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  1. Biologics Research Projects

Safety and Efficacy of Immune Globulins and Alpha-1 Proteinase Inhibitors

Principal Investigator: Dorothy Scott, MD
Office / Division / Lab: OTAT / DPPT / PDB

General Overview

Our research program focuses on safety and effectiveness of Immunoglobulin Products (IG) and Alpha-1 Proteinase Inhibitor (A1PI). IG are essential to prevent serious infections in people with primary immunodeficiencies and other conditions, and for certain neurological diseases. A1PI is the only licensed treatment for people with A1PI deficiency and emphysema.

  1. Preventing hemolytic complications of IG therapy
    Immune Globulin products, especially in high doses, can cause destruction of red blood cells. Severe hemolysis causes anemia and temporary kidney failure. The main cause is antibodies against blood groups A and B that copurify with therapeutic antibodies in IG. Despite FDA-required limits on A/B antibodies IG, hemolytic reactions occur.
    We developed a hemolysis test for IG products, which helps FDA evaluate products implicated in hemolysis, new products, and manufacturing changes that may change hemolytic activity, thus improving product safety. We are currently identifying characteristics of hemolytic antibodies, to provide information that may help in their removal from IGs.
  2. Biological product protein aggregation: detection and characterization to enhance product safety
    Aggregation of proteins during biotherapeutic manufacturing/storage is a common problem. Aggregates may cause serious side effects, including allergic reactions or development of antibodies in patients that block therapeutic effects. It's unclear what levels and types of aggregates are unsafe. We are comparing advanced analytical methods for measuring aggregates, characterizing aggregate standards in multi-lab studies, and collaborating with an academic institution to measure biological effects of aggregates. We also participate in investigations of aggregated products that have caused serious adverse events. This project allows FDA to develop tests to better understand consequences of aggregation, to measure aggregate qualities, and to rapidly investigate possible aggregate-related clinical events.
  3. Influenza Immune Globulin: from Plasma to Product
    Providing safe, effective, timely interventions during an influenza pandemic is challenging. Vaccine production requires time, and antiviral treatments are sometimes ineffective. Studies suggest that antibodies from people who had influenza or influenza vaccination might help patients with severe influenza. We tested samples from plasma donations of self-identified vaccinated or convalescent donors from the 2009 pandemic. The plasma was commercially manufactured into FLUIGIV, which protected mice from 09pdmH1N1. Analysis of antibody levels from individual donors showed enrichment in self-selected donors, which could be further improved by plasma testing. Current efforts are focused on developing a rapid, virus-free test that could be used to screen plasma donations for flu antibodies, to product a more potent product.
  4. Reference Materials and New Tests to Measure Ebola Virus Antibodies
    This project addresses the need for anti-Ebola antibody standards, to measure potency of investigational antibody therapies, and standardization of neutralization and serological tests. We are currently generating and evaluating anti-Ebola antibody preparations using BSL-2 neutralization assays and ELISA tests. We are developing complement-mediated cytotoxicity (CMC) and antibody dependent cellular cytotoxicity (ADCC) assays to measure antiviral effects of Ebola antibodies that are not conventionally tested but may be important for Ebola treatment. This work should further development potency tests for Ebola antibody therapies, and provide insights into how antibody preparations might be improved.

Scientific Overview

Hemolytic complications of IG therapy.
Developing a hemolysis assay for IGIV products: Complement-dependent hemolysis assays to detect levels of hemolytic antibodies attempt to model intravascular transfusion reactions. In our assay, test or positive control IGIV are added to type A1rr rbc (hemolytic targets) followed by human complement, incubation, and measurement of free hemoglobin in resulting supernatant. We identified critical parameters for this assay: pH, mixing time and method, papain presence/absence,complement source, centrifugation speed, and buffer composition/ionic strength. A reliable method of preserving rbc droplets minimizes experimental variation, and active complement collected from whole blood donations by a special protocol provided a stable long-lasting supply. IG-mediated hemolytic events may be intravascular (complement mediated), and/or extravascular (cell-mediated). In the coming year, we will develop an in vitro cell-based model for extravascular hemolysis, with the aim of understanding IG characteristics and cellular conditions that predispose to extravascular hemolysis.

Biological product protein aggregation:
In the past year, we published a collaboration with Purdue University to study effects of product aggregates on THP-1 cell inflammatory cytokine release, which revealed differences in cellular responses to IG products subjected to different types of stressors that can occur during manufacturing. We also published a study that correlated protein aggregates with serious adverse reactions to a license product. In the coming year, we plan to develop a surface plasmon-resonance based method to detect aggregate binding to ligands that can stimulate monocyte/macrophage cells. We will also continue to test advanced methods that purport to measures quantitative and qualitative properties of aggregates, and to continue to evaluate of products implicated in adverse events.

Influenza Immune Globulin: From Plasma to Product.
We used hemagglutination inhibition testing to measure anti-influenza antibodies in over 200 sera from plasma donors who self-identified as having been infected by or vaccinated with the 2009 pandemic strain. Fifty-four per cent of vaccinated donors and 37% of convalescent donors had HAI titers >= 1:64, whereas 16% of random donors had titers this high. However, Low titer (<= 1:16) donations were most prevalent in the random donor group (83%) followed by the convalescent (47%) and vaccinated (41%) donors, suggesting that if feasible, screening for high titer lots in a pandemic should yield more potent immune plasma and FLUIGIV product. Our current goal is to optimize a surface plasmon-resonance based test that is rapid and virus-free, to measure serum antibodies that interfere with influenza binding to receptors on cells.

