Authors:Prof. János Szebeni, M.D.1 and Peter Haima, Ph.D.2

1. NanomedicineResearchandEducationCenter,SemmelweisUniversityandSeroScienceLtd.,Budapest,Hungary2. Life-Forcebiomedicalcommunication,Netherlands

TECOmedical Clinical & Technical Review

December 2013

Hemocompatibility of medical devices, blood products,

nanomedicines and biologicals

Testing activation of the C-system

2

ABSTRACTHemocompatibility testing is the evaluation of critical interactions of foreign material with blood to explore possible adverse effects arising from the exposure of the foreign material to blood cells and proteins. Because such adverse effects are frequent and may represent serious health risks, hemocompatability testing is very important for the in-troduction of new medical devices, medicines, blood products and diagnostic agents. Stimulation of the immune system can lead to an allergy-like syndrome called hypersensitivity or infusion reaction, a major and potentially lethal hemo-incompatibility whose symptoms involve almost all organ systems. Infusion reactions can represent a real allergy (IgE-mediated) or a “pseudoallergy”, one that involves no IgE and may arise as a consequence of activation of the complement (C) system. Non-IgE-mediated anaphylactoid, or pseudoallergic reactions are frequent side effects of i.v. administered nanomedicines (e.g. drug carrier systems like liposomes) and biologicals (e.g. monoclonal antibodies). These new drugs are in the frontline of modern pharmacotherapy, but their unique toxicity problem has not been solved to date. Regulatory guidance on the assessment of C-mediated infusion hypersensitivity has been published recently for the case of generic liposome production. In this review we outline the theoretical foundation of C-mediated infusion hypersensitivity to medical devices, nanomedicines and biologicals, and offer practical methods for using C assays to predict their hemo-incompatibility.

We recommend that essentially all intravenously applied drug candidates should be tested with regard to direct C-activation as a risk factor for infusion hypersensitivity.

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CONTENT

1 INTRODUCTION 41.1 TheComplementsystem 41.2 HemocompatibilityandC-activation 6 Infusion hypersensitivity caused by activation of the C-system 61.3 RegulatoryguidancesfortestingforC-activation 7 Medical Devices 8 Nanomedicines and biologicals 8

2 ACTIVATIONOFTHEC-SYSTEMBYARTIFICIALSURFACES,NANOMEDICINES ANDBIOLOGICALS 92.1 MechanismofinfusionhypersensitivitycausedbyC-activation 92.2 C-activationbyartificialsurfaces 92.3 C-activationbynanomedicines 92.4 C-activationbybiologicals 102.5 PrevalenceofC-activation-relatedpseudoallergy 112.6 Marketednanomedicines&biologicalsinducingC-activation-relatedpseudoallergy 11 Marketed Liposomal drugs 13 Marketed therapeutic monoclonals 13 Other drugs and agents on the market inducing complement activation-related pseudoallergy 142.7 RecommendationstoassesstheC-activatingcapabilityofdrugs anddrugcarriersystems 14

3 METHODSANDASSAYSTOMEASUREACTIVATIONOFTHEC-SYSTEM 153.1 InvitroC-testingforhemocompatabilityinhumansera 15 Experimental design of in vitro C-testing for hemocompatability 16 Protocols for in vitro C-testing for hemocompatability 173.2 Testmodelsformedicaldevices 20 Static models with serum 20 Dynamic models with fresh whole blood 203.3 FlowcytometricexaminationstoevaluatebindingofC-proteinstoartificialsurfaces 203.4 Personalizedmedicine:testingforhypersensitivityreactionstoanticancerdrugs 203.5 TestingC-activationinanimalmodels 20 CxH50 21 Species-Independent C-testing for hemocompatability in animals 21 The porcine model 21 Other animal models 233.6 Testingofmonoclonalantibodiesforcomplementdependentcytotoxicity 24

4 REFERENCES 25

5 TECHNICALDATASHEETSOFCOMPLEMENTPRODUCTS 27

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1 INTRODUCTION1.1 TheComplementSystemThe Complement (C-) system helps, or “complements” the ability of antibodies and phagocytic cells to clear invading cellular pathogens (e.g. bacteria) [1-3]. It is part of the immune system called the innate immune system that is not adaptable and responds to foreign challenges in a non-specific manner. When stimulated by one of several triggers, proteases in the system cleave other C proteins and initiate an amplifying cascade of further cleavages. The end-result of this activation cascade is massive amplification of the response, release of cytokines, massive acute inflammatory reactions and activation of the cell-killing membrane attack complex (MAC).

Figure 1The complement cascade (figure was adaped from Rutkowski et al. (2010)) [4].

The classical pathway is activated by the Fc portion of immunoglobulins bound to antigen, apoptotic cells, Gram-negative bacteria, and viruses.

The C1 complex, made up of C1q, C1r, and C1s subunits, initiates the downstream classical cascade. Upon binding of C1q to an inciting stimulus,

C1r catalyzes breakage of a C1s ester bond, resulting in its activation and subsequent cleavage of C2 and C4 into their respective “a” and “b” frag-

ments. The formation of C2a4b creates C3 convertase, which cleaves C3 into C3a and C3b. C3b binds to other C3 convertases, forming C2a4b3b,

also known as C5 convertase. It facilitates the final steps of the cascade by splitting C5 into C5a and C5b. The latter fragment is the critical first

protein that combines with C6, C7, C8, and multiple C9 proteins to form the MAC, the terminal, pore-forming c-protein complex responsible for

lysis of cells and pathogens. The MBL pathway is activated by surfaces bearing mannose groups or other pathogen-associated molecular pat-

terns. MBL or ficolin activation of mannose-associated serine proteases (MASP) results in cleavage of C2 and C4 similar to the C1 complex, with

subsequent production of C3 convertase and C- cascade activation resembling the classical pathway. Lastly, the alternative pathway is activated

by a multitude of infectious agents including various bacteria, viruses, and fungi, as well as neoplastic cells. This pathway exhibits a unique

“tickover” effect whereby low-level C3 cleavage occurs continuously. Generated C3b binds Bb, a cleavage fragment of factor B, and properdin, re-

sulting in the formation of the alternative pathway C3 convertase. Binding of additional C3b to the alternative pathway C3 convertase renders it

capable of C5 cleavage, and forms the basis for the amplification loop of the alternative pathway. Additionally, C3b generated by the alternative

pathway C3 convertase can attach to target surfaces and bind Bb, forming a C3 convertase that amplifies downstream C proteins locally at the

target surface. Although the activation and amplification of the three pathways differ initially, they commonly cleave C3 into C3a and C3b, finally

resulting in terminal formation of the MAC.

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The cascade is composed of some 30 plasma and cell membrane proteins and provides the first line of defense against microbial or other pathogenic attacks. C-activation can proceed via three pathways (classical, alternative and lectin) which are activated in different ways (Figure 1 [4]). They all converge in one final common pathway (Terminal Pa-thway) at the pivotal protein C3. Mast cells, basophils, platelets and other inflammatory cells are activated liberating inflammatory mediators (histamine, PAF, prostaglandins, etc.), which in turn, set in motion a complex cascade of res-piratory, hemodynamic, and hematological changes, assisting the body’s self-defense. Final endpoint of the terminal pathway is C5b to C9 being assembled into the Terminal Complement Complex (TCC, C5b-9) referred to as Membrane Attack Complex (MAC), which causes direct lysis of invader cells by creating membrane protein channels.

While fulfilling its basic function in innate immunity, the C-system may also have serious adverse effects. The C cascade contains some of the most powerful pro-inflammatory molecules in the body, including most notably the anap-hylatoxins C3a, C4a and C5a [5-8] which are recognized pathogenic factors in a wide spectrum of chronic inflam-matory diseases, including rheumatoid arthritis, glomerulonephritis, atherosclerosis, asthma, and multiple sclerosis [9-14]. Evidence is accumulating that C proteins may also facilitate some basic processes in carcinogenesis, including sustained cellular proliferation, angiogenesis, insensitivity to apoptosis, invasion and metastasis, and escape from immunosurveillance [4].

Monitoring the development of C-activation is necessary to detect C-related diseases in patients and predict possible adverse effects arising from the exposure of foreign material (medical devices, i.v. medicines, etc.) to blood cells and proteins. A list of analytical methods available for the investigation of C-activation is shown in Figure 2.

Figure 2 Analytical methods for Complement diagnostics.

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1.2 HemocompatibilityandC-activationHemocompatibility testing, a major part of biocompatibility testing, is the evaluation of critical interactions of foreign material with blood to explore possible adverse effects arising from the exposure of the foreign material to blood cells and proteins. Because such adverse effects are frequent and may represent serious health risks, testing of new me-dical devices, intravenously applied medicines, blood products and diagnostic agents for hemocompatability is very important. As for the hemocompatibility testing of medical devices, ISO 10993-4 [15] provides a list of recommended assays (Figure 3). This list includes the testing of C-activation, as the above devices often expose large surfaces to blood which provide surface for C deposition and, hence, C-activation.

A new class of i.v. medicines, called “nanomedicines” are of particular importance, as the hemo-incompatibility pro-blems they can cause often arises from C-activation. Nanomedicines include a wide variety of synthetic and semi-synthetic drugs, agents and drug carrier systems (liposomal drugs, micellar systems, polymer-conjugates of proteins, imaging agents, drug carrier nanosystems) whose complexity and size in the nanometer range (5-250 nm) distinguis-hes them from the traditional (Lipinski-type), low molecular weight medicines [16]. Biologicals, or biopharmaceuticals (antibodies, cytokines, protein fragments and synthetic peptides) are also large molecular weight medicines; their size range (8 – 20 nm) and molecular complexity would also qualify them as nanomedicines, however, for practical purposes, only functionally modified (e.g., pegylated or conjugated) biologicals are considered as nanomedicines. A common feature of nanomedicines and biologicals is that -while they are in the frontline of modern pharmacothe-rapy-, they also have a unique toxicity problem: stimulation of the immune system. It is a frequent side effect, leading to an allergy-like syndrome called infusion or hypersensitivity reaction (HSR). It is a major and potentially lethal hemo-incompatibility whose symptoms involve almost all organ systems (Figure 4) [17].

