TT he number of therapeuticproteins (ThPs) approvedfor marketing is not aslarge as the number ofsmall therapeutic

molecules. However, ThPs arebecoming increasingly significant ascurrent candidates in preclinical andclinical studies are licensed. Mostare human or “humanized”recombinant proteins. Evidencesuggests that most ThPs cause somelevel of immune response, which insome cases might produce seriousside effects. Hence, immunogenicityis a concern for professionalsworking in the ThP field.

Some recent journal articles havediscussed the issue ofimmunogenicity with biologicalsand biotechnologically deriveddrugs (1, 2). Immunogenicity ofThPs is nothing new; the issue has

been around since the dawn of thesetechnologies. However, the issueheated up when several cases of redblood cell aplasia were attributed tothe Eprex drug (3). It was not longbefore people in the industry madethe connection betweenimmunogenicity and cleaningvalidation (CV). The connectionmay appear logical but it should notbe cause for concern wherever arobust cleaning validation program(CVP) is in place.

To most people in the biologics/biotechnology industry, CV mayseem complicated and esoteric, butit should not. So I begin with abrief introduction to CV beforediscussing the immunogenicityissues involved.

What Is It? According to theFDA, cleaning validation isdocumented evidence that cleaningprocedures will consistently reduceactive pharmaceutical ingredients(APIs), cleaning aids (CAs), andbacterial endotoxin (BET) residues,as well as microbial bioburden(MBB), on equipment and productcontact surfaces to acceptably safelevels for the processing of drugproducts and APIs (4). Simply said,cleaning validation in manufacturingfacilities ensures that the productsmanufactured or cleaning aids useddo not leave residues that willadulterate the final product. Incleaning validation, the types ofresidue listed above are considered

to be contaminants when theyexceed acceptable levels.

A contaminant is defined by boththe detection of a “foreign”substance and the concentration ofthat substance in a drug product orAPI. With state-of-the-art analyticalmethods, it is possible to measurecontaminants at ultralow levels.Therefore, the limit of detection(LOD) and limit of quantitation(LOQ) grow smaller as newtechniques and technologies areimplemented. It would beimpossible to prove that nocontaminants are present. Of course,there are exceptions to every rule,

PRODUCT FOCUS: PROTEINS

PROCESS FOCUS: CLEANING

VALIDATION (MANUFACTURING)

WHO SHOULD READ: QA/QC,PRODUCTION, VALIDATION, TECHNICAL

SERVICES, AND LABORATORY STAFF

KEYWORDS: CLEANING VALIDATION,PROTEINS, CONTAMINATION CONTROL,IMMUNOGENICITY, DENATURATION

LEVEL: INTERMEDIATE

B I O P R O C E S S TECHNICAL

GEA liquid processing cGMP standardsingle-use CIP/WIP skid-mountedsystem from Niro, Inc. (WWW.NIROINC.COM)

Immunogenic Potential ofTherapeutic Protein Residues After CleaningJosé E. Martínez

38 BioProcess International OCTOBER 2004

and EMEA (PIC/S) requires thatfor certain allergenic ingredients,penicillins, cephalosporins, and

potent steroids and cytotoxics, levelsshould be below the LOD of thebest available analytical methods (5).

In practice, dedicated plants areused for manufacturing suchproducts.

Table 1: Examples of typical immunogen doses

Immunogen Concentration Estimated Minimum Immunogenic(Reference) per Injection Typical Adult Weight Dose per Body Weight

Soluble/membrane Mice: 10–100 µg Male: 22 g Female: 18 g 0.45 µg/g (450 µg/kg)proteins (14) Rabbits: 50–250 µg Male/Female: 1–6 kg 50 µg/kg

Sheep: 250 µg – 10 mg Male: 33 kg Female: 28 kg 8 µg/kgHumans1: 530 µg minimum Male/Female: 70 kg 8 µg/kg extrapolation

Conjugated Mice: 100 µg Male: 22 g Female: 18 g 6 µg/g (6000 µg/kg)peptides/haptens (15) Rabbits: 100–500 µg Male/Female: 1–6 kg 17 µg/kg

