Biochemical Characterization of a New 10% IVIG Preparation [IgG Next Generation (BT595)/Yimmugo®] Obtained from a Manufacturing Process Preserving IgA/IgM Potential of Human Plasma.

BACKGROUND AND OBJECTIVE: Human plasma is used for the generation of several life-saving drugs and contains valuable antibodies from the immunoglobulin classes IgG, IgM and IgA. Purified intravenous IgG solutions (IVIGs) form the majority of plasma-derived medicine to treat patients with various forms of immunodeficiencies. In conventional IVIG manufacturing processes, immunoglobulin classes IgM and IgA are often discarded as contaminants, but these antibody classes have been proven to be effective for the treatment of acute bacterial infections. Considering the increase in demand for human plasma-derived products and the ethical value of the raw material, a more resource-saving usage of human plasma is needed. Intensive research over the last decades showed that adverse reactions to IVIGs depend on the presence of thrombogenic factors, partially unfolded proteins, non-specific activation of the complement system, and blood group specific antibodies. Therefore, new IVIG preparations with reduced risks of adverse reactions are desirable. METHOD: A new manufacturing process that yields two biologics was established and quality attributes of the new IVIG solution (Yimmugo®) obtained from this process are presented. RESULTS: Here, we provide a biochemical characterization of Yimmugo®, a new 10% IVIG preparation. It is derived from human blood plasma by a combined manufacturing process, where IgM and IgA are retained for the production of a new biologic (trimodulin, currently under investigation in phase III clinical trials). Several improvements have been implemented in the manufacturing of Yimmugo® to reduce the risk of adverse reactions. Gentle and efficient mixing by vibration (called "vibromixing") during a process step where proteins are at risk to aggregate was implemented to potentially minimize protein damage. In addition, a dedicated process step for the removal of the complement system activator properdin was implemented, which resulted in very low anticomplementary activity levels. The absence of measurable thrombogenic activity in combination with a very high degree of functional monomeric antibodies predict excellent efficacy and tolerability. CONCLUSION: Yimmugo® constitutes a new high quality IVIG preparation derived from a novel manufacturing process that takes advantage of the full therapeutic immunoglobulin potential of human plasma.

Analysis of filtration behavior using integrated column chromatography followed by virus filtration.

We evaluated filtration behavior and virus removal capability for a monoclonal antibodies (mAb) and plasma IgG under constant flow rate directly following flow-through column chromatography in an integrated process. mAb solution with quantified host cell protein (HCP) content processed in flow-through mode on in-series mixed-mode AEX and modified CEX columns connected to the Planova BioEX filter (pool-less) achieved HCP logarithmic reduction value (LRV) of 2.3 and 93.9% protein recovery, demonstrating comparable or higher HCP LRV with high protein recovery compared to previous reports. For 5-15 mg/ml plasma IgG run to 100 L/m2 , similar filtration behavior was achieved for flux of 10-100 LMH, and lower flux runs remained well below the maximum operating pressure, suggesting that higher throughput in continuous processing is achievable. Comparison of fit of plasma IgG and mAb filtration behavior to four blocking models showed little differences but slightly better fit to the cake filtration model. Viral clearance of the filtration step tested by in-line spiking X-MuLV or MVM into purified plasma IgG following the chromatography step showed robust removal at low flux. Integrating the Planova BioEX filter into continuous processes with column chromatography can achieve efficient downstream processing with reduced footprint and process time.

Machine learning and statistical analyses for extracting and characterizing "fingerprints" of antibody aggregation at container interfaces from flow microscopy images.

Therapeutic proteins are exposed to numerous stresses during their manufacture, shipping, storage and administration to patients, causing them to aggregate and form particles through a variety of different mechanisms. These varied mechanisms generate particle populations with characteristic morphologies, creating "fingerprints" that are reflected in images recorded using flow imaging microscopy. Particle population fingerprints in test samples can be extracted and compared against those of particles produced under baseline conditions using an algorithm that combines machine learning tools such as convolutional neural networks with statistical tools such as nonparametric density estimation and Rosenblatt transform-based goodness-of-fit hypothesis testing. This analysis provides a quantitative method with user-specified type 1 error rates to determine whether the mechanisms that produce particles in test samples differ from particle formation mechanisms operative under baseline conditions. As a demonstration, this algorithm was used to compare particles within intravenous immunoglobulin formulations that were exposed to freeze-thawing and shaking stresses within a variety of different containers. This analysis revealed that seemingly subtle differences in containers (e.g., glass vials from different manufacturers) generated distinguishable particle populations after the stresses were applied. This algorithm can be used to assess the impact of process and formulation changes on aggregation-related product instabilities.