InfusionhypersensitivitycausedbyactivationoftheC-systemInfusion reactions can represent a “real” allergy, one that arises after prior exposure of the reactogenic drug to blood and involves immune memory in the form of specific IgE formation. The other type of infusion reactions involves no IgE and may arise, at least in part, as a consequence of activation of the C-system. Non-IgE-mediated infusion reactions are also referred to as non-IgE-mediated anaphylactoid or pseudoallergic reactions or C-activation-Related Pseudoal-lergy (CARPA) [8]. In fact C-activation can be the sole cause of infusion reactions or may be a contributing factor. Most important differences between IgE- and non-IgE mediated infusion reactions is that true allergies are observed only after repeated exposure of the reactogenic drug to blood and they get stronger upon repeated administration, while pseudoallergies develop at the first exposure and the reaction loses strength with time and repetition (Figure 5) [18].

Figure 3 Hemocompatability assays for medical devices [15].

Figure 4Symptoms of infusion-induced hypersensitivity

reactions [17].

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1.3 RegulatoryguidancesfortestingforC-activationFigure 4 gives an overview of current regulatory guidance papers related to hemocompatibility and testing for C-activation.

Figure 5Distinguishing features and grading of true

and pseudoallergy [18].

Guideline

ISO 10993-4 (2009) [15]

ASTMF2567. StandardPracticefortestingforclassicalpathwayC-activationinserumbysolidmaterials(2010).ASTMF2065.StandardPracticefortestingforalternativepath-wayC-activationinserumbysolidmaterials(2010).

ASTMF1984.StandardPracticefortestingforwholeC-activationinserumbysolidmaterials(2010).U.S. Pharmacopeia <1031>.Thebiocompatibilityofmaterialsusedindrugcontainersandme-dicaldevicesandimplants.European pharmacopeia 6.0.MeasurementofC-activationbyintravenousimmunoglobulinpreparations.

Recommendedassays:C3a,C5a,Bb,iC3b,C4d,SC5b-9,CH50,C3&C5convertase.DescribesproceduresforexposingstandardlotofhumanserumtosolidmaterialsandmeasuringC4depletion.OthervalidatedtestsforspecificComplementcomponentsorsplitproductsmaysubstitutetheassaysdescribedhere.DescribesproceduresforexposingstandardlotofC4-deficientguineapigserumtosolidmaterialsandmeasuringactivationofalternativeC-activation.OthervalidatedtestsforspecificComplementcomponentsorsplitproductsofthealternativepathwaymaysubstitutetheassaysdescribedhere.DescribesproceduresforexposinghumanserumtosolidmaterialsandmeasuringComplementactivityremainingbyclassicalpathwaymediatedlysisofsensitizedredbloodcells.OthervalidatedC-testsmaysubstitutethemethoddescribedhere.USPmonographonhemocompatabilityisunderdevelopment.

DefinedamountoftestmaterialisincubatedwithadefinedamountofguineapigComplementandtheremainingComplementistitrated;theantiComplementaryactivityisexpressedasthepercentageconsumptionofComplementrelativetotheComplementconsideredas100percent.

Comments on C testing

Medical devices

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MedicaldevicesExposure of foreign material to blood cells and proteins frequently has adverse effects and may represent a serious health risk. Therefore, introduction of blood-exposed materials in clinical use is highly regulated. Official guidances by the USA Food and Drug Administration (FDA), European Medicines Agency (EMA), ASTM International (American Society for Testing and Materials) and other regulatory agencies are available, giving recommendations on assays that need to be conducted by the manufacturers to assure the lack of harm caused by blood-exposed foreign material. As for the hemocompatibility testing of medical devices, such as endovascular grafts, shunts, rings, patches, heart valves, balloon pumps, stents, pacemakers, hemopheresis filters, ISO 10993-4 [15] provides a list of recommended assays for testing C-activation (Figure 3). As for the C tests evaluating the hemocompatibility of i.v. medicines and diagnostic agents, the same assays can be applied as recommended for medical devices, with appropriate adaptation for dispersed, water soluble or insoluble molecules [18].

NanomedicinesandbiologicalsDetailed guidance on the assessment of C-mediated hypersensitivity reactions to nanomedicines and biologicals has not been included in any available regulatory document, although they can be lethal and it is possible to measure and predict these reactions as part of the safety evaluation of i.v. applied nanomedicines and biologicals. However, the phenomeon is gaining increasing attention. For example, the EMA released a final paper on data requirements for intravenous liposomal products (Figure 6) [48] in which it is stated that use of in vitro and in vivo immune reactogenicity assays such as complement (and/or macrophage/basophil activation assays) and testing for C-activation-related pseudoallergy (CARPA) in sensitive animal models should be considered to evaluate the extent of potential adverse event” (Figure 4).

Guideline

USDHHS, FDA, CDER.GuidanceforIndustry,Immuno-toxicologyevaluationofinvestigationalnewdrugs(2002).

European Medicines Agency.CommitteeforHumanMedicinalProducts(CHMP),reflectionpaperonthedatarequirementsforintravenousliposomalproductsdevelopedwithreferencetoaninnovatorliposomalproduct.EMA/CHMP/806058/2009/Rev.02,21February2013“[48]USDHHS, FDA, CDER, CBER. GuidanceforIndustry,Immuno-genicityAssessmentforThera-peuticProteinProducts(2013)

Ifsignsofanaphylaxisareobservedinanimalstudies,follow-upstudiesshouldbeconsidered.Biochemicalmarkersofananaphy-lactoidreactioncanbeobservedinnonclinicaltoxicologystudies(e.g.,detectionofserumanaphylacticComplementproductsinanimalsshowingsignsofanaphylaxis)(Szebeni,2001).Carefulevaluationofthesereactionshasresultedinvaluableinformationonbiochemicalmarkersusedinclinicaltrials.UseofinvitroandinvivoimmunereactogenicityassayssuchasComplement(and/ormacrophage/basophilactivationassays)andtestingforC-activation-relatedpseudoallergy(CARPA)insensitiveanimalmodelsshouldbeconsideredtoevaluatetheextentofpotentialadverseevent.

Immunologicallybasedadverseevents,suchasanaphylaxis,cytokinereleasesyndrome,so-called“infusionreactions,”andnonacuteimmunereactionssuchasimmunecomplexdiseasehavecausedsponsorstoterminatethedevelopmentoftherapeuticproteinproducts.NorecommendationsaremadetotestforC-activationinpre-clinicalphase.

Comments on C testing

Intravenous Drugs

Figure 6Overview of current regulatory guidance papers related to hemocompatability and testing for C-activation.

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2 ACTIVATIONOFTHEC-SYSTEMBYARTIFICIAL SURFACES,NANOMEDICINESANDBIOLOGICALS2.1 Mechanismofinfusionhypersensitivitycausedbycomplement activationThe immune cells responsible for true allergy are also responsible for CARPA [17]. These include mast cells, basophils and macrophages that express a group of G-protein coupled receptors which bind anaphylatoxins (i.e., C3a/C5a/C5L2 receptors). Binding of C-activation byproducts, C3a and C5a to these receptors can trigger essentially the same intra-cellular signal cascade that is activated upon the engagement of allergen to membrane-bound IgE, leading to the release of a battery of secondary vasoactive mediators (so called “allergomedins“), including histamine, tryptase, PAF, leukotrienes (LTB2, LTB4, LTC4, LTD4, LTE4, TXA2, PGD2 and TXD4). In the next step allergomedins bind to their respec-tive receptors on endothelial and smooth muscle cells, modifying their function in ways that lead to the symptoms of CARPA, which involve almost all organ systems (Figure 3). Different individuals and tissues may have very different patterns of allergomedin receptors, and these receptors mediate different functions in different tissues. For example, skin and cardiac mast cells respond to different allergomedin stimuli [19]. Increased vascular permeability, a hallmark sign of severe (Grade IV) CARPA may entail the transfer of up to 50 % of intravascular fluid into the extravascular space within 10 minutes [21].

2.2 C-activationbyartificialsurfacesC-activation by artificial surfaces can occur via the alternative and/or classical pathway (Figure 7) [21]. The classical pathway is initiated by activation of the C1 complex, leading to assembly of the classical pathway C3 convertase and generation of C3a, C5a, and the terminal complement complex. The alternative pathway is initiated when water-reacted C3 (which is constantly being formed at a low level) interacts with a receptive surface to form bound C3b. The Bb fragment (the result of cleavage of Factor B by Factor D) binds to bound C3b to form the alternative pathway C3 and C5 convertases capable of generating C3a and C5a. The convertases are short lived and Bb is likely released from the surface. The cleavage of C5 also releases C5b, which can ultimately lead to formation of the terminal complement complex.

Figure 7C-activation by artificial surfaces

(adapted from Gemmell [38]).

2.3 C-activationbynanomedicinesIn the case of non-proteinaceous nanomedicines, which consist of normally non-immunogenic molecules or poly-mers, it is their size (in the 50 – 200 nm range) and surface characteristics (molecular arrays or repetitive elements which are recognized by pattern recognition receptors on immune cells) which make them resemble human patho-genic viruses recognizable by the C-system. Figure 8 illustrates the sizes of drug carrier nanosystems, in relation to the “window of immune vision” , i.e., the size and molecular weight thresholds of cellular and humoral immunity in

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recognizing particles as foreign [17]. The Figure suggests that liposomes and certain carbon nanotubes are within the spotlight of immune surveyance (blue triangle), while fullerenes, micelles, dendrimers, conjugated polymers, poly-meric micelles or vesicles, aptamers, quantum dots, superparamagnetic iron oxide nanoparticles (SPIONs), polymeric micelles, nanocrystals all fall beyond immune recognition, at least in monomeric form. Consequently, the immune reactogenicity of these theoretically stealth particles probably involve interactions with blood elements or other ef-fects which make them recognizable by the immune system.

On the other hand, most pathogenic human virus classes in the 40 – 300 nm range look very much like small unilamel-lar (SUV) and multilamellar (MLV) liposomes, with Doxil, the first FDA-approved nanomedicine, being almost indistin-guishable from HIV-1 [17]. In addition to their similarity to viruses in terms of size, there is another major reason why nanomedicines are recognized by C as foreign; the absence of membrane proteins that protect cells from C attack [17] or surface camouflaging that viruses use for C evasion.

Figure 9Humanized antibodies activate C, after binding to their target.

Figure 8Drug carrier nanosystems and their visibility by the

immune system [17].

