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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.


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

AAPS J 2014 May;16(3):440-51
Analyzing Subvisible Particles in Protein Drug Products: a Comparison of Dynamic Light Scattering (DLS) and Resonant Mass Measurement (RMM).
Panchal J, Kotarek J, Marszal E, Topp EM

PDA J Pharm Sci Technol 2014 May-Jun;68(3):215-20
Meeting report-workshop on spike characterizations and virus removal by filtration: trends and new developments.
Willkommen H, Blumel J, Brorson K, Chen D, Chen Q, Groner A, Hubbard BR, Kreil TR, Ruffing M, Ruiz S, Scott D, Silvester G

PDA J Pharm Sci Technol 2014 May-Jun;68(3):193-214
Meeting Report: 2013 PDA Virus & TSE Safety Forum.
Willkommen H, Blumel J, Brorson K, Chen D, Chen Q, Groner A, Hubbard BR, Kreil TR, Ruffing M, Ruiz S, Scott D, Silvester G

Am J Hematol 2013 Dec;88(12):1035-40
Hyperimmune globulins and same-day thrombotic adverse events as recorded in a large healthcare database during 2008-2011.
Menis M, Sridhar G, Selvam N, Ovanesov MV, Divan HA, Liang Y, Scott D, Golding B, Forshee R, Ball R, Anderson SA, Izurieta HS

Influenza Other Respir Viruses 2013 Jan;7(1):46-54
Mucosal antibody responses are directed by viral burden in children with acute influenza infection.
He Y, Abid A, Fisher R, Eller N, Mikolajczyk M, Welliver RC Sr, Bonner AB, Scott DE, Reed JL

PLoS One 2012;7(11):e51078
Cytosolic, autocrine alpha-1 proteinase inhibitor (A1PI) inhibits caspase-1 and blocks IL-1beta dependent cytokine release in monocytes.
Wang Y, He Y, Abraham B, Rouhani FN, Brantly ML, Scott DE, Reed JL

Transfusion 2012 Oct;52(10):2113-21
Immune globulins and thrombotic adverse events as recorded in a large administrative database in 2008 through 2010.
Daniel GW, Menis M, Sridhar G, Scott D, Wallace AE, Ovanesov MV, Golding B, Anderson SA, Epstein J, Martin D, Ball R, Izurieta HS

J Pharm Sci 2012 Oct;101(10):3555-9
Workshop on predictive science of the immunogenicity aspects of particles in biopharmaceutical products.
Marszal E, Fowler E

Virol J 2012 Sep 24;9:217
Epitope mapping by random peptide phage display reveals essential residues for vaccinia extracellular enveloped virion spread.
He Y, Wang Y, Struble EB, Zhang P, Chowdhury S, Reed JL, Kennedy M, Scott DE, Fisher RW

Clin Infect Dis 2012 Mar;54(6):832-40
Eczema vaccinatum.
Reed JL, Scott DE, Bray M

IEEE Trans Nanotechnol 2012 Jan;11(1):88-96
Pandemic Influenza Detection by Electrically Active Magnetic Nanoparticles and Surface Plasmon Resonance
Kamikawa TL, Mikolajczyk MG, Kennedy M, Zhong LL, Zhang P, Setterington EB, Scott DE, Alocilja EC

Arch Virol 2011 Oct;156(10):1877-81
Infection of cynomolgus macaques with a recombinant monkeypox virus encoding green fluorescent protein.
Goff A, Mucker E, Raymond J, Fisher R, Bray M, Hensley L, Paragas J

Virol J 2011 Sep 20;8:441
Polyclonal antibody cocktails generated using DNA vaccine technology protect in murine models of orthopoxvirus disease.
Golden JW, Zaitseva M, Kapnick S, Fisher RW, Mikolajczyk MG, Ballantyne J, Golding H, Hooper JW

Vox Sang 2011 Jul;101(1):83-9
An international collaborative study to establish the WHO 1st international standard for alpha-1-antitrypsin.
Thelwell C, Marszal E, Rigsby P, Longstaff C

PLoS Pathog 2011 Jun;7(6):e1002081
Cross-neutralizing antibodies to pandemic 2009 H1N1 and recent seasonal H1N1 influenza A strains influenced by a mutation in hemagglutinin subunit 2.
Wang W, Anderson CM, De Feo CJ, Zhuang M, Yang H, Vassell R, Xie H, Ye Z, Scott D, Weiss CD