Humans2: 1190 µg minimum Male/Female: 70 kg 17 µg/kg extrapolation

M. tuberculosis– Baboons: 500–2000 µg Male: 18 kg Female: 10 kg 28 µg/kgsecreted protein (16) Humans3: 1960 µg minimum Male/Female: 70 kg 28 µg/kg extrapolation

E. coli heat-labile Mice: 25–100 µg Male: 22 g Female: 18 g 1.4 µg/g (1400 µg/kg)enterotoxin (17)

M. tuberculosis Rhesus macaques: 1200 µg Male: 9 kg Female: 8 kg 133 µg/kgor MTP (18) Humans4: 9310 µg minimum Male/Female: 70 kg 133 µg/kg extrapolation

Influenza Virus Mice: 75–750 µg Male: 22 g Female: 18 g 4.2 µg/g (4200 µg/kg)Hemagglutinin (19)

Humanized anti- Cynomolgus monkeys: 10,000 µg/kg, Male: 6 kg Female: 3 kg 16,666 µg/kginterleukin Ab (20) subcutaneous, 10,000 µg/kg and

100,000 µg/kg intravenousHumans5: 1167 mg minimum Male/Female: 70 kg 16,666 µg/kg extrapolation

1 Extrapolation based on sheep 3 Extrapolation based on baboons 5 Extrapolation based on cynomolgus monkeys2 Extrapolation based on rabbits 4 Extrapolation based on rhesus macaques

Residues must be removed to apredetermined level becausecleaning is not an absolute. Despitethe technology we have, it cannotguarantee that no residues are lefton an equipment surface. That isbeyond our capability. In addition,all substances are toxic at highenough levels (even water,excipients, APIs, and so on). Sospecifying the target residue levelsfor which we are sampling andtesting is critical. Manufacturersmust specify a predetermined levelfor residues in CV not only for thatreason, but also to quantify theamount of residues that can becarried over to the next productmanufactured using the sameequipment. Such residues can comefrom APIs or CAs. If the carryoverresidue cannot be quantified, then aCVP is incomplete.

Regulatory agencies worldwiderequire that manufacturers of APIsand drug products have CVPs inplace. And a CVP is nothing morethan the integration of tasks anddeliverables required to providedocumented proof that following

the standard operating procedures(SOPs) for cleaning will consistentlyand effectively clean the target pieceof equipment, utensil, or instrumentto preselected levels.

Building a CVP requiresaddressing certain questions: Whatcontaminants are removed? How arethey removed? What level ofresidues (contaminants) will beallowed on the next product? Onequipment surfaces? How areresidues measured? When will theybe measured?

As Flickinger wrote inPharmaceutical Formulation andQuality (6), “It [CV] sounds simpleenough, but it is deceptively so.Because if the tremendous amount ofanalytical work that goes intoproperly validated cleaningprocedures is not carried out withadequate planning and scientificjustification, then the work could befor naught and the final results lessthan what is intended.” If you don’tdo it right the first time, you will haveto do it again. And that translates tomore time and money spent.

Acceptance criteria for MBB andBET are commonly based on USPharmacopeia specifications for WFI(water-for-injection) or PW (purifiedwater), on historical data, and onspecific product attributes.Acceptance criteria for API and CAresidues are mostly based on a 1993article by Fourman and Mullen (7)and other subsequent contributions:

• dose criterion (pharmacological) • limits based on the toxicity of

the residue (ID50) • lack of observable effect levels

(NOEL values) • process capabilities • visual confirmation of

cleanliness.Cleaning validation for

developmental, clinical, and stabilitylots is discussed in reference 8. Forfurther discussion and insights intoCV, consult references 4, 5, 7, and 8.

THP RESIDUES AFTER CLEANING

Most biotechnology products areparenterally administered proteins.Besides the risk of crosscontamination, there is a subsequenttheoretical risk of immune reponse

to protein residues. So this aspect ofCV for biotechnology facilities mustbe addressed as part of the CVP.

