiple myeloma (MM). However, several analytical challenges remain unresolved. We compared the clinical performances of the Sebia FLC assay in MM to the Freelite assay. PATIENTS AND METHODS: A total of 177 patients from the IFM DFCI 2009 trial were enrolled onto this study, with a total of 368 samples analyzed. At baseline, concordance of the involved to noninvolved FLC ratio (iFLC/niFLC) was evaluated. During therapy, comparison of the disease response assessments according to International Myeloma Working Group criteria was performed. RESULTS: Compared to Freelite, the Sebia FLC assay demonstrated lower results, with a proportional bias with increased values. We demonstrated that the Sebia equivalent of the iFLC/niFLC ratio of 100 was 16. During follow-up, agreement in response assessment was moderate (for light chains MM) to good (for intact immunoglobulin MM). In the context of relapse, the concordance was moderate, but longitudinal follow-up showed a similar kinetics. CONCLUSION: The Sebia FLC assay provides inequivalent absolute results from the Freelite assay. Despite lower absolute FLC values, the kinetics of response and relapse is exactly the same. As with other FLC assays available, follow-up of MM with the same method is advisable.

Early use of PEPA dialyzer for light chains removal and for the recovery from myeloma cast nephropathy: A case report.

Chemotherapy and extracorporeal treatment reduce serum free light chains (FLCs) allowing the recovery of acute kidney injury (AKI) caused by myeloma cast nephropathy (MCN). We report the first case of recovery from AKI in a patient with MCN who underwent the removal of FLCs using the PEPA filter, with an undisclosed cut-off, combined with chemotherapy for multiple myeloma (MM).

Isolation and purification of recombinant immunoglobulin light chain variable domains from the periplasmic space of Escherichia coli.

Immunoglobulin light chain amyloidosis is the most common form of systemic amyloidosis. However, very little is known about the underlying mechanisms that initiate and modulate the associated protein aggregation and deposition. Model systems have been established to investigate these disease-associated processes. One of these systems comprises two 114 amino acid light-chain variable domains of the kappa 4 IgG family, SMA and LEN. Despite high sequence identity (93%), SMA is amyloidogenic in vivo, but LEN adopts a stable dimer, displaying amyloidogenic properties only under destabilising conditions in vitro. We present here a refined and reproducible periplasmic expression and purification protocol for SMA and LEN that improves on existing methods and provides high yields of pure protein (10-50mg/L), particularly suitable for structural studies that demand highly concentrated and purified proteins. We confirm that recombinant SMA and LEN proteins have structure and dimerization capabilities consistent with the native proteins and employ fluorescence to probe internalization and cellular localization within cardiomyocytes. We propose periplasmic expression and simplified chromatographic steps outlined here as an optimized method for production of these and other variable light chain domains to investigate the underlying mechanisms of light chain amyloidosis. We show that SMA and LEN can be internalised within cardiomyocytes and were observed to localise to the perinuclear area, assessed by confocal microscopy as a possible mechanism for underlying cytotoxicity and pathogenesis associated with amyloidosis.

Serum Free Light Chains Removal by HFR Hemodiafiltration in Patients with Multiple Myeloma and Acute Kidney Injury: a Case Series.

BACKGROUND/AIMS: Multiple myeloma (MM) represents 10% of all haematologic malignancies. Renal involvement occurs in 50% of MM patients; of them, 12-20% have acute kidney injury (AKI), with 10% needing dialysis at presentation. While hemodialysis (HD) has no effect upon circulating and tissue levels of monoclonal proteins, novel apheretic techniques aim at removing the paraproteins responsible for glomerular/tubular deposition disease. High cut-off HD (HCO-HD) combined with chemotherapy affords a sustained reduction of serum free light chains (FLC) levels. One alternative technology is haemodiafiltration with ultrafiltrate regeneration by adsorption on resin (HFR-SUPRA), employing a "super high-flux" membrane (polyphenylene S-HF, with a nominal cut-off of 42 kD). Aim of our pilot study was to analyze the effectiveness of HFR-SUPRA in reducing the burden of FLC, while minimizing albumin loss and hastening recovery of renal function in 6 subjects with MM complicated by AKI. METHODS: Six HD-dependent patients with MM were treated with 5 consecutive sessions of HFR-SUPRA on a Bellco® monitor, while simultaneously initiating chemotherapy. Levels of albumin and FLC were assessed, calculating the rates of reduction. Renal outcome, HD withdrawal and clinical follow-up or death were recorded. RESULTS: All patients showed a significant reduction of FLC, whereas serum albumin concentration remained unchanged. In three, HD was withdrawn, switching to a chemotherapy alone regimen. The other patients remained HD-dependent and died shortly thereafter for cardiovascular complications. CONCLUSION: Our study suggests that HFR-SUPRA provides a rapid and effective reduction in serum FLC in patients with MM and AKI, while minimizing the loss of albumin. When started early in combination with chemotherapy, blood purification by HFR-SUPRA was followed by the recovery of renal function in half of the patients treated.