Enhanced Pro-apoptotic Effects of Fe(II)-Modified IVIG on Human Neutrophils.

Mild modification of intravenous immunoglobulin (IVIG) has been reported to result in enhanced polyspecificity and leveraged therapeutic effects in animal models of inflammation. Here, we observed that IVIG modification by ferrous ions, heme or low pH exposure, shifted the repertoires of specificities in different directions. Ferrous ions exposed Fe(II)-IVIG, but not heme or low pH exposed IVIG, showed increased pro-apoptotic effects on neutrophil granulocytes that relied on a FAS-dependent mechanism. These effects were also observed in human neutrophils primed by inflammatory mediators or rheumatoid arthritis joint fluid in vitro, or patient neutrophils ex vivo from acute Crohn's disease. These observations indicate that IVIG-mediated effects on cells can be enhanced by IVIG modification, yet specific modification conditions may be required to target specific molecular pathways and eventually to enhance the therapeutic potential.

Anti-A/B isoagglutinin reduction in an intravenous immunoglobulin product and risk of hemolytic anemia: a hospital-based cohort study.

BACKGROUND: Intravenous immunoglobulins (IVIG) are derived from large human plasma pools. IVIG-associated hemolytic anemia (HA) is a known class effect, likely attributed to dose-dependent passive transfer of anti-A/B isoagglutinins. Two isoagglutinin reduction steps were implemented in the manufacturing process of Privigen (human 10% liquid IVIG): exclusion of high-anti-A-titer donors in 2013, replaced by specific immunoaffinity chromatography in 2015. We aim to estimate the clinical effectiveness of both measures. STUDY DESIGN AND METHODS: Using the US hospital-based Premier Healthcare Database, three Privigen cohorts were generated based on calendar periods indicative of manufacturing changes: Period 1 (baseline) January 2008 to December 2012, Period 2 (high-anti-A-titer donor exclusion) October 2013 to December 2015, and Period 3 (immunoaffinity chromatography) October 2016 to April 2019. HA within a 10-day at-risk period after Privigen administrations was identified from review of patient record summaries. Incidence rate ratios (IRRs) were estimated from Poisson regression (Period 1 reference) adjusting for hospital setting, sex, age, Privigen indication, dose, and first use. RESULTS: Crude incidence rates of HA were 1.49 per 10,000 person-days in Period 1 (38 HA, 9439 patients), 1.01 in Period 2 (20 HA, 7710 patients), and 0.14 in Period 3 (3 HA, 7759 patients). Adjusted IRR for HA in Period 2 was 0.71 (95% confidence interval [CI], 0.41-1.23), and in Period 3 was 0.10 (0.03-0.33) compared with Period 1. The IRR for HA in Period 3 compared with Period 2 was 0.14 (95% CI, 0.04-0.47). CONCLUSION: Implementation of immunoaffinity chromatography in Privigen manufacturing resulted in a significant 90% reduction of HA risk. HA has become a rare event in association with Privigen use.

Isoagglutinin reduction in intravenous immunoglobulin (IgPro10, Privigen) by specific immunoaffinity chromatography reduces its reporting rates of hemolytic reactions: an analysis of spontaneous adverse event reports.