Any substance that can elicit animmune response is said to beimmunogenic and is called animmunogen. A distinction is madebetween antigens (Ags) andimmunogens. An antigen is anysubstance that can bind to a specificantibody. All Ags therefore have thepotential to activate specificantibodies, but some need to beattached to immunogens to do so.All immunogens are Ags; not all Agsare immunogenic.

The best-known and -studied Agsare proteins. Purified proteins,however, are not always highlyimmunogenic. To provoke animmune response, some purifiedproteins must be administered alongwith an adjuvant (9–12).Carbohydrates, nucleic acids, andother types of molecules arepotential antigens that will ofteninduce an immune response onlywhen attached to protein carriers.So the immunogenicity of proteinantigens determines the outcome ofvirtually every immune response.

Although almost any structurecan be recognized by an antibody(Ab) as an Ag, usually only proteinselicit strong and long-actingimmune responses. That is becausethey can engage T cells, whichcontribute to inducing mostantibody responses and are requiredfor immunological “memory.”Proteins engage T cells because thecells recognize as Ags peptidefragments of proteins bound tomajor histocompatibility complex(MHC) molecules.

The larger and more complex aprotein, and the more distant its

40 BioProcess International OCTOBER 2004

Table 2: 0.1% the lowest therapeutic dose(LTD) of selected therapeutic proteins andminimum immunogenic doses for specifiedlarge mammals

Therapeutic Protein 1/1000 LTD

Darbepoetin-� 0.0315 µgPEGfilgrastim (SD01) 6 µgInterferon alfacon-1 0.009 µgEpoetin alfa 0.0168 µgFilgrastim (GCSF) 0.350 µgEtanercept 25 µgAdalimumab (Ref 23) 20 µgInfliximab (Ref 24) 5 µg

MinimumSpecies Immunogenic Dose

Sheep 250 – 10000 µg ofsoluble/membrane proteins

Baboons 500 – 2000 µg of M.tuberculosis–secreted protein

Rhesus 1200 µg of Influenza Virusmacaques hemagglutinin

Cynomolgus 10,000 µg/kg subcutaneous,monkeys 10,000 and 100,000 µg/kg

intravenous of humanizedanti-interleukin-4 Ab

All immunogensare antigens; notall antigens areimmunogenic.

����

relationship to “self” proteins, themore likely it is to elicit an immuneresponse. That is because suchresponses depend on the proteinsbeing degraded into peptides thatcan bind to MHC molecules — andon the subsequent recognition ofthose peptide-MHC complexes by T cells. The larger and more distincta protein Ag, the more likely it is tocontain such peptides. SpecializedAg-presenting cells responsible forinitiating an immune reaction willrespond better to particulate oraggregated Ags, which are taken upmore efficiently by the body. In fact,small soluble proteins are unable toinduce immune responses unlessthey are made to aggregate in someway. Many vaccines, for example,use aggregated protein Ags toinitiate an immune response (9–13).

Haptens are low–molecular-weightmolecules that contain antigenicsites but are not antigenic unlessattached covalently to proteincarriers. Examples include

dinitrophenols, dextrans, and phenylarsonates. Antihapten Abs areimportant medically because theymediate allergic reactions topenicillin and other compounds that

elicit Ab responses when attached toself-proteins. Thus, haptens areincomplete Ags that can stimulateAb production only in combinationwith particular proteins.

Table 3: Summary of immunogenicity for selected therapeutic proteins (adapted and expandedfrom references 21–24)

AntibodyProtein Incidence Immunogenicity

Recombinant human Rare No reports for most products. Infrequent erythropoietin (Epoetin) observations of anti-epoetin Abs. Reports of

pure red cell aplasia (PRCA) cases are rare(<1:10,000 patient-years). Anti-erythropoietin Absdetected in most (but not all) patients with PRCA.

Human granulocyte- Very Rare Low titers of Abs observed did not affect clinicalcolony stimulating efficacy. No Ab production reported byfactor (G-CSF) independent sources except in Neulasta package

insert.