Effects of Hemodialysis Therapy Using Dialyzers with Medium Cut-Off Membranes on Middle Molecules.

The removal of larger middle molecules, such as free immunoglobulin light chains (FLC), is poor with most currently used dialysis technologies. While hemodiafiltration (HDF) provides enhanced clearance of middle molecules compared to high-flux hemodialysis (HD), this technique is currently not approved in some regions and, hence, not accessible for all patients. The retention of middle molecules is thought to be one factor, which contributes to excessive morbidity and mortality in HD patients. The development of medium cut-off (MCO) dialysis membranes is aimed at a more efficient clearance of larger uremic toxins while retaining albumin and may extend the benefit of enhanced solute removal to more patients. In 2 pilot studies, the removal of middle molecules using HD with an MCO dialyzer prototype was compared to (1) high-flux HD and (2) high-flux HD and HDF. The primary outcome was the overall clearance of λ FLC, and the secondary outcome was the clearance of other middle molecules and safety. Pre-to-post reduction ratios and instantaneous clearances during HD were also assessed. In both trials, the overall λ FLC clearance with MCO HD was significantly larger than with high-flux HD and HDF. Accordingly, instantaneous clearances at 30 and 120 min showed significantly higher removal of λ FLC compared to high-flux HD and HDF. MCO HD provides a more efficient removal of larger middle molecules compared to high-flux HD and HDF. A potential drawback is slightly increased albumin loss, yet preliminary data suggest that this does not lead to permanently decreased albumin levels. Thus, MCO HD may present a promising approach to further improve middle molecule removal in maintenance dialysis patients and to extend its benefit to more patients.

2A4 binds soluble and insoluble light chain aggregates from AL amyloidosis patients and promotes clearance of amyloid deposits by phagocytosis †.

Amyloid light chain (AL) amyloidosis is characterized by misfolded light chain (LC) (amyloid) deposition in various peripheral organs, leading to progressive dysfunction and death. There are no regulatory agency-approved treatments for AL amyloidosis, and none of the available standard of care approaches directly targets the LC protein that constitutes the amyloid. NEOD001, currently in late-stage clinical trials, is a conformation-specific, anti-LC antibody designed to specifically target misfolded LC aggregates and promote phagocytic clearance of AL amyloid deposits. The present study demonstrated that the monoclonal antibody 2A4, the murine form of NEOD001, binds to patient-derived soluble and insoluble LC aggregates and induces phagocytic clearance of AL amyloid in vitro. 2A4 specifically labeled all 21 fresh-frozen organ samples studied, which were derived from 10 patients representing both κ and λ LC amyloidosis subtypes. 2A4 immunoreactivity largely overlapped with thioflavin T-positive labeling, and 2A4 bound both soluble and insoluble LC aggregates extracted from patient tissue. Finally, 2A4 induced macrophage engagement and phagocytic clearance of AL amyloid deposits in vitro. These findings provide further evidence that 2A4/NEOD001 can effectively clear and remove human AL-amyloid from tissue and further support the rationale for the evaluation of NEOD001 in patients with AL amyloidosis.

A novel method of preparing the monoform structure of catalytic antibody light chain.

Along with the development of antibody drugs and catalytic antibodies, the structural diversity (heterogeneity) of antibodies has been given attention. For >20 yr, detailed studies on the subject have not been conducted, because the phenomenon presents many difficult and complex problems. Structural diversity provides some (or many) isoforms of an antibody distinguished by different charges, different molecular sizes, and modifications of amino acid residues. For practical use, the antibody and the subunits must have a defined structure. In recent work, we have found that the copper (Cu) ion plays a substantial role in solving the diversity problem. In the current study, we used several catalytic antibody light chains to examine the effect of the Cu ion. In all cases, the different electrical charges of the molecule converged to a single charge, giving 1 peak in cation-exchange chromatography, as well as a single spot in 2-dimensional gel electrophoresis. The Cu-binding site was investigated by using mutagenesis, ultraviolet-visible spectroscopy, atomic force microscope analysis, and molecular modeling, which suggested that histidine and cysteine residues close to the C-terminus are involved with the binding site. The constant region domain of the antibody light chain played an important role in the heterogeneity of the light chain. Our findings may be a significant tool for preparing a single defined, not multiple, isoform structure.