BACKGROUND: Hemolysis is an infrequent but recognized and potentially serious adverse effect of intravenous immunoglobulin (IVIG). Relatively elevated hemolysis reporting rates were seen with some IVIG products with high anti-A/B isoagglutinin content, among which IgPro10 (Privigen, CSL Behring). For IgPro10, two isoagglutinin reduction measures were successively implemented: 1) anti-A donor screening and 2) immunoaffinity chromatography (IAC; Ig IsoLo)-based isoagglutinin reduction step included in the production process. The aim of this analysis was to investigate the effects of these isoagglutinin reduction measures on the reporting rates of IgPro10 hemolysis worldwide. STUDY DESIGN AND METHODS: Between February 2008 and December 2018, hemolysis reports from the CSL Behring Global Safety Database were analyzed in relationship to changes in IVIG IgPro10 production methods. Further analysis classified hemolysis reports by indication and blood group. RESULTS: Median (minimum-maximum) anti-A/anti-B titers were 32 (8-64)/16 (8-32) at baseline, 32 (8-64)/16 (8-32) after donor screening, and 8 (8-32)/4 (2-8) after implementation of IAC. The reporting rate of hemolytic reactions per 1000 kg IgPro10 sold was 4.05 cases at baseline, 2.00 after donor screening, and 0.50 after implementation of IAC. In 2018, there were seven reports of hemolytic reactions; representing 0.18 cases per 1000 kg IgPro10 sold, with a reduction of 95.6% versus baseline. CONCLUSION: Following implementation of the IAC isoagglutinin reduction step, spontaneous reports of hemolytic events with IgPro10 were significantly and consistently reduced versus IgPro10 without isoagglutinin reduction, offering patients a more favorable benefit-risk profile.

Long-term efficacy, safety, and tolerability of recombinant human hyaluronidase-facilitated subcutaneous infusion of immunoglobulin (Ig) (fSCIG; HyQvia(®)) in immunodeficiency diseases: real-life data from a monocentric experience.

Humoral immunodeficiency diseases represent a heterogeneous group of disorders that require long-term therapies. Thus, the treatment provided must not only be effective but also safe and well tolerated. In this paper, we report our data on the efficacy, safety, and tolerability of recombinant human hyaluronidase-facilitated subcutaneous infusion of immunoglobulin (Ig) (fSCIG; HyQvia(®)) in immunodeficiency patients. We collected retrospective data from 30 patients with primary and secondary immunodeficiency diseases in therapy with fSCIG from September 2014 to December 2019. We evaluated the efficacy of the therapy, taking into account serum IgG values during follow-up and the number of annual infectious events and serious bacterial infections reported by patients. Safety was assessed on the basis of the number and intensity of adverse events (AEs) and local reactions reported. Our real-life data suggest that long-term repeated self-administration of recombinant human hyaluronidase-facilitated subcutaneous infusion of immunoglobulins results in a reduced rate of infectious events if compared to the pre-treatment rate. Both AEs and local reactions are mild to moderate and were never reasons for treatment discontinuation. Therapy with HyQvia shows prolonged efficacy and good tolerability; these aspects, together with the possibility of self-administration at home, minimize the impact the illness has on patients.

False-positive Aspergillus galactomannan immunoassays associated with intravenous human immunoglobulin administration.

OBJECTIVES: Evidence of false-positive galactomannan enzyme immunoassay (GM-EIA) results associated with intravenous immunoglobulin (IVIG) administration is scarce. Here, we aimed to determine the false-positive rate of GM-EIA after IVIG administration and to identify the related factors. METHODS: Standard GM-EIA was performed using diluted and pure human IVIG samples with and without heat treatment. We also included adult patients who had at least one GM-EIA result within 1 week of IVIG administration for analysis. Those who had prior invasive aspergillosis within 1 year before IVIG therapy were excluded. The clinical characteristics and galactomannan index (GMI) kinetics between patients with false-positive and true-positive GMI were compared. RESULTS: All diluted and pure IVIG samples tested positive for GM. Heat treatment resulted in the considerable elevation of GMI. Of 48 patients with positive GM-EIA results within 1 week of IVIG administration, 22 (45.8%) were considered to have false-positive antigenaemia (false-positive group, FPG). After the completion of IVIG administration, a decline in GMI was observed in all FPG patients but in only 18 out of 26 patients (69.2%) with true-positive results (true-positive group, TPG). By 7, 14, and 18 days of IVIG administration, GMI reverted to negative values in 7/15 (46.7%), 18/20 (90%) and 22/22 (100%) FPG patients, respectively, and 6/24 (25%), 14/24 (58.3%), and 16/26 (61.5%) of TPG patients, respectively. The TPG was more likely to have two or more consecutively positive GMIs after IVIG administration than the FPG (adjusted odds ratio, 9.01; 95% confidence interval, 1.99-40.9). CONCLUSIONS: IVIG treatment may produce false-positive GM-EIA results. A positive GMI among patients receiving human IVIG should be interpreted with caution.