Etanercept (tumor 16% 16% of recipients reported to develop necrosis factor receptor nonneutralizing Ab; clinical effects; injection site fusion protein) reactions reported.

Adalimumab (23) 6% Low-titer neutralizing Abs. Serious allergic adversereactions were not reported with subcutaneousadministration during clinical trials.

Infliximab (24) 14% Abs to Infliximab were detected in 14% of patientswith immunosuppressant therapy and 24% without.Some patients with high Ab titers had evidence ofreduced efficacy.

»Virusesare

purifiedextremely fastand maintainInfectivity and

Immunogenicity.«Professor Alois Jungbauer,

University of Natural Resources,Austria

Plasmid DNApurification is one order

of magnitude more efficientthan using traditional resins.

Dr. Roman Necina, Head of BiopharmaceuticalProduction, Boehringer Ingelheim*

»The specific activity of Factor IX wasincreased by one order of magnitude.«Professor Djuro Josi}, Brown University, RI, USA

*Genet Eng News, 2003, 19 (23), 50–51.

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Adjuvants: Small polypeptides(<10 kDa) and nonprotein Agsusually need to be conjugated tolarge immunogenic carrier proteinsto become good immunogens. Sothose and most other protein Ags(especially when administered insmall quantity) are administeredwith adjuvants to ensure Abresponse (14). Strong immuneresponses to protein Ags almostalways must be injected in suchmixtures (9–14).

An adjuvant is any substance thatenhances the immunogenicity ofother substances mixed with it.Adjuvants differ from proteincarriers in that they do not formstable linkages with theimmunogens. Furthermore,adjuvants are needed primarily forinitial immunizations, whereascarriers are required to elicit notonly primary, but also subsequentresponses to haptens.

Effects of Immunogen Dose: Themagnitude of an immune responsedepends on the dose of theimmunogen administered. Ingeneral, milligram to gramquantities of a protein immunogenare needed to elicit Ab response inlaboratory animals. That range maydiffer from Ag to Ag and fromspecies to species and is called the“window of immunogenicity” (14).Below a certain threshold dose,most proteins do not elicit anyimmune response (9–14). Above thatdose, the response graduallyincreases as the dose of immunogenincreases. In general, secondary andsubsequent immune responses occurat lower immunogen doses. For

laboratory animals to sustain an Abresponse, a continual or intermittentsupply of the immunogen is needed.It is understood that adjuvants mayhelp the formation of Abs byforming pools of immunogens incirculation.

Table 1 lists examples of typicalimmunogen doses. Dose values arebased on animal experimentalmodels because no studies havebeen designed to determine thelower limit of ThPs required toinduce an immune response inhumans. Experimenting on humansfor such a purpose would beunethical. Some values wereextrapolated to apply to a typical70-kg person.

In Table 2, the resultingimmunogenic doses are comparedwith the 0.1% dose of some ThPs.In CV, 0.1% of the lowesttherapeutic dose (LTD) iscommonly used to establish apharmacological acceptancecriterion. From that comparison, wehave a good idea that the 0.01% ofLTD should be below the minimumimmunogenic dose reported forlarge- and medium-size mammalssuch as sheep, baboons, cynomolgusmonkeys, and rhesus macaques. It isscientifically sound to expect thatthe minimum immunogenic dosefor proteins in humans would behigher because humans are larger.And it is worth mentioning that inmost published studies ofexperimental immunology, adjuvantsare used to elicit the immunogenicresponse (9–14).

As stated in review articles byKoren et al. (21) and Schellekens(22), those proteins that look more

like “self” — or those with fewmutations compared with wild types— are generally less immunogenicthan those that look foreign to animmune system. Furthermore, asSchellekens says: “It is reasonable topredict that proteins isolated fromhuman tissues or sera are lessimmunogenic than nonhumanproteins and, in general, this hasproved to be the case” (22).