Rapid quantitative analysis of monoclonal antibody heavy and light chain charge heterogeneity.

An alternative method to traditional 2-dimensional gel electrophoresis (2D-PAGE) and its application in characterizing the inherent charge heterogeneity of chromatographically isolated monoclonal antibody heavy and light chains is described. This method, referred to as ChromiCE, utilizes analytical size-exclusion chromatography (SEC), performed under reducing and denaturing conditions, followed by imaged capillary isoelectric focusing (icIEF) of the chromatographically separated heavy and light chains. Under conditions suitable for the subsequent icIEF analysis, the absolute and relative SEC elution volumes of the heavy and light chains were found to be highly pH dependent, a phenomenon that can be exploited in optimizing chromatographic separation. Compared to 2D-PAGE, the ChromiCE method substantially decreases the time and labor needed to complete the analysis, improves reproducibility, and provides fully quantitative assessment of charge heterogeneity. The ChromiCE methodology was applied to a set of diverse monoclonal antibodies to demonstrate suitability for quantitative charge variant analysis of heavy and light chains. A typical application of ChromiCE in extended characterization and stability studies of a purified antibody is shown.

Evaluation of strategies to control Fab light chain dimer during mammalian expression and purification: A universal one-step process for purification of correctly assembled Fab.

Fabs are an important class of antibody fragment as both research reagents and therapeutic agents. There are a plethora of methods described for their recombinant expression and purification. However, these do not address the issue of excessive light chain production that forms light chain dimers nor do they describe a universal purification strategy. Light chain dimer impurities and the absence of a universal Fab purification strategy present persistent challenges for biotechnology applications using Fabs, particularly around the need for bespoke purification strategies. This study describes methods to address light chain dimer formation during Fab expression and identifies a novel CH 1 affinity resin as a simple and efficient one-step purification for correctly assembled Fab.

Plant expression systems for early stage discovery and development of lead therapeutic antibodies.

BACKGROUND: Antibodies for human clinical applications are predominantly produced in mammalian expression systems, with Chinese hamster ovary (CHO) cells being the gold standard. CHO cells are ideal for the manufacturing of the IgG class of antibodies, but not for the production of complex antibodies like secretory IgAs (SIgAs) and IgMs, which remain unavailable for clinical use. In contrast, plant seeds and leaves hold the promise to produce SIgAs, IgMs and similar complex antibody formats to scalable amounts. Using transient transformation of Nicotiana benthamiana leaves, complex antibody formats can be produced up to milligram amounts in less than a month. OBJECTIVE: Based on these merits, we propose a model for early-phase exploration and designing of innovative antibody formats for therapeutic application. Further in this essay, we elaborate how the model was followed during the selection of novel antibodies for seed-based production. RESULT: This exploratory model led to the engineering of novel light-chain devoid porcinized-llama antibodies (VHH-Fc) of the IgG (VHH-IgG) and IgA (VHH-IgA) isotypes and also tetravalent dimeric and SIgAs. CONCLUSION: The proposed strategy may lead to plant-based rapid engineering of innovative antibodies more apt and efficacious for therapy, and in the coarse also add to the understanding of their mode of action.

A novel monoclonal antibody against the constant region of goose immunoglobulin light chain.

A monoclonal antibody (MAb) against the antigenic determinant of the constant region of goose immunoglobulin light chain (GoIgCL) was produced and characterized for the first time here. Goose immunoglobulin (Ig) in serum was purified by immunoaffinity chromatography and the resulting protein was used as immunogen to immunize BALB/c mice. At the same time, the GoIgCL gene was expressed and purified as the screening antigen for selecting MAb against GoIgCL. One hybridoma that produces antibodies against GoIgCL was selected by indirect ELISA. Then the characterization of the MAb was analyzed by ELISA, Western blot, and flow cytometry. It was found to be IgG1 with κ light chain; the MAB has high specificity to Ig in goose serum, bile, and B lymphocytes from peripheral blood, reacts only with the light chain of goose Ig, and can distinguish Ig from other birds. Therefore, the MAb generated in this study can be used as a specific reagent for detection of goose disease-specific antibodies and as a powerful tool for basic immunology research on geese.

Using mass spectrometry to monitor monoclonal immunoglobulins in patients with a monoclonal gammopathy.