Measurement of isoagglutinins in immunoglobulins for intravenous application by flow cytometry.

Isoagglutinins present in intravenous immunoglobulin (IVIG) products have been linked to haemolysis. Therefore, accurately assessing isoagglutinin content in IVIG products is important. The standard European Pharmacopoeia (Ph.Eur.) direct assay is limited by low precision. Here, we describe the development of a fluorescence-activated cell sorting (FACS) method for assessing isoagglutinin levels. Serially diluted IVIG samples were incubated with red blood cells (RBCs), RBC-bound anti-A and anti-B antibodies were detected using a fluorescently-labelled antibody and the median fluorescence intensity of samples was assessed by FACS. Results were compared with the Ph.Eur. direct assay. The method was used to determine isoagglutinins in commercial products produced with and without isoagglutinin reduction steps. Assay precision, reported as the coefficient of variation, for the FACS method was 14% and 8% for anti-A and anti-B, respectively versus 33% and 20% with the Ph.Eur. direct assay. Application of the method on commercially available IVIGs revealed differences in isoagglutinin content between products produced with and without isoagglutinin reduction steps. This FACS assay allows for quantification of isoagglutinin concentrations in IVIGs with higher precision than the Ph.Eur. direct assay. Also the FACS assay confirms differences in isoagglutinin levels between IVIG products and the efficacy of isoagglutinin reduction measures.

DEHP Nanodroplets Leached From Polyvinyl Chloride IV Bags Promote Aggregation of IVIG and Activate Complement in Human Serum.

Concerns regarding the impact of subvisible particulate impurities on the safety and efficacy of therapeutic protein products have led manufacturers to implement strategies to minimize protein aggregation and particle formation during manufacturing, storage, and shipping. However, once these products are released, manufacturers have limited control over product handling. In this work, we investigated the effect of di(2-ethylhexyl) phthalate (DEHP) nanodroplets generated in polyvinyl chloride (PVC) bags of intravenous (IV) saline on the stability and immunogenicity of IV immunoglobulin (IVIG) formulations. We showed that PVC IV bags containing saline can release DEHP droplets into the solution when agitated or transported using a pneumatic tube transportation system in a clinical setting. We next investigated the effects of emulsified DEHP nanodroplets on IVIG stability and immunogenicity. IVIG adsorbed strongly to DEHP nanodroplets, forming a monolayer. In addition, DEHP nanodroplets accelerated IVIG aggregation in agitated samples. The immunogenicity of DEHP nanodroplets and IVIG aggregates generated in these formulations were evaluated using an in vitro assay of complement activation in human serum. The results suggested DEHP nanodroplets shed from PVC IV bags could reduce protein stability and induce activation of the complement system, potentially contributing to adverse immune responses during the administration of therapeutic proteins.

Effects of Tubing Type, Formulation, and Postpumping Agitation on Nanoparticle and Microparticle Formation in Intravenous Immunoglobulin Solutions Processed With a Peristaltic Filling Pump.

The main goal of this study was to use state-of-the-art instruments for nanoparticle (nanoparticle tracking analysis and resonant mass measurement) and microparticle counting (flow imaging) to assess the effects of peristaltic filling pump operation on particle formation in formulations of intravenous immunoglobulin. Microparticle levels were also measured with light obscuration. Postpumping agitation was studied as an accelerated degradation method, 3 different commercial peristaltic tubing types were tested, and the effects of formulation pH and inclusion of polysorbate 80 were determined. Overall, the results documented that nanoparticle measurements, as well as microparticle determinations with flow imaging, were essential to gain rigorous insights into impacts of processing and formulation parameters on pumping- and agitation-induced particle formation. In addition, light obscuration was a relatively insensitive method and failed to detect large increases in protein particles caused by pumping and postpumping agitation. Formulation studies showed that the presence of polysorbate 80 or increasing protein colloidal stability with appropriate choice of buffer generally reduced particle formation. The results highlight the need for filling pump assessments in formulation development studies. Combining such assessments with appropriate analytical methods should help assure that particle levels are controlled during filling pump operation and that the highest quality products are manufactured.