Table 3 summarizes theimmunogenicity of some ThPs. Asindicated above, the amount ofresidue from the first-made productin the next one manufactured usingthe same equipment is expected tobe less than 0.01% of the LTD.Adding up the reportedly lowimmunogenicity of ThPs and theextremely low dose that patientsmight receive as a carryover, the riskof immune response as aconsequence of a residue aftercleaning is deemed very low.

Denaturation is the loss ofsecondary, tertiary, and/orquaternary structures of proteins,usually accompanied by a loss ofbiological function. It can haveseveral causes:

• high temperatures weakeningor disrupting side-chain (covalent)interactions (25–28)

• mechanical agitation (26, 27) • detergents disrupting side-

chain hydrophobic interactions (25,26)

• pH extremes, as with alkalinesolutions and detergents (25–28).

Current research shows thatchemical modifications (e.g.,deamidation, cysteine oxidation, andpeptide bond hydrolysis) mainlytake place once a protein has beenunfolded by high temperatures(25–28). Accelerated at elevatedtemperatures, chemicalmodifications can makedenaturation irreversible (25–28). Inaddition, hot water itself behaves asa detergent by promotingsaponification and hydrolysisreactions (26, 27).

The primary effect of alkalinehydrolysis with NaOH or KOH onproteins is to break peptide bondsand generate sodium or potassiumsalts of free amino acids, with

42 BioProcess International OCTOBER 2004

Table 4: Major degradation processes as determined by experiments on selected enzymes (28)

Enzyme Conditions Prevalent Degradation Processes

Cellobiohydrolase I 70 °C, pH 4.8 Aggregation, deamidation

Lysozyme 100 °C, pH 6 Deamidation

Ribonuclease A 90 °C, pH 4 Deamidation, hydrolysis at Asp

�-Amylase from 90 °C, pH 8 DeamidationBacillus amyloliquefaciens

�-Amylase from 90 °C, pH 8 Oxydation of CysB. stearothermophilus

oligopeptides as intermediates. Certain amino acids(arginine, asparagine, cysteine, cystine, glutamine,serine, and threonine) are destroyed in the process. The L-amino acids that are normal protein constituents areracemized to a D–L mixture, and carbohydrate sidechains are released from glycoproteins (26).

Effects of Common Cleaning Procedures: Mostmanufacturing equipment in a biotechnology facility iscleaned using clean-in-place (CIP) systems that use PWor WFI at �70 °C. Small pieces of equipment andchangeable parts are usually cleaned either by hand orin an ultrasonic washer using PW/WFI at �50–60 °C.Alkaline detergents are the cleaning aids mostly used insuch procedures.

Those cleaning procedures effectively degradeproteins using several degradation reactions at analkaline pH. It is common knowledge that peptidesdeamidate at acidic pH levels; however, a lesser knownreaction occurs between pH 5 and 12 for asparagineresidues. The deamidation reaction proceeds entirelythrough a succinimide intermediate and depends onOH– concentrations (nucleophiles appear to catalyzethe reaction) (28). Table 4 provides further detail ondegradation processes.

Cleaning procedures that use industry-proventechnology will render all common proteins inactive bydenaturation and degradation. CIP procedures achievethose effects through the combined action of causticand/or acid agents with hot water. In ultrasonic andmanual cleaning procedures, alkaline detergents and hotwater achieve nearly the same effect. In fact, the CIPprocedure is so harsh that only small peptides and singleamino acids are expected to survive it. As discussedabove, small peptides are nonimmunogenic bythemselves. Moreover, no reports in current literaturehave shown antibodies against a degraded proteininterfering with the actions of a parenteral drug.

Cleaning procedures are normally followed by steamor dry heat sanitization/sterilization anddepyrogenation processes using temperatures from 121to 300 °C. The high heat ensures that any remainingprotein fragments are further degraded. To beimmunogenic, proteins as a rule need a molecular massgreater than 2000 Da, so it is expected that onlynonimmunogenic peptides will remain after sanitation/sterilization (9).