2.4 C-activationbybiologicalsAs for the cause of C recognition of biologicals, non-self proteins normally carry numerous antigenic epitopes to which the body responds with antibody production. Once such antibodies are formed, they bind to the foreign pro-teins and activate C, which is one of their roles in immune defense. Different antibody classes have different capability to activate C, with IgM being the most potent. The C binding function of IgGs is well-known in immunology as well as in the industry of biologicals, leading developers to produce “C-stealth” humanized proteins (humanized antibodies) for intravenous use. Nevertheless, once they bind to their target, humanized antibodies also activate C, as C binding is an intrinsic function of certain IgG antibodies, regardless of their origin. This very basic principle is illustrated in Figure 9, reminding of the mechanism by which even totally humanized, immunologically fully matched monoclonal antibodies activate C if they bind to their target epitopes.

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2.5 PrevalenceofC-activation-relatedpseudoallergyIt has been estimated that as many as 30 % of hospitalized patients may have a drug reaction of some type, with the incidence of severe and fatal reactions being approximately 7 % and 0.3 % , respectively [22]. These statistics imply roughly 2 million serious reactions per year with ~ 100,000 fatalities, making adverse drug reactions the fourth to sixth leading cause of death in the USA [22]. Another analysis pointed out that about 25 % of all adverse drug reactions are of allergic nature [39], of which about 77 % is non-IgE-mediated, i.e., represent pseudoallergy [23]. These statistics imply approximately 400,000 severe and 20,000 fatal C-activation-related pseudoallergy events each year for the USA only.

2.6 Marketednanomedicines&biologicalsinducingC-activation-related pseudoallergyThere are a great number of nanomedicines and therapeutic antibodies reported to cause infusion reactions [17] for liposomal-, micellar-, antibody based-, conjugated- and miscellaneous other drugs, respectively. There are also some drug carrier systems whose C activating capabilities have been shown in vitro (e.g., poloxamers, carbon nanotubes) [24-30].

MarketedliposomaldrugsLiposomes or other types of phospholipid assemblies are increasingly used in medicine for targeted or controlled release of various drugs and diagnostic agents. At present, more than a dozen liposomal drugs are in the market, and more in advanced clinical trials. Figure 10 lists those liposomal drugs in the market that have been reported to cause CARPA. The frequency of reactions reported to different drugs varies between 3 % and 45 % [31]. Out of these, the reactions to Doxil have been studied in most detail, in humans as well as animals. C-activation by Doxil, documented in several studies [32,33] have been correlated with clinical symptoms in humans [34] and pigs [25,33,35]. The main conclusions about C-activation by Doxil and other liposomes are summarized in Figure 11.

Figure 10 Marketed Liposomal drugs inducing C-activation-related pseudoallergy.

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Figure 11 Features of C-activation-related pseudo-

allergy induced by liposomes.

Figure 12 Marketed therapeutic monoclonal antibodies inducing C-activation-related pseudoallergy [17].

TherapeuticmonoclonalsMost, if not all of more than 20 mAb-based pharmaceuticals approved to date carry a risk for causing infusion reacti-ons. Figure 12 provides specific information on the symptoms caused by these agents, which are essentially the same as for liposomes, i.e. typical symptoms of infusion reactions (Figure 4). However, there are differences between mAb-induced and nanoparticle-induced CARPA, one of which is that some mAb reaction may start later, mostly after 30 min, compared to the immediate start of symptoms in the case of liposomes and micellar drugs. This difference can most easily be rationalized by the different kinetics of C-activation in the case of nanoparticles and mAbs. Namely, while nanoparticles bind C almost immediately on their surface, mAbs need to undergo steric changes to become C activators (see Figure 9). It is widely known in molecular immunology that when antibodies bind to foreign surfa-ces, steric changes in the hinge region free up the C binding site on the Fc region. With mAb therapy, the relatively slow kinetics of target binding will most likely control the rate of C-activation and other immune consequences of Ab binding.

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Figure 13 Marketed protein conjugates inducing C-activation-related pseudoallergy [17].

OtherdrugsandagentsonthemarketinducingC-activation-relatedpseudoallergyMarketed protein conjugates that have been reported to cause CARPA are listed in Figure 13. Other marketed intra-venously applied pharmaceuticals, including micellar drugs, radio and ultrasound contrast agents, that have been reported to cause CARPA, are described by Szebeni et al. [17].

2.7 RecommendationstoassesstheC-activatingcapabilityofdrugs anddrugcarriersystemsWorldwide numerous nanomedicines, biologicals and other drugs with or without carrier systems are in various sta-ges of development or are being evaluated in preclinical or clinical phase. In Figure 14 we have listed the various categories of drug and drug carrier systems, estimate their C-activating capability and as a consequence the need for C-testing to predict C-mediated infusion hypersensitivity. It is recommended that all drugs and carrier systems are tested.

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LiposomesCarbonNanotubesSpidersilkBeads(Amsilk)

FullerenesMicellesDendrimersPolymerConjugatesPolymericvesiclesAptamersQuantumdotsSPIONsPolymericmicellesNanocrystals

drugdelivery

Yes

ParticlesizeiswithinimmunesurveyanceandmembraneproteinsthatprotectfromCattackareabsent.ManyliposomaldrugscauseC-activation(Fig.10,11).Inmonomericform,particlesizefallsbeyondimmunerecognition.Nevertheless,Cattackcannotbeaprioriexclu-ded,asinteractionswithplasmacomponentsorcellsmayentailC-activa-tion,asdescribedformicelles[17].

C-testinginanimalmodelsandhumanseraforsafetytopredictC-mediatedinfusionhypersensitivity.

Drug, carrier or combination

Theoretical foundation

RecommendationAction Indication C-activation

carrier systems

antibodies

PreventstriggeringofCcascade.

Strategybelievedtoenhanceantitumorcomplement-dependentcytotoxicityBlockCprotectionbytumorcells

BlockT-cellactivation

CancerNon-Hodgkin’sLymphoma,Chroniclymphoidleukemia(CLL)(CD20)

Renalcellcarci-noma,colorectalcancer

T1diabetes,autoimmune

Yes

YesYes(bydesign)

Yes

C-testingfordrugefficacyC-testinginanimalmodelsandhumanseraforsafetyC-testinginanimalmodelsandhumanseraforsafety

Monoclonalstargetingspe-cificsubtypesofFcreceptorsorIL-3.(SuppreMol)Monoclonalantibodiesagainstmembrane-boundproteinsdisplayedbyvarioustumors.E.g.anti-CD20(e.g.Rituxan).

Monoclonalantibodiesagainstmembraneboundcomplementregulatoryproteinsdisplayedbyvarioustumors.E.g.anti-CD46,CD55andCD59).

Monoclonalantibodiesthatareimmunosuppressive(eg.antiCD3)

mAbsmayactivatetheC-system,asCbindingisanintrinsicfunctionofIgGandIgMantibodies.mAbsmayactivatetheC-system,asCbindingisanintrinsicfunctionofIgGantibodies

mAbsmayactivatetheC-system,asCbindingisanintrinsicfunctionofIgGandIgMantibodiesSuchstrategiesignorethepossibilitythattheC-systempromotesneo-plasticdevelopmentandprogressionratherthanexclusivelyretardingit[4].mAbsmayactivatetheC-system,asCbindingisanintrinsicfunctionofIgGantibodies.

various

various

Yes

Particlesizeiswithinimmunesurveyance.

C-testinginanimalmodelsandhumanseraforsafety.

Various

Improvingimmunity

various

Yes

ActivevaccinesactivateC,butonlylocally.PassivevaccinesareantibodiesandthereforeC-activating

C-testinginanimalmodelsandhumanseraforsafety

Various

Interfe-renceatearlystageoftheimmunereactionpreventsthetriggeringofthecascade.

Autoimmunedisease

Unknown

Duetoitssize(40-72kDa),itmayactivateC.

C-testingfordrugefficacyC-testinginanimalmodelsandhumanseraforsafety.

SolubleFcγreceptorscom-petingwithFcγRsexpressedonimmunecells(Suppre-mol)

Variousactions

Various,e.g.cancer

Yes

Yes

Somemembrane-boundpeptidesandnucleicacidsareknowntoactivateC.DNAvectorsarestrongCactivators.Canformchargedrepe-titiveunitsthatbindC1qandactivateC

C-testinginanimalmodelsandhumanseraforsafety

Peptides,DNAvector-basedvaccines,receptoragonists/antagonistsandinhibitors

>2000Dapolymers

Receptors

Small drugs

Small drugs

(Conjugated ) proteins

Figure 14 C-inducing potential of various drugs in development or (pre)clinical evaluation and recommendations for measurement of C-

activation.

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3 METHODSANDASSAYSTOMEASUREACTIVATION OFTHEC-SYSTEM3.1 InvitroC-testingforhemocompatabilityinhumanseraThere are several methods to quantitate C-activation in man (Figure 2). Some are based on the measurement of activation products (activation markers) while others measure consumption of (precursor) C proteins that can be activated. Both approaches can be done by way of immunochemical assays, ELISA, RIA, (rocket) electrophoresis, Western blot or hemolytic assays (which measure red blood cell (RBC) hemolysis caused by the formation of the TCC. C-fragments express unique neo-epitopes that are detectable by specific monoclonal antibodies. Exposure of serum (C source) to a test sample (biomaterial or therapeutic) results in production of these fragments, which can be characterized quantitatively under standard conditions. The steps below (Figure 15) will facilitate investigation of C-activation and identification of the exact underlying route of activation.

Step1 TestforC3cleavageComplement component 3 (C3) is the pivotal protein of the C-system. The two C3 convertases, C2a4b (classical and lectin pathway) and C3bBb (alternative pathway) cleave C3 into C3a and C3b. This step exposes a reactive thioester group within the C3b molecule, which can result in C3b deposition on surfaces. C3a is an important anaphylatoxin and can be detected in the fluid phase. Thus, C3 activation can be tested by detecting surface bound C3b and/or fluid phase C3a.

C3:SpecialcasesIn some cases, C3 cleavage products may be masked in the C-sample. For example, C3a may be adsorbed on to specific biomaterials like polyacrylonitrile (PAN) and C3b may adhere to the surface of reactive materials like cellulose acetate (e.g. hemodialysis circuits). Therefore it is recommended not only to test for both C3a and C3b, but to also test for SC5B-9 and C5a (see step 2).