Subvisible Particles in IVIg Formulations Activate Complement in Human Serum.

When administered intravenously, various particles and nanomedicines activate complement, potentially leading to infusion reactions and other adverse drug reactions. Particles form within formulations of therapeutic proteins due to stresses incurred during shipping, handling, and administration to patients. In this study, IVIg solutions were stored in multiple types of vials and prefilled syringes and exposed to agitation and freeze-thaw stresses to generate particles. The stressed samples were added to human serum to determine whether these particles activated complement. Subvisible IVIg particles ranging in size between 2 and 10 microns activated complement in a fashion that was linearly dependent on the number of particles dosed, whereas little correlation was found between doses of larger particles (>10 microns) and complement activation. Activation of complement by subvisible particles of IVIg followed the alternative pathway, as shown by the release of complement cascade factor Bb and the production of the anaphylatoxins C3a and C5a without generation of C4a. The number and the morphology of subvisible particles formed depended on the applied stress, formulation, and on the container material. But the capacity of the 2- to 10-micron-sized particles to activate complement in human serum appeared to depend only on particle concentration.

Automatic Identification of the Stress Sources of Protein Aggregates Using Flow Imaging Microscopy Images.

A novel approach to identify 5 types of simulated stresses that induce protein aggregation in prefilled syringe-type biopharmaceuticals was developed. Principal components analyses of texture metrics extracted from flow imaging microscopy images were used to define subgroups of particles. Supervised machine learning methods, including convolutional neural networks, were used to train classifiers to identify subgroup membership of constituent particles to generate distribution profiles. The applicability of the stress-specific signatures for distinguishing stress source types was verified. The high classification efficiencies (100%) precipitated the collection of data from more than 20 independent experiments to train support vector machines, k-nearest neighbors, and ensemble classifiers. The performances of the trained classifiers were validated. High classification efficiencies for friability (80%-100%) and heating at 90°C (85%-100%) are indicative of high reliability of these methods for stress-stability assays while extreme variations in freeze-thawing (2%-100%) and heating at 60°C (2.25%-98.25%) indicate the unpredictability of particle composition profiles for these forced degradation conditions. We also developed subvisible particle classifiers using convolutional neural network to automatically identify silicone oil droplets, air bubbles, and protein aggregates. The developed classifiers will contribute to mitigating aggregation in biopharmaceuticals via the identification of stress sources.

Heparin chromatography as an in vitro predictor for antibody clearance rate through pinocytosis.

The pharmacokinetic (PK) properties of therapeutic antibodies directly affect efficacy, dose and dose intervals, application route and tissue penetration. In indications where health-care providers and patients can choose between several efficacious and safe therapeutic options, convenience (determined by dosing interval or route of application), which is mainly driven by PK properties, can affect drug selection. Therapeutic antibodies can have greatly different PK even if they have identical Fc domains and show no target-mediated drug disposition. Biophysical properties like surface charge or hydrophobicity, and binding to surrogates for high abundant off-targets (e.g., baculovirus particles, Chinese hamster ovary cell membrane proteins) were proposed to be responsible for these differences. Here, we used heparin chromatography to separate a polyclonal mix of endogenous human IgGs (IVIG) into fractions that differ in their PK properties. Heparin was chosen as a surrogate for highly negatively charged glycocalyx components on endothelial cells, which are among the main contributors to nonspecific clearance. By directly correlating heparin retention time with clearance, we identified heparin chromatography as a tool to assess differences in unspecific cell-surface interaction and the likelihood for increased pinocytotic uptake and degradation. Building on these results, we combined predictors for FcRn-mediated recycling and cell-surface interaction. The combination of heparin and FcRn chromatography allow identification of antibodies with abnormal PK by mimicking the major root causes for fast, non-target-mediated, clearance of therapeutic, Fc-containing proteins.

Treatment potential of pathogen-reactive antibodies sequentially purified from pooled human immunoglobulin.