Clin Vaccine Immunol 2011 Jan;18(1):67-74
Postexposure prevention of progressive vaccinia in SCID mice treated with vaccinia immune globulin.
Fisher RW, Reed JL, Snoy PJ, Mikolajczyk MG, Bray M, Scott DE, Kennedy MC

Biosens Bioelectron 2010 Dec 15;26(4):1346-52
Nanoparticle-based biosensor for the detection of emerging pandemic influenza strains.
Kamikawa TL, Mikolajczyk MG, Kennedy M, Zhang P, Wang W, Scott DE, Alocilja EC

Biologicals 2010 Sep;38(5):602-11
Meeting report on protein particles and immunogenicity of therapeutic proteins: filling in the gaps in risk evaluation and mitigation.
Carpenter J, Cherney B, Lubinecki A, Ma S, Marszal E, Mire-Sluis A, Nikolai T, Novak J, Ragheb J, Simak J

Transfusion 2009 Jun;49(6):1050-8
Investigation of whether the acute hemolysis associated with Rh(D) immune globulin intravenous (human) administration for treatment of immune thrombocytopenic purpura is consistent with the acute hemolytic transfusion reaction model.
Gaines AR, Lee-Stroka H, Byrne K, Scott DE, Uhl L, Lazarus E, Stroncek DF

J Infect Dis 2009 Apr 15;199(8):1128-1138
Innate Immune Signals Modulate Antiviral and Polyreactive Antibody Responses during Severe Respiratory Syncytial Virus Infection.
Reed JL, Welliver TP, Sims GP, McKinney L, Velozo L, Avendano L, Hintz K, Luma J, Coyle AJ, Welliver Sr RC

J Infect Dis 2008 Dec 15;198(12):1783-93
Macrophage Impairment Underlies Airway Occlusion in Primary Respiratory Syncytial Virus Bronchiolitis.
Reed JL, Brewah YA, Delaney T, Welliver T, Burwell T, Benjamin E, Kuta E, Kozhich A, McKinney L, Suzich J, Kiener PA, Avendano L, Velozo L, Humbles A, Welliver Sr RC, Coyle AJ

Clin Infect Dis 2008 May 15;46(10):1555-61
Severe eczema vaccinatum in a household contact of a smallpox vaccinee
Vora S, Damon I, Fulginiti V, Weber SG, Kahana M, Stein SL, Gerber SI, Garcia-Houchins S, Lederman E, Hruby D, Collins L, Scott D, Thompson K, Barson JV, Regnery R, Hughes C, Daum RS, Li Y, Zhao H, Smith S, Braden Z, Karem K, Olson V, Davidson W, Trindade G, Bolken T, Jordan R, Tien D, Marcinak J

J Virol 2007 Jul;81(14):7449-62
Temporal analysis of Andes virus and Sin Nombre virus infections of Syrian hamsters.
Wahl-Jensen V, Chapman J, Asher L, Fisher R, Zimmerman M, Larsen T, Hooper JW

Cancer Res 2007 May 15;67(10):5059
Regulatory B cells inhibit antitumor immunity.
Inoue S, Scott D, Golding B, Leitner WW

J Infect Dis 2006 Sep 15;194(6):781-9
Measles-virus-neutralizing antibodies in intravenous immunoglobulins.
Audet S, Virata-Theimer ML, Beeler JA, Scott DE, Frazier DJ, Mikolajczyk MG, Eller N, Chen FM, Yu MY

Cancer Res 2006 Aug 1;66(15):7741-7
Inhibitory effects of B cells on antitumor immunity.
Inoue S, Leitner WW, Golding B, Scott D

Curr Opin Biotechnol 2005 Oct;16(5):561-7
The clearance of viruses and transmissible spongiform encephalopathy agents from biologicals.
Farshid M, Taffs RE, Scott D, Asher DM, Brorson K

Nat Med 2005 Jul;11(7):740-7
Smallpox vaccine-induced antibodies are necessary and sufficient for protection against monkeypox virus.
Edghill-Smith Y, Golding H, Manischewitz J, King LR, Scott D, Bray M, Nalca A, Hooper JW, Whitehouse CA, Schmitz JE, Reimann KA, Franchini G

J Virol 2005 Jun;79(11):6791-800
Identification of a Linear Peptide Recognized by Monoclonal Antibody 2D7 Capable of Generating CCR5-Specific Antibodies with Human Immunodeficiency Virus-Neutralizing Activity.
Khurana S, Kennedy M, King LR, Golding H

Vaccine 2005 Feb 25;23(14):1730-8
Programming of CTL with heat-killed Brucella abortus and antigen allows soluble antigen alone to generate effective secondary CTL.
Inoue S, Golding B, Scott D