Figure 15Investigation of C-activation and identification of the exact underlying route of activation.

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Step 2 SC5B and C5a: Confirming resultsC5 cleavage and subsequent formation of Terminal Complement Complexes (TCC) is evidence of activation of the common terminal pathway in response to C-activation. There are several reasons to test for Terminal Pathway activation:1. Biomaterials which do not activate C3 should be confirmed (see C3: special cases).2. To check the extent of C-activation when C3 cleavage (step 1) is positive.

SC5B-9 and C5a are unique fragments of the terminal pathway. After activation of C3, the two C5-convertases, C4b2aC3b (Classical and Lectin pathways) and C3bBbC3b (Alternative pathway) cleave C5 into C5a (the most potent anaphylatoxin) and C5b. C5b binds Component C6 and C7, which in turn bind C8 and C9, thereby forming the lytic C5b-9 complex. The soluble, non-lytic complex, SC5B-9, is also formed and can be detected fluid phase together with C5a.

Step 3 Understanding the process: test for C4d and BbIf prior studies are positive and if the method of activation needs to be defined (for example surface characterization) testing can be done for C4d and Bb. C4d is a fragment of C4b formed through activation of the Classical and Lectin pathway after cleavage of C4. C4b is deposited on a reactive surface; C4d may be released by action of complement regulatory proteins in the serum. Bb, a fragment of the alternative pathway, can be detected when Factor B is cleaved into Bb andBa. Bb is indirectly bound to a surface via binding to C3b. Testing for these fragments will provide insight into chemicalmodifications or other bioengineered approaches for circumventing the problem.

Experimental design of in vitro C-testing for hemocompatabilityFor a proper design of experiments, there are three elements to consider:1. Complement source2. Controls3. Standardization

1 Complement sourceHuman serum is the best Complement source to test. However, the serum has to be fresh, frozen serum with low exis-ting levels of activation fragments. EDTA, low dose Heparin and recalcified plasma can be used for C testing, citrated plasma should be avoided. Quidel provides human serum, which is standardized and collected specially to preserve active C (product No A113 5.0 ml or A112 2.5 ml). This human serum has been tested for infectious diseases, for C-activation markers, and for the CH50. After testing, specimen stabilizer (Quidel A9576) must be added to the plas-ma/blood samples to prevent any further C- activation.

2 ControlsAn adequate number of controls and reference materials must be used for the respective experimental test system. Most kits include internal controls and standards. C-activation takes place on the boundary layer of the blood-material contact. Therefore, the relation between the size of the test surface and the volume used (serum, plasma, blood) is an absolutely essential parameter for the standardization of the test system. In addition to the controls provided by the kit, the following experimental controls (CONTROL) must be considered:• CONTROL 1 Background: Serum source only Measures the impact of the system alone on Complement• CONTROL 2 Positive control: Serum + activator Confirms C-activity and demonstrates strong positive signal• CONTROL 3 Negative control: Serum + inhibitor (EDTA) Negative controls shows any direct interference in the assay• CONTROL 4 Buffer control (NEG): Buffer which contains the therapeutics Confirms buffer does not interfere

Activator – Since the complete sample handling is never entirely possible without artificial activations, the back-ground activation can be quantified by using control samples and then compared to the test samples. Based on the experimental condition, one can decide which activator to use to function as a positive control:

TECOmedical 17

• HAGG: Heat Aggregated Gamma globulin (Quidel A114). Activator of the classical pathway – C4d, however also C3a will be activated (application therapeutic antibodies, passive gamma globulin therapies).• Zymosan: yeast lipopolysachararide. Activator of the alternative pathway - iC3b – C3a - C5a - SC5b-9 – Bb (application antisense oligonucleotides, charged biomaterial surfaces.)• CVF: Cobra Venom Factor (Quidel A600). Alternative pathway: iC3b – C3a - C5a - SC5b-9 – Bb. Acts directly on C3 and is an extremely potent C-activator to fully activate the C-system.• Certain biomaterials (e.g. Cuprophan™). A membrane that can activate. Maybe used as activator for material comparison - should only be used if the surface area of the test material is similar.Note that for inhibition experiments, titrating an activator is challenging to find the right balance between activation and inhibition using CVF. In this case, HAGG is a better option.Temperature has an impact on C-activation. So to control for temperature and for related activators created by thesystem (e.g. “nephritic” factors), keep one sample of each control (CONT 1-4) on ice.

3 Material or therapeuticIt is important to standardize for size & surface area for volume/concentration of therapeutic/biomaterial. Please refer to figures 16a and 16b for guidelines.

Figure 16a Biomaterial Testing Decision Tree.

18

Figure 16b Experimental design flow charts.

Protocols for in vitro c-testing for hemocompatability

PROTOCOLSComplement source• Neat normal human Complement serum (A113)• EDTA, EDTA-treated and citrated plasma are not suitable

Therapeutics• Neat and dilutions 1:2 to 1:32

Controls• Activator HAGG (A114) for postitive control 2: 10 μl for 1 ml (normal human Complement serum) = Complete activation after 30 Minutes at 37 °C• Activator CVF (A600) for positive control 2: App. 8 Units for 1 ml (normal human Complement serum) = Complete activation after 30 – 90 Minutes at 37 °C• Activator Zymosan for positive control 2: 1 mg Zymosan for 1 ml (normal human Complement serum) = Complete activation after 60 Minutes at 37°C• Inhibitor for negative Control 3: • EDTA 10 mmol final concentration

One aliquot of all controls on ice = Background activationIncubation• At 37 °C, Move alliquots to ice every 15 minutes and dilute 1:2 with sample stabilizer to block Complement reaction

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Timepoints• 15 – 30 – 45 – 60 – 120 – 240 minutes

Assay• Dilute aliquots according to protocol – Sample is already diluted 1:2 with stabilizer• e.g. dilution protocol 1:100 = dilute Aliquot 1:50• Calculate concentration, include dilution factor• Run controls included in the Kit• Run additional external controls A115

RefiningtheProtocol• Determining the proper therapeutic concentration• Impact on activator titration• Optimizing the incubation parameters – Time, temperature• Optimizing dilution profiles for activators and non activators –Impact of assay type• Additional Controls

INTERPRETINGRESULTS–CONTROLS

CONTROL 1 Background: Serum source only, measures the impact of the system alone on Complement• Control 1 – on ice = Value as QC Sheet• Control 1 – 37 °C = Background activation due to Temperature = 2 – 3 times higher value as QC Sheet

CONTROL 2 Positive control: Serum + activator in the experimental protocol, Confirms Complement activity and demonstrates strong positive signal• Control 2 – on ice = Background = app. value as QC Cheet• Control 2 – 37 °C = Total activation = app. 10 times higher value as QC Sheet

CONTROL 3 Negative control: Serum + inhibitor (EDTA), Negative controls shows any direct interference in the assay• Control 3 – on ice = Value as QC Sheet• Control 3 – 37 °C = Should be negative/low – Value as QC Sheet

CONTROL 4 Buffer control (NEG): Buffer which contains the therapeutics, Confirms buffer (therapeutics) does not interfere• Control 4 – on ice = app. Value as QC Sheet• Control 4 – 37 °C = Should be app. value as QC Sheet. Influence of buffer only

ProtocolsforthesetupofassaysforthemeasurementofC-activationofdifferentbiomaterialsareavaila-blefromTecoMedical.

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3.2 TestmodelsformedicaldevicesSeveral in vitro or ex vivo test models may be used for preclinical assessment of new components (artificial devices) regarding their clinical usability for blood contact. The quality of the blood components used (whole blood, platelet rich plasma, plasma, serum) and the choice of suitable anticoagulants (Citrate, EDTA, heparin, hirudin) are of decisiveimportance for all these models. Therefore, the following elimination criteria must be observed when selecting test individuals: no smokers and no intake of medication 14 days prior to blood donation (especially haemostasis- influen-cing pharmaceuticals such as acetylsalicylic acid (ASA), oral contraceptives, non-steroidal antiphlogistics, etc). Equally important are gentle blood drawing and rapid processing. Dynamic models using fresh human whole blood should be used for testing complete hemocompatibility, including C-activation. Blood, plasma, serum extraction and hepa-rinization to test heart-lung machines, etc., a large amount of blood is needed. For other tests a defined serum that has been prepared and tested accordingly should be used. If no fresh whole blood is available, frozen NHS (normal human serum) should be used (e.g. A113 or A112). This serum should be specially harvested to preserve the C factors and tested for infectious diseases, C-activation and CH50. EDTA plasma or recalcificated plasma are not suitable.

Note: NHS must be placed on ice at all times prior to activation.

Heparin (low dose) or hirudin are to be used for plasma or whole blood. After testing, EDTA must be added to the plasma/blood samples to rule out any further in vitro activation.

Use of fresh blood

Figure 17 Chandler loop model for testing new coatings stents etc. (http://chandlerloop.com)

Dynamic models with fresh whole bloodChandler loop modelIn an in vitro “closed loop” model (modified Chandler loop), 30 ml heparinized (1 IE/ml Heparin) fresh human blood is recirculated (20 U/min, 37 °C) in circular closed tube sections. This test arrangement can be employed through various modifications; for testing new coatings, new pharmaceuticals (anticoagulants, inhibitors, etc.), stents, etc. This modelcan be used to quantify activation products directly via traditional ELISAs (C3a, C5a, SC5b-9) and the adsorbed plasma proteins can be detected directly on the surface using a newly developed modi-fied ELISA technique (e.g. C3a). For this purpose, the tube sections to be examined will be rinsed directly after completion of the Chandler circulation, fixed (4 % para-formaldehyde in PBS [w/v], pH 7.4), cut into 2 cm long pieces and frozen. The tube sections will then be closed on one side and used directly as large “ELISA well”. Using appropriate antibodies, different plasma proteins can be detected directly on the test surface (e.g. C3a). Using incubation times of different length in the Chandler loop model, the kinetics of the adsorption behavior of plasma proteins can be determined. In addition, the soluble C-activation markersC3a, C5a, SC5b-9, etc. can be measured in the systemic blood.

perfecthealth,noinfectiousdiseasesnoknownfactordeficiencynocoagulationdisorder

nomedication(especiallyhaemostasis-influencingpharmaceuticals)

skindisinfectionbriefcongestiongentlepunctureofthelargecubitalveinonthearmbyaninjectioncannula(e.g.VenisystemsButterfly-M9)useofpre-preparedneutralmonovettes(Heparinlowdose,Hirudin,etc.)rejectionofthefirst3mlblood

Important criteria for blood collection: Important criteria for blood donors:

TECOmedical 21

Heart-lung machine modelIn this rather complex model, fresh human blood is recirculated in a short-circuited system, using oxygenation, defined tube length and a build-up of an arterial counter pressure (i.e. the same conditions as for a patient during cardiopulmonary bypass surgery). The activation potential of foreign surfaces with respect to the different parame-ters of haemostaseological systems can be checked, without endogenous counterbalances trying to compensate for these effects [36]. Concentrations of C3a, C5a, SC5b-9, etc. can be determined from the blood drawn at different times and thus the kinetics of the C

3.3 FlowcytometricexaminationstoevaluatebindingofC-proteinsto artificialsurfacesAssessment of C-activation by artificial surfaces can be performed by using ELISAS to measure the products of C- activation. However, measurement in the fluid phase only could be misleading as was the case for acrylonitrile hol-low-fiber and plate dialyzers [37] where C3a andC5a were surface adsorbed which explained for the lack of incre-ase of plasma C3a and C5a during hemodialysis. Using artificial surfaces in the form of microspheres, Gemmell [38] demonstrated that there are significant levels of surface bound Bb andC4d, C3d, and iC3b and that the membrane attack complex (SC5b-9) was surface bound on all surfaces with its concentration increasing to levels as high as 0.5 mg/cm2. Further, the surface bound represented a substantial percentage of the total generated.