OBJECTIVE: Intravenous immune globulin (IVIG), pooled from human blood, is a polyspecific antibody preparation that inhibits the super-antigenic proteins associated with streptococcal and staphylococcal toxic shock, and the Shiga toxin. In addition to this toxin-neutralising activity, IVIG contains other pathogen-reactive antibodies that may confer additional therapeutic benefits. We sought to determine if pathogen-reactive antibodies that promote opsonophagocytosis of different organisms can be sequentially affinity-purified from one IVIG preparation. RESULTS: Antibodies that recognise cell wall antigens of Streptococcus pyogenes, Staphylococcus aureus, and vancomycin-resistant enterococcus (VRE) were sequentially affinity-purified from a single preparation of commercial IVIG and opsonophagocytic activity was assessed using a flow cytometry assay of neutrophil uptake. Non-specific IgG-binding proteins were removed from the S. aureus preparations using an immobilised Fc fragment column, produced using IVIG cleaved with the Immunoglobulin G-degrading enzyme of S. pyogenes (IdeS). Affinity-purified anti-S. aureus and anti-VRE immunoglobulin promoted significantly higher levels of opsonophagocytic uptake by human neutrophils than IVIG when identical total antibody concentrations were compared, confirming activity previously shown for affinity-purified anti-S. pyogenes immunoglobulin. The opsonophagocytic activities of anti-S. pyogenes, anti-S. aureus, and anti-VRE antibodies that were sequentially purified from a single IVIG preparation were undiminished compared to antibodies purified from previously unused IVIG.

Rapid Quantification of Protein Particles in High-Concentration Antibody Formulations.

Current technologies for monitoring the subvisible particles that may be generated during fill-finish operations for protein formulations are cumbersome. Measurement times are generally too long for real-time analysis, and the high protein concentrations that are characteristic of many antibody products interfere with common optical techniques for particle analysis. To rapidly monitor protein particle levels in high-concentration protein solutions, we developed a fluorescence-based method that uses extrinsic fluorescent dyes such as 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid that are sensitive to the presence of aggregated protein. To test the method, antibody formulations containing various concentrations of protein particles were generated by application of various mechanical and freeze-thaw stresses. After addition of fluorescent dyes, fluorescence intensities were measured and compared to fluorescence intensities in particle-free formulations. The differences in fluorescence intensities were linearly proportional to protein particle levels, which for calibration purposes were measured offline by fluid imaging microscopy and protein assays. Protein particle levels could be measured without requiring sample dilution, even in high-concentration (e.g., 40 mg/mL) antibody formulations.

Analysis of sialic acid levels in Chinese intravenous immunoglobulins by high-performance liquid chromatography with fluorescence detection.

Intravenous immunoglobulin (IVIg) is increasingly used for the treatment of autoimmune and systemic inflammatory diseases with both licensed and off-label indications. Recent studies indicated that IVIg-mediated immunomodulation and anti-inflammation are closely associated with the IgG sialylation, especially with IgG crystallizable fragment (Fc) sialylation. The sialic acid levels of the IgG molecules and Fc fragments in 12 IVIg preparations from six Chinese manufacturers were evaluated. The Fc fragments were derived from the papain digestion of IVIg, followed by affinity and size exclusion chromatography. The sialic acid levels in Fc fragments and IVIg preparations were determined by high-performance liquid chromatography with fluorescence detection, after the sialic acid residues were released from the proteins. The results showed that the sialic acid levels in Chinese IVIg preparations ranged from 0.875 (mol/mol IgG) to 1.085 (mol/mol IgG), and the sialic acid levels in Fc fragments were from 0.321 (mol/mol Fc) to 0.361 (mol/mol Fc). Furthermore, the sialic acid levels of IVIg preparations and Fc fragments from different Chinese manufactures were significantly different. These findings will contribute to an increased understanding of Chinese IVIg preparations and the relationship between the sialic acid levels in IVIg preparations and their clinical efficacy in future clinical studies.

Collaborative Study for Analysis of Subvisible Particles Using Flow Imaging and Light Obscuration: Experiences in Japanese Biopharmaceutical Consortium.