A monoclonal gammopathy is defined by the detection a monoclonal immunoglobulin (M-protein). In clinical practice, the M-protein is detected by protein gel electrophoresis (PEL) and immunofixation electrophoresis (IFE). We theorized that molecular mass could be used instead of electrophoretic patterns to identify and quantify the M-protein because each light and heavy chain has a unique amino acid sequence and thus a unique molecular mass whose increased concentration could be distinguished from the normal polyclonal background. In addition, we surmised that top-down MS could be used to isotype the M-protein because each immunoglobulin has a constant region with an amino acid sequence unique to each isotype. Our method first enriches serum for immunoglobulins followed by reduction using DTT to separate light chains from heavy chains and then by microflow LC-ESI-Q-TOF MS. The multiply charged light and heavy chain ions are converted to their molecular masses, and reconstructed peak area calculations for light chains are used for quantification. Using this method, we demonstrate how the light chain portion of an M-protein can be monitored by molecular mass, and we also show that in sequential samples from a patient with multiple myeloma the light chain portion of the M-protein was detected in all samples, even those negative by PEL, IFE, and quantitative FLC. We also present top-down MS isotyping of M-protein light chains using a unique isotype-specific fragmentation pattern allowing for quantification and isotype identification in the same run. Our results show that microLC-ESI-Q-TOF MS provides superior sensitivity and specificity compared to conventional methods and shows promise as a viable method of detecting and isotyping an M-protein.

Production and characterization of murine monoclonal antibody against synthetic peptide of CD34.

BACKGROUND: The treatment of hematologic malignancies and immunodeficiency diseases are offered by hematopoietic stem cells (HSCs) as a unique self-renewal and differentiation source which most commonly is selected by CD34 surface marker for HSC. OBJECTIVE: The purpose of this study was to develop and characterize monoclonal antibody against CD34 antigen for detection of hematopoietic stem cells. METHODS: Balb/c mice were immunized with two synthetic peptides of CD34 and Spleen cells were fused with SP2/0.Fused cells were grown in hypoxanthine, aminopterine and thymidine (HAT) selective medium and cloned by limiting dilution. Large scale of monoclonal antibodies was produced by mouse ascites production of mAb (in vivo) method. Monoclonal antibody was purified by chromatography. Then reactivity of these antibodies was evaluated in different immunological assays including ELISA, immunofluorescence (IF), western blot (WB) and flowcytometry. RESULTS: In this study, between five positive clone wells, two clones were chosen for limiting dilution. Limiting dilution product was one monoclone (3-D5 monoclone) with absorbance about 2. Isotype of this mAb was identified as IgG1 class with Kappa (κ) light chain. CONCLUSIONS: This antibody is highly specific and functional in biomedical applications such as ELISA, flowcytometry, immunofluorescence, and western blot assays.

Characterization of deamidation at Asn138 in L-chain of recombinant humanized Fab expressed from Pichia pastoris.

A method was previously established for evaluating Asn deamidation by matrix-assisted laser desorption/ionization time of flight-mass spectrometry using endoproteinase Asp-N. In this study, we demonstrated that this method could be applied to the identification of the deamidation site of the humanized fragment antigen-binding (Fab). First, a system for expressing humanized Fab from methylotrophic yeast Pichia pastoris was constructed, resulting in the preparation of ∼30 mg of the purified humanized Fab from 1 l culture. Analysis of the L-chain derived from recombinant humanized Fab that was heated at pH 7 and 100°C for 1 h showed the deamidation at Asn138 in the constant region. Then, we prepared L-N138D Fab and L-N138A Fab and examined their properties. The circular dichroism (CD) spectrum of the L-N138D Fab was partially different from that of the wild-type Fab. The measurement of the thermostability showed that L-N138D caused a significant decrease in the thermostability of Fab. On the other hand, the CD spectrum and thermostability of L-N138A Fab showed the same behaviour as the wild-type Fab. Thus, it was suggested that the introduction of a negative charge at position 138 in the L-chain by the deamidation significantly affected the stability of humanized Fab.

Exploring the amyloid proteome in immunoglobulin-derived lymph node amyloidosis using laser microdissection/tandem mass spectrometry.

Amyloidosis affecting lymph nodes (LN) may occur in the setting of systemic amyloidosis or as an entity localized to the site of production (peritumoral). Why some LN amyloid remains peritumoral is unknown. We speculated that the composition of amyloid in these two presentations differs. We analyzed the amyloid proteome in LN amyloid samples to identify differences between the systemic and peritumoral subtypes. In immunoglobulin-derived LN amyloidosis (N = 26), 70% had heavy chain amyloid (AH or mixed AH/AL). True localized LN amyloidosis was rare, with only 2 patients without a monoclonal protein component. Nineteen patients (73%) had typical amyloid syndromes (100% of AL vs 67% of AH/AL, P = 0.02). A trend to improved survival for the AH/AL group in comparison to AL (median 5-year survival 48 vs. 19 months, P = 0.06) was seen. Mass spectrometric amyloid analysis is a powerful tool for characterizing amyloid and may provide additional prognostic information.