After incubation of the microparticles in serum, C proteins adsorbed on the surface were marked using fluorescent dyes. The fluorescence intensity, in addition to other parameters, could be detected in the flow cytometer. Compared with standardized fluorescent particles, Gemmell obtained information on the absolute amount of adherent C proteins and their composition after various incubation times.

3.4 Personalizedmedicine:testingforhypersensitivityreactionsto anticancerdrugsAnticancer drugs that contain nanoparticles (liposomes, micelles), as well as therapeutical monoclonal antibodies (mAbs) often cause hypersensitivity reactions (HSRs) as a consequence of their Complement (C) activating effect. Liposomes (e.g., Doxil), micellar drugs (e.g., Taxol and Taxotere), and monoclonal antibodies (mAbs), such as rituximab (Rituxan, MabThera) are known to induce hypersensitivity reactions (HSRs) in a relatively high percentage (10-80 %) of patients [15, 39]. A study by Szebeni et al. suggested a relation between C-activation caused by these reactogenic anticancer drugs in vitro (in sera of cancer patients), and the HSRs they cause in vivo upon infusion. Study conclusions were following:1. Measurement of SC5b-9 in human serum or whole blood in vitro following incubation with reactogenic drugs may serve as a predictive test for HSRs. Details and validation of the assays need to be elaborated for each drug.2. In case of Rituxan and other drugs, whose therapeutic targets are blood cells, whole blood C assays may better mimic the in vivo C response than serum assays. 3. FACS analysis of the deposition and subsequent proteolysis of C3b on lymphocytes provides direct evidence for, and a measure of cell-associated C-activation by Rituxan. 4. On the basis of the known role of C-activation in the rise of specific immunity, in vitro C tests might also be used to predict the immunogenicity of Rituxan and other protein therapeutics.

3.5 TestingC-activationinanimalmodelsTo quantify the C activating capability of test materials (e.g. liposomes, other nanoparticles, antibodies, etc), experi-ments can be performed using animal models. For example, the EMA recommends to use immune reactogenicity assays such as C assays to test intravenous liposomal products for C-activation-related pseudoallergy (CARPA) in sen-sitive animal models. Rat, mouse and porcine C5a can be measured by specific commercially available ELISA kits. However, the use of C5a as a marker for in vivo C-activation is problematic because of the rapid clearance of C5a from blood by C5aR-carrying cells (WBC, platelets, macrophages, etc), while specific ELISAs for the measurement of stable byproducts are not available. The classical C hemolytic (CH50) assay can be used to measure total C-activation through the ability of the C-system to lyse sensitized sheep red blood cells. The CH50 reflects the ability of the C in a sample to activate; the more activation, the higher the CH50 signal. However this signal is not a direct measurement of C-activation. Moreover, the technique

22

is very sensitive to serum matrix irregularities, requires normalization of the serum, shows high inter-lab and inter-assay variation and is very insensitive.

CxH50The traditional hemolytic assay can be made more specific assay by focusing on a single protein. This assay, called CxH50, detects the functional activity of a specific Complement component (for example,. C3H50 for C3 function).The method utilizes specific Complement depleted sera as a source of C proteins. A test specimen can reconstitute the specifically depleted protein in the test matrix, thereby restoring the C activity. Thus, all lysis of the erythrocyte (minus background lysis of depleted serum and erythrocytes) is a function of the specific C protein from the test specimen. Studies have shown that non-human serum can serve to replace the depleted protein and reconstitute Complement function. The CxH50 is available in many test systems such as:• C2 (C2H50)• C3 (C3H50)• C4 (C4H50)• C5 (C5H50)• C1q (C1qH50)

Protocolsfortheset-upofCxH50assaysareavailablefromTecoMedical.

Species independent c-testing for hemocompatability in animalsUnfortunately, most of the commercially available ELISAs are human specific ([40] leaving an expanding need for animal testing of C-activation largely unmet. To fill this gap Tecomedical and Quidel have recently introduced a pan-specific C3 (PS-C3) converter kit (MicroVue Pan-Specific C3 kit) represents a novel approach of animal-specific C ELISAs (patent pending). The C3 Complement Matrix (CCM) and the C3 Converter Reagent (CCR) in the kit convert the activity of C3 in the animal specimen to human SC5b-9 that is detectable with the traditional human SC5b-9 ELISA kit. Thus the method allows sensitive and quantitative measurement of C3 in animal blood, plasma or serum, which, to the extent C3 had been consumed prior to the assay, also provides a measure of prior C-activation. The MicroVue Pan-Specific C3 kit expands the methodical arsenal of C analysis in animals, enabling, among many applications, preclinical immune toxicology testing of C-mediated (pseudoallergic) adverse drug effects. The PS-C3 method is a three-step procedure. In the first step animals are treated – or their sera or plasma incubated - with the test drugs or device to explore possible C-activation. Samples are collected at appropriate times for the second step:conversion of animal C3 to human SC5b-9. C3 conversion is performed as specified in the MicroVue Pan-Specific C3 kit. In the third step SC5b-9 is measured using the human SC5b-9 ELISA kit. Figure 18 gives an example of results obtained with this new approach

Figure 18 C3 consumption in rats measured by the

Pan-Specific C3 kit. Rats were injected

with cobra venom factor and zymo-

san, known activators of the C-system

in animals. The graph shows the effects

of these agents, triggering major C3

consumption with kinetics consistent

with the C activating effect of these

agents in vivo.

More information can be found in the Tecomedical review: Species Independent Measurement ofC-activationinAnimals(August2013).

TECOmedical 23

The porcine modelPigs are particularly sensitive for nanoparticle induced cardiopulmonary distress and cutaneous changes, a feature that may be related to the presence of pulmonary intravascular macrophages (PIM cells) in this, as well as in other even-toed (hoofed) animals (Artiodactyla), including sheep and goats [41].

Protocol[17]• Animals. Pigs (15– 40 kg) of both sexes are sedated within intramuscular ketamine (500 mg) and anesthetized with 2 % isoflurane, using an anesthesia machine, or with intravenous nembutal (30 mg/kg).• A pulmonary artery catheter is advanced via the right internal jugular vein through the right atrium into the pulmonary artery to measure pulmonary artery pressure (PAP).• A catheter is advanced in the femoral artery to measure systemic arterial pressure (SAP).

TreatmentLiposomes or other test materials are diluted in phosphate buffered saline (PBS) and injected into the pulmonary artery as a bolus, via the pulmonary arterial catheter. The bolus is flushed into the circulation with 5 – 10 mL PBS.

Endpoints

Note:• The cardiopulmonary symptoms of pig CARPA mimics the most severe, grade IV or V allergic reactions in humans(Figure 4) which involves hypotensive shock with cardiac arrhythmias, ventricular fibrillation and cardiac arrest, as well as the skin symptoms (flushing and rash). Thus, the model is appropriate for studying the mechanism and conditions of severe HSRs in hypersensitive man.• The individual variation of hemodynamic reaction in pigs is relatively small.• The pig CARPA model is uniquely capable to screen out immune reactive nanoparticles that may cause severe reactions in hypersensitive individuals.• Although its oversensitivity implies false-positivity in terms of predicting reactions in normal humans, it needs to be remembered that infusion reactions cause real harm on lying hypersensitive individuals, who are also not “normal ” in their response to nanoparticles.

CASscore.

ClassicalC-hemo-lyticassay(CH50).

PorcineC5aassay.

MicroVuePan-SpecificC3kit.

CAS0,noresponse;CAS1,minimal;CAS2,mild;CAS3,moderate;CAS4,severe;CAS5,lethal.Totalcomplementactivityisabnor-malifC-systemisactivated.

%increaseismea-sureofactivationofC-activation.

%consumptionofC3providesmea-sureofC-activation.

Thesymptomsspe-cifyingdifferentCASvaluesaredescribedinSzebenietal.[25].

Assayisinsensitive,cumbersomeandshowshighinter-labandinter-assayvariation.UseofC5aasamarkerisproblematicbecauseoftherapidclearanceofC5afrombloodbyC5aR-carryingcells(WBC,platelets,macropha-ges,etc).Species-independentsensitivemethod.

Measureoftheseverityofcardiacelectric,circulatory(systemicandpulmonary),andskinchanges.

Traditionalmethodfordeterminationoffunctionalcomplementactivity.Measurestheabilityofthetestsampletolyse50%ofastandardizedsuspensionofsheeperythrocytescoatedwithanti-erythrocyteantibody.Boththeclassicactivationandtheterminalcomplementcomponentsaremeasuredinthisreaction.MeasureincreaseofactivationfragmentC5aasmeasureofformationofTerminalComplementComplex,indicatingactivationofthecommonterminalpathway.