Reference Materials and New Tests to Measure Ebola Virus Antibodies:
In the past year, we have identified and synthesized potentially immunogenic/neutralizing epitopes on Ebola Virus (EboV) glycoproteins. Immunogenicity will be evaluated in small animal studies, and neutralizing antibodies measured by pseudotype virus assay. We will test protocols to measure ADCC and CMC antibody function, using EboV-GP expressing MRC-5 cells and flow cytometric methods. Rabbit and equine polyclonal anti-EboV sera will be characterized for suitability as standards in BSL-2 neutralizing, CMC, and ADCC assays.


J Interferon Cytokine Res 2019 May;39(5):283-92
S27 of IFNalpha1 contributes to its low affinity for IFNAR2 and weak antiviral activity.
Sharma N, O'Neal AJ, Gonzalez C, Wittling M, Gjinaj E, Parsons LM, Panda D, Khalenkov A, Scott D, Misra S, Rabin RL

Transfusion 2018 Dec;58 Suppl 3:3121-4
Assuring immune globulin potency in a world of changing pathogen challenges.
Scott DE

Transfusion 2018 Dec;58 Suppl 3:3051-3
Immune globulin potency: challenges and opportunities.
Scott DE

Transfusion 2018 May;58(5):1108-16
Characterization of source plasma from self-identified vaccinated or convalescent donors during the 2009 H1N1 pandemic.
Khalenkov A, He Y, Reed JL, Kreil TR, McVey J, Norton M, Scott J, Scott DE

PLoS One 2018 Apr 9;13(4):e0195525
The use of plant lectins to regulate H1N1 influenza A virus receptor binding activity.
Lee N, Khalenkov AM, Lugovtsev VY, Ireland DD, Samsonova AP, Bovin NV, Donnelly RP, Ilyushina NA

FEMS Microbiol Lett 2018 Feb 1;365(4):fny005
Vitamin K5 is an efficient photosensitizer for ultraviolet A light inactivation of bacteria.
Xu F, Li Y, Ahmad J, Wang Y, Scott D, Vostal JG

J Allergy Clin Immunol 2018 Jan;141(1):180-8
A distinct biomolecular profile identifies monoclonal mast cell disorders in patients with idiopathic anaphylaxis.
Carter MC, Desai A, Komarow HD, Bai Y, Clayton ST, Clark AS, Ruiz-Esteves KN, Long LM, Cantave D, Wilson TM, Scott LM, Simakova O, Jung MY, Hahn J, Maric I, Metcalfe DD

Biologicals 2017 Nov;50:35-41
Binding and neutralizing anti-cytomegalovirus activities in immune globulin products.
Wang X, Xu Y, Scott DE, Murata H, Struble EB

Lancet Respir Med 2017 Jun;5(6):462-4
Serotherapy for patients with severe influenza.
Scott D, Epstein JS, Hayden FG

Am J Hematol 2017 Apr;92(4):E44-5
Association of immune globulin intravenous (IGIV) and thromboembolic adverse events (TEEs).
Ovanesov MV, Menis MD, Scott DE, Forshee R, Anderson S, Bryan W, Golding B

Br J Haematol 2016 Jun;173(6):876-83
Monoclonal gammopathy-associated pure red cell aplasia.
Korde N, Zhang Y, Loeliger K, Poon A, Simakova O, Zingone A, Costello R, Childs R, Noel P, Silver S, Kwok M, Mo C, Young N, Landgren O, Sloand E, Maric I

PLoS One 2016 Mar 23;11(3):e0151902
AFM imaging reveals topographic diversity of wild type and Z variant polymers of human alpha1-Proteinase inhibitor.
Gaczynska M, Karpowicz P, Stuart CE, Norton MG, Teckman JH, Marszal E, Osmulski PA

J Pharm Sci 2016 Mar;105(3):1023-7
Subvisible particle content, formulation, and dose of an erythropoietin peptide mimetic product are associated with severe adverse postmarketing events.
Kotarek J, Stuart C, De Paoli SH, Simak J, Lin TL, Gao Y, Ovanesov M, Liang Y, Scott D, Brown J, Bai Y, Metcalfe DD, Marszal E, Ragheb JA

PDA J Pharm Sci Technol 2016 Mar-Apr;70(2):177-88
Meeting Report: 2015 PDA Virus & TSE Safety Forum.
Willkommen H, Blumel J, Brorson K, Chen D, Chen Q, Groener A, Kreil T, Ruffing M, Ruiz S, Scott D, Silvester G

J Infect Dis 2016 Feb 1;213(3):403-6
Sera from middle-aged adults vaccinated annually with seasonal influenza vaccines cross-neutralize some potential pandemic influenza viruses.
Wang W, Facundo EA, Chen Q, Anderson CM, Scott D, Vassell R, Weiss CD

Transfusion 2015 Jul;55(S2):S2-5
Hemolytic adverse events with immune globulin products: product factors and patient risks.
Scott DE, Epstein JS

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