RECOMMENDATIONS

Cleaning validation acceptance criteria for ThPmanufacturing facilities would ideally be based on thepharmacological dose data from toxicology andimmunogenicity studies after cleaning. However, suchdata are unknown for protein degradation products. Ihaven’t seen any studies in the literature designed todetermine the lower limits of ThPs or their degradationproducts that will induce an immune response. Asmentioned, experimenting on humans for such purposeswould be unethical. Therefore, addressing the issue incleaning validation exercises is not possible.

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Fortunately, current researchimplies that ThP cleaning residuesare unlikely to initiateimmunological reactions in patientswho receive the drugs. No reportsin the literature have shownantibodies against a degradedprotein interfering with the actionsof a parenteral ThP. It is extremelyunlikely that any common proteinwill retain its biological activity afterbeing subjected to the cleaning andsterilization procedures used in theindustry. For that reason, ThPresidues after cleaning should be aminor issue. Nevertheless, testingfor residues after cleaning withspecific and nonspecific analyticalassays is recommended to ensurethat any degraded residues areaccounted for. Acceptance criteriafor such tests should be based on0.1% of the LTD for the target ThP.

CV is a field in which regulatoryenforcement is increasing as theagencies grow more knowledgeableon the subject. Actions that wereconsidered acceptable some yearsago are now inadequate. Companiesshould continuously updatethemselves on current regulatoryrequirements. In the end, the goalof any CVP is to contribute to theoverall safety, quality, and purity ofpharmaceutical products bypreventing undesirable residues andcross-contamination betweenproducts.

REFERENCES1 Gerrard TL. Possible Regulatory

Changes As a Consequence ofImmunogenicity Concerns. BioProcessInternational 1(9) 2003: 64–69.

2 Thorpe R, Wadhwa M. UnwantedImmunogenicity of Therapeutic BiologicalProducts. BioProcess International 1(9) 2003:60–62.

3 Breckenridge A. EPREX® (EpoetinAlfa) and Pure Red Cell Aplasia:Contraindication of SubcutaneousAdministration to Patients with ChronicRenal Disease. Memo to NHS Trusts MedicalDirectors, DHSC Directors of Public Health,Public Health Link, Strategic HealthAuthorities (England) Directors of PublicHealth, and territorial CMOs; 12 December2002; www.info.doh.gov.uk/doh/embroadcast.nsf/vwDiscussionAll/35595645F6304BFA80256C8D00456DCD

4 Office of Regulatory Affairs. Guide toInspections of Validation of Cleaning Processes.US Food and Drug Administration, July1993; www.fda.gov/ora/inspect_ref/igs/valid.html.

5 PIC/S Secretariat. Recommendationson Validation Master Plan, Installation andOperational Qualification, Non-Sterile ProcessValidation, Cleaning Validation.Pharmaceutical Inspection Convention /Pharmaceutical Inspection Co-OperationScheme Guideline PI 006-1, 3 August 2001;www.picscheme.org/pubs/pubs.htm.

6 Flickinger B. Charting a Course forCleaning Validation. The Best ofPharmaceutical Formulation and Quality(1996): www.pharmaquality.com/validatn.html.

7 Fourman G, Mullen M. DeterminingCleaning Validation Acceptance Limits forPharmaceutical Manufacturing Operations.Pharm. Tech. 17(4) 1993: 54–60.

8 Martinez JE. Cleaning Validation forDevelopmental, Stability, and Clinical Lots.Pharm. Tech. 26(11) 2002: 62–74.

9 Liddell E. Antibodies. In The ImmunoAssay Handbook. 2nd Ed. David W, Ed.Nature Publishing Group: London, 2001:121.

10 Aucouturier J, Dupuis L, Ganne V.Adjuvants Designed for Veterinary Use andHuman Vaccines. Vaccine 19, 2001:2666–2672.

11 Buchan GS, et al. Targeting EarlyEvents in T Cell Activation to ConstructImproved Vaccines. Mol. Immunol. 37, 2000:545–552.