SensitiveandquantitativemeasurementofC3(%consumptionofC3).

endpoint score commentmeasurement

24

Other animal modelsC-activation-related pseudoallergy can be induced by i.v. injection of liposomes and other nanoparticles in many other species, with sensitivity and symptoms differing from those seen in pigs [27y]. The sensitivity to liposomal CARPA usually decreases in the following order: pig, dog, human (hypersensitive), rabbit, sheep, rat and mouse. The methods of detection and endpoints are similar or the same as described above, with obvious adaptations to the size differences among different animals. Figure 19 tabulates some characteristic features of CARPA in pigs, dogs, goats, rats, rabbits and mice.

Figure 19 Liposome-induced CARPA in different

species: Dose dependence and symptoms.

Up and down arrows indicate the rise or fall

of the parameter specified,while the size of

arrows is proportionate with the strength of

response [42-45].

Figure 21Scoring of Complement-Dependent Cytotoxicity reactions [47].

3.6 Testingofmonoclonalantibodiesfor ComplementdependentcytotoxicityAntibodies against defined cell surface antigens are being widely explored as the-rapies for cancer and other disease. Complement-Dependent Cytotoxicity (CDC) is a mechanism of killing cells in which the C protein C1q binds an antibody trig-gering the C cascade, leading to the formation of the membrane attack complex (MAC) at the surface of the target cell. The activation of the Classical pathway results in lysis of the target cell (Figure 20).

Testing for CDC potential of monoclonal antibodies is performed by plating target cells and treating them with serial dilutions of the antibody. Normal Human Serum Complement (Quidel P/N A112 or A113) is also added to the cells as a qualified source of intact C. Following incubation, cell lysis is measured and used to calculate percent toxicity [46].

Protocol• Plate target cells (Raji, WIL2-2, etc.), 50 μL of 10 X 106 cells/mL density per well• Prepare serial 3 –fold dilutions of test antibody, Add 50 μL/well• Dilute Normal Human Serum Complement 1:4, Add 50 μL/well• Incubate 2 hours at 37°C (7.5% CO2 incubator)• Measure cell lysis (using Alamar Blue, 51Cr release, etc)

EndpointsCalculate toxicity based on percent lysis (Figure 21)

Figure 20 Complement-Dependent

Cytotoxicity.

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4 REFERENCES[1] Liszewicki MK and Atkinson JP. The C-system In: Fundamental Immunology. Paul, WE (ed.) (1993).[2] Botto M et al. Complement in human diseases: Lessons from Complement deficiencies Mol. Immunol. 46(4):2774-2783 (2009).[3] Wallis R et al. Paths reunited: initiation of the classical and lectin pathways of C-activation Immunobiol. 215(1):1-11 (2010).[4] Rutkowski MJ, Sughrue ME, Kane AJ, et al. Cancer and the Complement Cascade Mol Cancer Res 8:1453-1465 (2010).[5] Guo RF, Ward PA. Role of C5a in inflammatory responses Annu Rev Immunol 23:821–52 (2005).[6] Kohl J. Anaphylatoxins and infectious and non-infectious inflammatory diseases Mol Immunol 38:175–87 (2001).[7] Gennaro R et al. C5a fragment of bovine Complement. Purification, bioassays, amino-acid sequence and other structural studies Eur. J. Biochem. 155 (1): 77–86 (1986).[8] Rosa PA et al. Sequence of the gene for murine Complement component C4 J. Biol. Chem. 264 (28): 16565–16572 (1989).[9] Linton SM et al. C-activation and inhibition in experimental models of arthritis Mol Immunol 36:905–14 (1999).[10] Welch TR. Complement in glomerulonephritis Nat Genet 31:333–4 (2002).[11] Niculescu F, Rus H. The role of C-activation in atherosclerosis Immunol Res 30:73–80 (2004).[12] Hawlisch H et al. The anaphylatoxins bridge innate and adaptive immune responses in allergic asthma Mol Immunol 41:123–31 (2004).[13] Humbles AA et al. A role for the C3a anaphylatoxin receptor in the effector phase of asthma Nature 406: 998–1001 (2000).[14] Storch MK et al. Multiple sclerosis: in situ evidence for antibody- and Complement-mediated demyelination Ann Neurol 43:465–71 (1998).[15] Biological evaluation of medical devices Part 4: selection of tests for interaction with blood, ANSI/AAMI/ISO 10993-4:2002/(R) 10 March 2009, 2009.[16 Lipinski CA, Lombardo F, Dominy BW, Feeney PJ. Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings Adv Drug Deliver Rev 1997;23:3 – 25.[17] János Szebeni Hemocompatibility testing for nanomedicines and biologicals: predictive assays for Complement mediated infusion reactions. Eur. J. Nanomed. 2012;1(1):33–53. [18] Lenz HJ. Management and preparedness for infusion and hypersensitivity reactions Oncologist 2007;12:601 – 9.

[19] Genovese A, Stellato C, Patella V, Lamparter-Schummert B, de Crescenzo G, Adt M, et al. Contrast media are incomplete secretagogues acting on human basophils and mast cells isolated from heart and lung, but not skin tissue Eur J Radiol 1994;18(Suppl 1):S61 – 6.[20] Lieberman P. Anaphylactoid reactions to radiocontrastmateria Clin Rev Allergy 1991;9:319 – 38.[21] Gemmell CH. A flow cytometric immunoassay to quantify adsorption of C-activation products (iC3b, C3d, SC5b-9) on artificial surfaces J Biomed Mater Res. 1997 Dec 15;37(4):474-80.[22] Lazarou J, Pomeranz BH, Corey PN. Incidence of adverse drug reactions in hospitalized patients. A meta-analysis of prospective studies J Am Med Assoc 1998;279:1200 – 5.[23] Demoly P, Kropf R, Bircher A, Pichler WJ. Drug hypersensitivity: questionnaire EAACI interest group on drug hypersensitivity. Allergy 1999;54:999 – 1003.[24] Szebeni J. C-activation-related pseudoallergy:a new class of drug-induced immune toxicity Toxicology 2005;216:106 – 21.[25] Szebeni J, Baranyi L, S á vay S, Bodó M, Milosevits J, Alving CR, et al. C-activation-related cardiac anaphylaxis in pigs: role of C5a anaphylatoxin and adenosine in liposome induced abnormalities in ECG and heart function Am J Physiol 2006;290:H1050 – 8.[26] Moghimi SM, Hamad I, Andresen TL, Jörgensen K, Szebeni J. Methylation of the phosphate oxygen moiety of phospholi pidmethoxy(polyethylene glycol) conjugate prevents PEGylated liposome-mediated C-activation and anaphylatoxin production FASEB J 2006;20:2591 – 3.[27] Szebeni J, Bunger R, Baranyi L, Bedocs P, Toth M, Rosivall L, et al. Animal models of Complement-mediated hypersensitivity reactions to liposomes and other lipid-based nanoparticles J Liposome Res 2007;17:107 – 17.[28] Szebeni J, Moghimi SM. Liposome triggering of innate immune responses: a perspective on benefits and adverse reactions J Liposome Res 2009;19:85 – 90.[29] Johnson RA, Simmons KT, Fast JP, Schroeder CA, Pearce RA, Albrecht RM, et al. Histamine release associated with intravenous delivery of a fluorocarbon-based sevoflurane emulsion in canines J Pharm Sci 2011;100:2685 – 92.[31] Cavadas M. Pathogen-mimetic stealth nanocarriers for drug delivery: a future possibility Nanomedicine: nanotechnology, Biology and Medicine 2011;7:730–43.[32] Szebeni J. The interaction of liposomes with the C-system Crit Rev Ther Drug Carrier Syst 1998;15:57 – 88.[30] Szebeni J, Baranyi B, Savay S, Lutz LU, Jelezarova E, Bunger R, et al. The role of C-activation in hypersensitivity to pegylated liposomal doxorubicin (Doxil ®) J Liposome Res 2000;10:347 – 61.[33] Szebeni J, Bedöcs P, Rozsnyay Z, Weiszhár Z, Urbanics R, Rosivall L, et al. Liposome-induced C-activation and related cardiopulmonary distress in pigs: factors promoting reactogenicity of Doxil and AmBisome. Nanomedicine NBM 2012;8:176 – 84.

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[34] Chanan-Khan A, Szebeni J, Savay S, Liebes L, Rafique NM, Alving CR, et al. C-activation following first exposure to pegylated liposomal doxorubicin (Doxil): possible role in hypersensitivity reactions Ann Oncol 2003;14: 1430 – 7.[35] Szebeni J, Fontana JL, Wassef NM, Mongan PD, Morse DS, Dobbins DE, et al. Hemodynamic changes induced by liposomes and liposome-encapsulated hemoglobin in pigs: a model for pseudo-allergic cardiopulmonary reactions to liposomes. Role of Complement and inhibition by soluble CR1 and anti-C5a antibody Circulation 1999;99:2302 – 9.[36] Colman RW, Scott CF, Schmaier AH, Wachtfogel YT, Pixley RA, Edmunds LH, Jr. (1987) Initiation of blood coagulation at artificial surfaces In: Ann.N.Y.Acad.Sci. Leonard,E.F.; Turrito,V.T.; Vroman,L.; Edtrs.p. 253-67[37] A. Kandus, R. Ponikvar, J. Drinovec, S. Kladnik, and P. Ivanovich. Anaphylatoxins C3a and C5a adsorption on acrylonitrile membrane of hollow-fiber and plate dialyzer, an in vivo study Int. J. Artif. Organs, 13, 176–180 (1990).[38] Gemmell, C.H. (1997) A flow cytometric immunoassay to quantify adsorption of C-activation products (iC3b, C3d, SC5b-9) on artificial surfaces J Biomed Mater Res, 37, 474-48[39] Harker LA, Ratner BD, Didisheim P. Cardiovascular biomaterials and biocompatibility CardiovascPathol 993;2(Suppl):1S –224S.[40] Quidel. Assays for Complement Antigens http://quidelcom/products/product_ listphp?cat=10&by=product_family&group=2. (2013).[41] Winkler GC. Pulmonary intravascular macrophages in domestic animal species: review of structural and functional properties Am J Anat 1988;181:217 – 34.[42] Szebeni J, Bunger R, Baranyi L, Bedocs P, Toth M, Rosivall L, et al. Animal models of Complement-mediated hypersensitivity reactions to liposomes and other lipid-based nanoparticles J liposome Res 2007;17:107 – 17.[43] Hugli TE, Morgan EL. Mechanisms of leukocyte regulation by Complement- derived factors Contemp Top Immunobiol 1984;14:109 – 53.[44] Hugli TE. Structure and function of anaphylatoxins Spring SeminImmunopathol 1984;7:193 – 219.[45] Marceau F, Lundberg C, Hugli TE. Effects of anaphylatoxins on circulation Immunopharmacol 1987;14:67 – 84.[46] European Pharmocopoeia 6.0. 2.6.17: Test for antiComplementary activity of immunoglobulin (2008).19 International[47] Lebeck, KL and Garavoy, MR. Histocompatibility testing and organ sharing In: Immunogenetics Laboratory Manual, 3rd Edition, Chapter 8 (1997).[48] Committee for Human Medicinal Products (CHMP), Reflection paper on the data requirements for intravenous liposomal products developed with reference to an innovator liposomal product. EMA/CHMP/806058/2009/Rev. 02, 21 February 2013 “