The evaluation of subvisible particles, including protein aggregates, in therapeutic protein products has been of great interest for both pharmaceutical manufacturers and regulatory agencies. To date, the flow imaging (FI) method has emerged as a powerful tool instead of light obscuration (LO) due to the fact that (1) protein aggregates contain highly transparent particles and thereby escape detection by LO and (2) FI provides detailed morphological characteristics of subvisible particles. However, the FI method has not yet been standardized nor listed in any compendium. In an attempt to assess the applicability of the standardization of the FI method, we conducted a collaborative study using FI and LO instruments in a Japanese biopharmaceutical consortium. Three types of subvisible particle preparations were shared across 12 laboratories and analyzed for their sizes and counts. The results were compared between the methods (FI and LO), inter-laboratories, and inter-instruments (Micro Flow Imaging and FlowCam). We clarified the marked difference between the detectability of FI and LO when counting highly transparent protein aggregates in the preparations. Although FlowCam provided a relatively higher number of particles compared with MFI, consistent results were obtained using the instrument from the same manufacturer in all 3 samples.

Effect of Different Aß Aggregates as Antigen on the Measure of Naturally Occurring Autoantibodies against Amyloid-ß40/42 in IVIG.

BACKGROUND: The specific Intravenous Immunoglobulin (IVIG) for Alzheimer's Disease (AD) is developing, which contains a high level of naturally occurring autoantibodies against amyloid-ß (nAbs-Aß), and the measure of nAbs-Aß content is greatly essential. Though Enzyme-Linked Immunosorbent Assay (ELISA) has been widely used in detecting the nAbs-Aß content, the impact of Aß aggregates species chosen as antigen in ELISA on this measure has not been evaluated. OBJECTIVE: To clarify the influence of different Aß40/42 aggregates as antigen during ELISA on the content of nAbs-Aß40/42 measured in IVIG. METHOD: Preparation of various Aß40/42 aggregates was performed by different aggregation solutions and various lengths of time, and analyzed by western blot. Different Aß40/42 aggregates as antigen were adopted to measure the nAbs-Aß40/42 content in IVIG by ELISA, and the control was carried out to reduce interference of nonspecific binding. The Bonferroni and Dunnett's T3 were used for statistical analysis. RESULTS: The duration for the formation of Aß40/42 aggregates had more effect on detecting nAbs-Aß40/42 content in IVIG than the aggregation solution. Higher content of nAbs-Aß40/42 in the same IVIG was displayed when measured with Aß40/42 aggregates at day 3, instead of at day 0.5 and day 7.0. The nAbs- Aß40/42 contents in the same IVIG measured with Aß40/42 aggregates prepared in different solutions were obviously different, but there was no significant regularity among them. CONCLUSION: The nAbs-Aß40/42 content in the same IVIG is significantly different when measured with Aß40/42 aggregated under different conditions. The nAbs-Aß40/42 content in IVIG by antigen-dependent measures, like ELISA, is uncertain.

Binding Performance of Human Intravenous Immunoglobulin and 20(S)-7-Ethylcamptothecin.

A previous study showed that intravenous immunoglobulin (IVIG) could preserve higher levels of biologically active lactone moieties of topotecan, 7-ethyl-10-hydroxycamptothecin (SN-38) and 10-hydroxycamptothecin at physiological pH 7.40. As one of camptothecin analogues (CPTs), the interaction of 7-ethylcamptothecin and IVIG was studied in vitro in this study. It was shown that the main binding mode of IVIG to 7-ethylcamptothecin was hydrophobic interaction and hydrogen bonding, which is a non-specific and spontaneous interaction. The hydrophobic antigen-binding cavity of IgG would enwrap the drug into a host-guest inclusion complex and prevent hydrolysis of the encapsulated drug, while the drug is adjacent to the chromophores of IgG and may exchange energy with chromophores and quench the fluorescence of the protein. Also, the typical ß-sheet structure of IVIG unfolded partially after binding to 7-ethylcamptothecin. Additionally, the binding properties of IVIG and six CPTs with different substituents at A-ring and/or B-ring including camptothecin, topotecan, irinotecan, 10-hydroxycamptothecin, 7-ethylcamptothecin and SN-38 were collected together and compared each other. Synergizing with anti-cancer drugs, IVIG could be used as a transporter protein for 7-ethylcamptothecin and other CPTs, allowing clinicians to devise new treatment protocols for patients.