12 Comoy EE, Capron A, ThyphronitisG. In Vivo Induction of Type 1 and 2Immune Responses Against Protein Antigens.International Immunol. 9(4) 2000: 523–531.

13 Cox J, Coulter A. Adjuvants: AClassification and Review of Their Modes ofAction. Vaccine 15, 1997: 248–256.

14 Hanly WC, Bennett BT, Artwohl JE.Overview of Adjuvants. Biologic ResourcesLaboratory, College of Medicine, Universityof Illinois: Chicago, IL, 1995;www.nal.usda.gov/awic/pubs/antibody/overview.htm.

15 Beignon AS, et al. Immunization ontoBare Skin with Heat-Labile Enterotoxin ofEscherichia coli. Immunol. 102(3) 2001: 344.

16 Brandt D and Figard S. ImmunoassayDevelopment in the In Vitro DiagnosticIndustry. In The Immunoassay Handbook(2nd Ed.). Wild D, Ed. Oxford UniversityPress: Oxford, UK, 2001: 122.

17 Pehler K, et al. Mycobacteriumtuberculosis–Secreted Protein Antigens:Immunogenicity in Baboons. Immunol.20(4) 2000: 306–3161.

18 Attanasio R, Pehler K, and McClureHM. Immunogenicity and Safety ofMycobacterium tuberculosis Culture FiltrateProteins in Nonhuman Primates. Clin. Exp.Immunol. 119(1) 2000: 84.

19 Liblau RS, et al. Intravenous Injectionof Soluble Antigen Induces Thymic andPeripheral T-Cell Apoptosis. Immunol. 93,1996: 3031–3036.

20 Hart TK, et al. Preclinical Efficacy andSafety of Pascolizumab (SB 240683): AHumanized Anti-Interleukin-4 Antibody withTherapeutic Potential in Asthma. Clin. Exp.Immunol. 130(1) 2002: 93.

21 Koren E, Zuckerman LA, Mire-SluisAR. Immune Responses to TherapeuticProteins in Humans: Clinical Significance,Assessment and Prediction. Curr. Pharm.Biotechnol. 3(9) 2002: 349–360.

22 Schellekens H. Immunogenicity ofTherapeutic Proteins: Clinical Implicationsand Future Prospects. Clin. Therapeutics24(11) 2002: 1720–1740.

23 Kress S. Clinical Review of Abbott,Biologic Licensing Application, STN 125057:Adalimumab for Use in the Treatment ofRheumatoid Arthritis. Center for BiologicsEvaluation and Research, US Food and DrugAdministration; www.fda.gov/cder/biologics/review/adalabb123102r1p1.pdf.

24 European Medicines EvaluationAgency. Remicade: Summary of ProductCharacteristics. www.emea.eu.int/humandocs/PDFs/EPAR/Remicade/190199en4.pdf.

25 Vieille C, Zeikus GYJ.Hyperthermophilic Enzymes: Sources, Uses,and Molecular Mechanisms forThermostability. Microbiol. Mol. Biol. Rev.March 2001: 1–43.

26 Kaye GI, Weber PB. Treatment andDisposal of Biological, Biohazardous, andRegulated Medical Waste by AlkalineHydrolysis: The WR² Process. Presented atthe 17th Annual College and UniversityHazardous Waste Conference, Yale University,New Haven, CT, 8–10 August 1999;www.yale.edu/oehs/hwc1999/hwc/kaye.pdf

27 Mohammed ZH, Hill SE, andMitchell JR. Covalent Crosslinking in HeatedProtein Systems. J. Food Sci. 65(2) 2000:221–226.

28 Daniel RM, Dines M, and PetachHH. The Denaturation and Degradation ofStable Enzymes at High Temperatures.Biochem. J. 317, 1996: 1–11. ��

José E. Martínez is a biopharmaceuticalconsultant at JEM Consulting Services,PO Box 7526, Caguas, Puerto Rico00726, 1-787-258- 2684, mobile 1-787-349-3857, jemartin@coqui.net.

44 BioProcess International OCTOBER 2004