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5.2 TechnicalinformationonComplementelisa’s

CIC-C1qMicroVue™ Quidel®QUANTIFICATIONOFCIRCULATINGIMMUNECOMPLEXES

Cat. No. A001 Tests 96 Method ELISA Range 10 - 30 µg Eq/ml Sensitivity 1.0 µg Eq/ml Incubation time 2 hours Sample volume 10 µl (dilute 1:50) Sample type Serum and plasma

Sample preparation Specimens should be collected aseptically and prepared using standard techniques or clinical laboratory testing. Do not heat-inactivate the specimens. Sample may be stored at 2-8 ºC for up to 7 days. For longer periods store below -20 ºC.

Reference values One hundred six (106) sera were collected from normal, asymptomatic subjects. The average CIC concentration was 2.1 µg Eq/mL (S.D.=1.9)

Specifity Complement-CICs are bound to immobilized to C1q-Protein Species Human, African green monkey, Cynomolgus Macaque, Rhesus macaque, Baboon

Intended use The CIC-C1q Assay is designed for the detection of circulating immune complexes (CIC) in human serum or plasma. CIC have been measured in a variety of conditions: infections, autoimmune disorders, trauma and neoplastic proliferative diseases. In addition the measurement of CIC can be important for the evaluation of certain forms of rheumatoid arthritis and for monitoring the effectiveness of therapy. CIC Control Cat. No. A013 1 Set (2 levels)

5 COMPLEMENTELISA’S5.1 OverviewofavailableComplementelisa’s

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Cat. No.Tests

MethodRange

SensitivityIncubation timeSample volume

Sample type Sample preparation

Reference values

Specifity

Species

Intended use

A002 96ELISA5 - 48 µg Eq/mlS4 µg Eq/ml2 hours10 µl (dilute 1:50)Serum and plasma

All specimens should be collected aseptically. Do not heat-inactivate the specimens. Samples may be stored on ice for up to 6 hours. For longer periods store below -70 ºC.

Normal ≤ 15 µg Eq/mlAbnormal ≥ 20 µg Eq/ml

Immobilized monoclonal anti human C3 fragments capture C3d containing immunecomplexes.

Human

The CIC-RCR Assay is designed for the detection of circulating immune complexes (CIC) in human serum or plasma. CIC have been measured in a variety of conditions: infections, au-toimmune disorders, trauma and neoplastic proliferative diseases. In addition the measure-ment of CIC can be important for the evaluation of certain forms of rheumatoid arthritis and for monitoring the effectiveness of therapy.

CIC-C3dMicroVue™ Quidel®Raji-Cell-ReplacementQUANTIFICATIONOFCIRCULATINGIMMUNECOMPLEXESWITHC3ACTIVATIONFRAGMENTS

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Cat. No.Tests

MethodRange

Incubation timeSample volume

Sample type

Reference values

Specifity

Species

Intended use

A037 96ELISAConcentrations of Functional C1-INH are expressed as Mean Percentage. Range 35 - 100 %.2 hours10 µl (dilute 1:101)

Serum and EDTA plasmaSample preparation: Specimens should be collected aseptically and prepared. EDTA plasma sample may be held at room temperature (15-30 ºC) for up to 24 hours. Serum sample should not be stored at room temperature for longer than 6 hours. If exceeded, the plasma or se-rum must be stored frozen (-20 ºC or below). Avoid freezing and thawing of the sample.

Concentrations ≥ 68 % Mean Normal is considered normal.

C1-INH-Reactant, binds specific to functional active C1-INH.

Human, Rhesus macaque, Baboon

The C1-Inhibitor assay measures the amount of functional C1 inhibitor protein (C1-INH) in human plasma or serum. This protease inhibitor has enzyme regulating functions. A defi-ciency of functionally active C1-INH may lead to life-threatening angioedema. Two major forms of C1-INH deficiency have been reported: the congenital form, termed hereditary an-gioedema (HAE), and the acquired form, which is associated with a variety of diseases inclu-ding lymphoid malignancies. Hereditary angioedema is characterized by transient but recur-rent attacks of nonpruritic swelling of various tissues throughout the body.

C1-InhibitorPlusMicroVue™ Quidel®FUNCTIONALC1INHIBITORPROTEINS

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Cat. No.Tests

MethodRange

Incubation timeSample volume

Sample type Sample preparation

Specifity

Species

Intended use

A006 96ELISA0.2 - 2.0 µg/ml1.5 hours100 µl (after dilution 1:25 – 1:200)Serum and EDTA-plasma

The proper collection and storage of specimens is essential, since C3 is highly susceptible to proteolysis and hydrolysis. Serum or EDTA plasma specimens should be collected aseptically using standard techniques. They should be tested immediately or stored at 4 ºC or on ice until assayed. This should not exceed 4 hours. For long-term storage, freeze at -70 ºC within 2 hours after collection.

Anti-iC3b monoclonal anti body specifically binds iC3B and not to C3.

Human

The iC3b enzyme immunoassay measures the amount of the iC3b fragment in human plas-ma or serum, as well as in other biological fluids, experimental samples or mixtures.The levels of iC3b can be significantly elevated in the serum and plasma of some patients with complex-associated diseases such as rheumatoid arthritis and systemic lupus erythe-matodus. iC3b levels may also be elevated in body fluids from other patients with infections, burns, myocardial infarctions, glomerulonephritis, and acute respiratory distress syndrome.

iC3bMicroVue™ Quidel®QUANTIFICATIONOFTHEIC3BFRAGMENTOFC3PROTEIN

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Cat. No.Tests

MethodRange

Sensitivity

Incubation timeSample volume

Sample type Sample preparation

Reference values

Specifity

Species

Intended use

A027 96ELISA0.06 - 0.65 µg/mlLOD: 0.018 µg/mlLLOQ: 0.033 µg/ml1.5 hours100 µl (after dilution 1:10 for plasma, 1:20 for serum)

Serum and plasma

Specimens should be tested immediately or stored at 4 ºC or on ice until assayed. This should not exceed four hours. For long-term storage, freeze at -70ºC within two hours after collection.

in Plasma 0.49 - 1.42 µg/ml (+2 SD)in Serum 0.80 - 6.26 µg/ml (+2 SD)

Monoclonal mouse-antibody, specifically binds Bb.

Human, cynomolgus macaque, baboon, rhesus macaque

The Bb fragment enzyme immunoassay measures the amount of the Complement fragment of Bb, an activation fragment of Factor B in human plasma or serum, as well as in other biological fluids, experimental samples or mixtures. This measurement allows a quantitative assessment of the extent of activation of the alternative pathway of Complement in the test sample. Measurement of alternate pathway activation aids in the diagnosis of several kidney diseases (e.g. chronic Glomerulonephritis, lupus nephritis), as well as several skin diseases (e.g. dermatitis herpetiformis and pemphigus vulgaris). Other diseases in which activation of the alternate pathway of Complement has been observed include rheumatoid arthritis, sickle cell anemia, and gram-negative bacterial infections.

BbPlusMicroVue™ Quidel®BBFRAGMENTSOFFACTORBOFTHEALTERNATIVECOMPLEMENTPATHWAY

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Cat. No.Tests

MethodRange

Sensitivity

Incubation timeSample volume

Sample type Sample preparation

Reference values

Specifity

Species

Intended use

C4aMicroVue™ Quidel®QUANTIFICATIONOFTHEC4AFRAGMENT

A03696ELISA5 – 40 ng/mlLOD: 0.29 ng/ml LLOQ: 5 ng/ml ULOQ: 61 ng/ml2 hours 15 minutes10 µl (dilute 1:40 for plasma, 1:80 for serum)

Human and primate plasma or serum, other biological fluids.

Sample collection is critical. Care must be taken to avoid C4a generation in the sample. For optimal plasma results K2- or K3 EDTA collection tubes are recommended. Serum and EDTA plasma specimens should be collected aseptically using standard techniques. Samples should be tested immediately or stored on ice for no longer than 4 hours. For long-term storage samples should be frozen at -70 ºC, or below.

Monoclonal mouse-antibody, specifically binds human C4a and C4a-des Arg.

Human, primate

The C4a Enzyme Immunoassay Kit measures the amount of the Complement fragment C4a, an activation fragment of Complement protein C4 in human and primate plasma, serum and other biological fluids. Measurement of C4a in human plasma or serum provides evidence for the involvement of the classical or lectin pathway of Complement.Under normal conditions, activation of the classical or lectin Complement pathways results in the cleavage of the Complement protein C4 into C4a and C4b by the protease C1s. C4a is rapidly cleaved to its more stable, less active form C4a-des Arg by endogenous serum car-boxypeptidase N enzyme. Thus quantitation of C4a (C4a plus C4a-des Arg) should provide a reliable measurement of classical or lectin pathway activation that has occurred in the test samples.

• Rheumatoid Arthritis• Systemic Lupus Erythematosis (SLE)• Lyme Disease

Sample n Mean(ng/ml) Range(ng/ml)

EDTA-Plasma 32 1694.65 383.5 – 8168.17 Serum 44 1098.00 20.92 – 4437.24

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Cat. No.Tests

MethodRange

Sensitivity

Incubation timeSample volume

Sample type Sample preparation

Reference values

Specifity

Species

Intended use

A008 96ELISA0.025 - 0.25 µg/mlLOD: 0.01 µg/mlLLOQ: 0.022 µg/ml 1.5 hours10 µl (dilute 1:70 for normal Samples)Serum, EDTA plasma or other biological fluids

The proper collection and storage of specimens is essential, since C4d is highly susceptible to proteolysis. Serum or EDTA plasma specimens should be collected aseptically using standard techniques. They should be tested immediately or stored at 4 ºC or on ice until assayed. This should not exceed four hours. For long-term storage freeze at -70 ºC within two hours after collection.

EDTA plasma 0.7 µg – 6.3 µg/ml (+2SD)Serum 1.2 µg – 8.0 µg/ml (+2SD)

Monoclonal mouse-antibody, specifically binds C4d.

Human, cynomolgus macaque, baboon, rhesus macaque

The C4d fragment enzyme immunoassay measures the amount of the C4d-containing activation fragments of C4 (C4b, iC4b, and C4d) present in human serum, EDTA plasma and other biological or experimental samples.The levels of C4d, when normalized for the presence of endogenous C4, can be significant-ly elevated is plasma specimens obtained from some patients with rheumatoid arthritis, hereditary angioedema, systemic lupus erythematosis and other illnesses. Cd4 may also be elevated in body fluids and plasma samples obtained from patients in which classical Complement pathway activation is known to occur, e.g. from patients with a variety of humoral autoimmune diseases, septicemia, thermal injury, multiple organ trauma, myocar-dial infarction, hereditary angioedema, glomerulonephritis and acute respiratory distress syndrome.

C4dMicroVue™ Quidel®QUANTIFICATIONOFC4D-CONTAININGFRAGMENTSOFACTIVATEDC4OFTHECLASSICALCOMPLEMENTPATHWAY

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Cat. No.Tests

MethodRange

Sensitivity

Incubation timeSample volume

Sample type Sample preparation

Reference values

Specifity

Species

Intended use

A02996 ELISA10 -170 ng/ml LOD: 3.7 ng/ml LLOQ: 8.8 ng/ml2 hours 50 µl (dilute 1:40 for serum) 10 µl (dilute 1:10 for plasma)Serum, EDTA plasma, spinal fluid or other biological fluids

The proper collection and storage of specimens is essential since SC5b-9 may be generated in improperly handled specimens. Serum or EDTA plasma specimens should be collected aseptically using standard techniques. They should be tested immediately or stored at 4 ºC or on ice until assayed. This should not exceed four hours. For longer-term storage freeze at -70 ºC. Plasma concentrations better reflect in vivo concentrations in comparison to serum concentrations.

Serum 334 - 1672 ng/mlEDTA Plasma 127 - 303 ng/ml

Monoclonal mouse-antibody, specifically binds SC5b-9.

Human, African green monkey, cynomolgus macaque, baboon, rhesus macaque, Pigtail monkey

The SC5b-9 enzyme immunoassay measures the amount of SC5b-9 present in human plasma, serum and other biological or experimental samples.The Terminal Complement Complex (TCC, SC5b-9) is generated by the assembly of C5 through C9 as a consequence of activation of the Complement system by either the classical, lectin or alternative pathway. The membrane attack complex (MAC), a form of TCC, is a stable complex that mediates the irreversible target cell membrane damage associated with C-activation.

SC5b-9PlusMicroVue™ Quidel®QUANTIFICATIONOFTHESC5B-9COMPLEX

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Cat. No.Tests

MethodRange

Sensitivity

Incubation timeSample volume

Sample type Sample preparation

Reference values

Specifity

Species

Intended use

A03296ELISA0.05 – 5 ng/mlLOD: 0.012 ng/ml 0.023 ng/ml2 hours 15 minutes10 µl (dilute 1:200 for plasma, 1:5000 for serum)

Human plasma, serum or other biological fluids.

Sample collection is critical. Care must be taken to avoid C3a generation in the sample. For plasma, blood samples should be collected with disodium EDTA as anticoagulant and should be centrifuged at 2000xg at 2-8 °C.The entire operation must be completed immediately. Samples should be prepared and as-sayed immediately or stored on ice for up to 2 hours. For long-term storage freeze at -70 ºC with stabilizing solution.

Serum 71.0 – 589.2 ng/mlEDTA Plasma 33.8 – 268.1 ng/ml

Monoclonal mouse-antibody, specifically binds C3a-desArg.

Human, African green monkey, cynomolgus macaque, baboon, rhesus macaque

The C3a enzyme immunoassay measures the amount of C3a-desArg in human EDTA plasma, serum and other research samples.Under normal conditions, activation of the classical, alternative or lectin Complement pathways results in the formation of a C3 convertase muli-molecular enzyme capable of cleaving C3 to C3a and C3b. C3a is a low molecular weight (approximately 9 kD) protein frag-ment of 77 amino acids. C3a is rapidly metabolized by the serum enzyme, carboxypeptidase N, to the more stable, 76 amino acid form, C3a des-Arg.The quantitation of C3a des-Arg therefore provides a reliable measurement of the level of C-activation in the test sample.

C3aPlusMicroVue™ Quidel®QUANTIFICATIONOFTHEC3AFRAGMENT

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Cat. No.Tests

MethodRange

Sensitivity

Incubation timeSample volume

Sample type Sample preparation

Reference values

Specifity

Species

Intended use

A025 96ELISA0.1 - 1 ng/mlLOD: 0.01 ng/mlLLOQ: 0.050 ng/ml 2 hours 15 minutes20 µl (dilute 1:20 for plasma)10 µl (dilute 1:50 for serum)Serum, EDTA- and citrated plasma.

The proper collection, storage and shipment of specimens are essential. Test immediately or stored up to 4 hours at 2-8 ºC or on ice. For long-term storage the samples should kept frozen at -70 ºC with stabilizing solution (Cat. No. A9576), dilute samples 1:1 with stabilizing solution.

EDTA Plasma 0.37 – 74.33 ng/mlSerum 13.37 – 179.23 ng/ml

C5a and C5a des-Arg

Human

C5a is generated as a result of cleavage of the terminal Complement protein C5,during activation of the Complement system via the classical, alternative or lectin pathway. C5a is a low molecular weight (approximately 9 kD) protein fragment of 74 amino acids. C5 a is rapidly metabolized by the serum enzyme carboxypeptidase to more stable, less active, 73 amino acid form, C5a des-Arg.Research has associated elevated levels of fluid phase and adsorbed C5a with hemo-incom-patibility of some biomaterials, particularly in extracorporeal circuits. Levels of C5a have also been associated with pathogenesis of a variety of disease states, including myocardial infarc-tion, stroke, as well as vascular leak syndrome and associated kidney injury. The role of C5a in the pathogenesis of malaria and other infectious diseases, as well as sepsis, is likewise well documented.

C5aMicroVue™ Quidel®MEASUREMENT OFTERMINAL COMPLEMENT PATHWAY ACTIVATION IN EXPERIMENTALSAMPLES

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Cat. No.Tests

MethodRange

Incubation timeSample volume

Sample type Sample preparation

Reference values

Specifity

Species

Intended use

A018 96ELISA Appr. 0 - 300 U Eq/ml3.5 hours14 µl (dilute 1:200)Serum ONLY. Plasma CANNOT be used.

The proper collection, storage and shipment of specimens are essential, since Complement may be activated in improperly handled specimens. Assay immediately or keep on ice for testing within 4 hours, up to 3 days at 4°C. For long-term storage, freeze at -70 ºC. Maximum 6 freeze/thaw cycles.

133 ± 54 U Eq/ml

The monoclonal antibody specific to terminal Complement complexes arising as the result of the activation step in the test.

Human, cynomolgus macaque

The binding of C1q component of C1 to immune complexes triggers the classical Comple-ment pathway. This activation results in a cascade of enzymatic and non-enzymatic reacti-ons, culminating in the formation of terminal Complement complexes (TCC). Under standard conditions, the level of TCC that can be generated in serum is a quantitative expression of the serum’s total classical Complement activity. The MicroVue CH50 Eq EIA is designed to mea-sure the total classical Complement pathway activity in human serum samples. The measu-rement of CH50 allows detection of deficiencies of one or more Complement components (C1 through C9).

CH50EqMicroVue™ Quidel®TOTALCLASSICALCOMPLEMENTPATHWAYACTIVITY

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Cat. No.Tests

MethodRange

SensitivityIncubation timeSample volume

Sample typeSample preparation

Reference values

Specifity

Species

Intended use

Application

A03496 ELISA0.05 – 2.1 ng/mlLOD: 0.011 ng/mlLLOQ: 0.033 ng/ml3 hours10 µl (dilute plasma 1:1000, serum 1:2000), 25 µl urine (dilute 1:15)EDTA Plasma, serum, urineSerum / Plasma: The Ba fragment of Factor B is susceptible to proteolysis. For optimal plasma results, K2 EDTA should be used. Collect blood sample and centrifuge immediately at 2-8°C. Assay immediately, do not store longer than 2 hours at 2-8°C. For longer storage -70°C.Urine: Collect preservative-free first Morning void (FMV) or second morning void (SMV) before 10:00 am. Store sample refrigerated (2-8°C) for less than 1 day, or freeze the sample at -70°C for longer storage. Maximum 5 freeze and thaw cycles.

Monoclonal mouse-antibody, specifically to capture the Ba fragment.

Human, African green monkey, cynomolgus monkey, rhesus monkey, canine

By quantifying the amount of Ba, the extent of alternative pathway activation at the time of sample collection can be determined.Activation of the alternative pathway has been associated with a variety of disease states including SLE, chronic glomerulonephritis, rheumatoid arthritis, sickle cell anaemia and gram negative bacterial infections. The activation of the alternative Complement pathway can be triggered by a variety of substances including microbial polysaccharides or lipids, gram-negative bacterial lipopolys-accharides, surface determinants present on some viruses, parasites, virally infected mam-malian cells, and cancer cells. In autoimmunediseases, the alternative Complement pathway may contribute directly to tissue damage. Alternative Complement pathway activation may also be an indicator of haemo-incompati-bility of biomaterials.

• kidney diseases• chronic glomerulonephritis• lupus nephritis• skin diseases• dermatitis herpetiformis

BaMicroVue™ Quidel®QUANTIFICATIONOFTHECOMPLEMENTBAFRAGMENT

Sample n Mean(ng/ml) Range(ng/ml)

EDTA-Plasma 35 658 226 – 2153 Serum 29 1642 436 – 3362

Urine 167 7.7 0.6 – 27

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NotesNOTES

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