`
`Non-Penicillin Beta-Lactam
`Drugs:
`A CGMPFramework for
`Preventing Cross-
`Contamination
`
`U.S. Department of Health and HumanServices
`Food and Drug Administration
`Center for Drug Evaluation and Research (CDER)
`
`April 2013
`Current Good Manufacturing Practices (CGMPs)
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`Guidance for Industry
`
`Non-Penicillin Beta-Lactam
`Drugs:
`A CGMP Framework for
`Preventing Cross-
`Contamination
`
`Additional copies are available from:
`Office ofCommunications
`Division ofDrug information, WOSI, Room 2201
`Centerfor Drug Evaluation and Research
`Food and Drug Administration
`10903 New Hampshire Ave.
`Silver Spring, MD 20993-0002
`Phone: 301-796-3400; Fax: 301-847-8714
`druginfo @fda.hhs.gov
`
`
`
`itipGuay fide coyDryos CuldanceonmplianceReonighoryyy formationruidances/defaut,pin
`
`
`
`U.S. Department of Health and HumanServices
`Food and Drug Administration
`Center for Drug Evaluation and Research (CDER)
`
`April 2013
`Current Good Manufacturing Practices (CGMP)
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`TABLE OF CONTENTS
`
`I.
`
`INTRODUCTION........:ccssscrsssrssssesscesscsssesssessseosseessessnssssessessenssoassosssansssorssoessonsssnesseesseesseseses 1
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`TL. BACKGROUND 200... cccsssccssseensccesenecnscccsnscceseansnsccessenoesscessnnecessessenscesenaecsscenenesoensessesesetenes2
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`TIT. RECOMMENDATIONS. .......cccsscssssssssscsssscseccescssssssessonsceneescosssessssscensssonasesssessoessesoessosneesooes7
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`Guidancefor Industry’
`
`Non-Penicillin Beta-Lactam Drugs:
`A CGMPFrameworkfor Preventing Cross-Contamination
`
`numberlisted on the title page of this guidance.
`
`This guidance represents the Food and Drug Administration's (FDA's) current thinking on this topic. It
`does not create or confer anyrights for or on any person and does not operate to bind FDA orthe public.
`You can use an alternative approach if the approachsatisfies the requirements of the applicable statutes
`and regulations. If you want to discuss an alternative approach, contact the FDA staff responsible for
`implementing this guidance. If you cannot identify the appropriate FDAstaff, call the appropriate
`
`I.
`
`INTRODUCTION
`
`This guidance describes the importance of implementing manufacturing controls to prevent
`cross-contamination of finished pharmaceuticals and active pharmaceutical ingredients (APIs)
`with non-penicillin beta-lactam drugs. This guidance also provides information regarding the
`relative health risk of, and the potential for, cross-reactivity in the classes of sensitizing beta-
`lactams (including both penicillins and non-penicillin beta-lactams). Finally, this guidance
`clarifies that manufacturers generally should utilize separate facilities for the manufacture of
`non-penicillin beta-lactams because those compounds posehealth risks associated with cross-
`reactivity.
`
`Drug cross-contamination is the contamination of one drug with one or moredifferent drugs.
`Penicillin can be a sensitizing agent that triggers a hypersensitive exaggerated allergic immune
`response in some people. Accordingly, implementing methods for preventing cross-
`contamination of other drugs with penicillin is a key element of manufacturing penicillin and
`current good manufacturing practice (CGMP) regulations require the use of such methods. See,
`e.g., 21 CFR §§ 211.42(d), 211.46(d), and 211.176. Non-penicillin beta-lactam drugs also may
`be sensitizing agents and cross-contamination with non-penicillin beta-lactam drugs can initiate
`the same types of drug-induced hypersensitivity reactions that penicillins can trigger, including
`life-threatening allergic reactions. Therefore, manufacturers of non-penicillin beta-lactam drugs
`should employ similar control strategies to prevent cross-contamination, thereby reducing the
`potential for drug-induced,life-threatening allergic reactions.
`
`The information in this guidance is intended for manufacturers of finished pharmaceuticals and
`APIs, including repackagers. Other establishments that handle drugs, such as pharmacy
`compounders, may find this information useful.
`
`' This guidance wasdeveloped bythe Office of Compliance, Office of Manufacturing and Product Quality, in the
`Center for Drug Evaluation and Research (CDER)at the Food and Drug Administration.
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`FDA's guidance documents, including this guidance, do not establish legally enforceable
`responsibilities. Instead, guidance documents describe the Agency’s current thinking on a topic
`and should be viewed only as recommendations, unless specific regulatory or statutory
`requirements are cited. The use of the word should in FDA guidance means that something is
`suggested or recommended, but not required.
`
`I.
`
`BACKGROUND
`
`A. Regulatory Framework
`
`Section 501(a)(2)(B) of the Federal Food, Drug, and Cosmetic Act (21 U.S.C. 351(a)(2)(B))
`requires that, with few exceptions, all drugs be manufactured in compliance with current good
`manufacturing practices (CGMPs). Drugsthat are not in compliance with CGMPsare
`considered to be adulterated. Furthermore, finished pharmaceuticals are required to comply with
`the CGMP regulations at 21 CFR parts 210 and 211.
`
`Several CGMP regulations directly address facility and equipment controls and cleaning. For
`example, § 211.42(c) requires building and facility controls in general to prevent cross-
`contamination of drug products. Specifically, the regulation states, “[t]here shall be separate or
`defined areas or such other control systems for the firm’s operations as are necessary to prevent
`contamination or mix-ups” during manufacturing, processing, packaging, storage, and holding.
`
`With respect to penicillin, § 211.42(d) requires that “[o]perations relating to the manufacture,
`processing, and packing of penicillin shall be performed in facilities separate from those used for
`other drug products for human use.” However, FDA hasclarified that separate buildings may
`not be necessary, provided that the section of the manufacturing facility dedicated to
`manufacturing penicillin is isolated (i.c., completely and comprehensively separated) from the
`areas ofthe facility in which non-penicillin products are manufactured.” Under § 211.46(d),
`manufacturers must completely separate air handling systems for penicillin from those used for
`other drugs for human use. Additionally, § 211.176 requires manufacturers to test non-penicillin
`drug products for penicillin where the possibility of exposure to cross-contamination exists, and
`prohibits manufacturers from marketing such products if detectable levels of penicillin are
`found.”
`
`Although FDA has not issued CGMPregulations specific to APIs, the Agency has provided
`guidance to API manufacturers in the guidance for industry, ICH* Q7, GoodManufacturing
`
`? Preamble to the final rule, “Current Good Manufacturing Practice, Processing, Packing, or Holding.” 43 FR 45014
`at 45038 (September 29, 1978).
`
`* See “A Review of Procedures for the Detection of Residual Penicillins in Drugs” (Appendix I, Proceduresfor
`Detecting and Measuring Penicillin Contamination in Drugs, FDA By-Lines No. 8 (November 1977)), available at
`bp /Avno Als sov/downloads/Abont®DA/CentersOffices/CDER/UCMOGSS8 12 pdf NB: This link worksas of
`
`5/18/2012.
`
`“ International Conference on Harmonization.
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`Practice Guidancefor Active Pharmaceutical Ingredients (ICH Q7 guidance).° Because some
`APIs are sensitizing compounds that may cause anaphylactic shock, preventing cross-
`contamination in APIs 1s as important as preventing cross-contamination in finished products.
`The ICH Q7 guidance recommends using dedicated production areas, which can include
`facilities, air handling equipment and processing equipment, in the production of highly
`sensitizing materials, such as penicillins and cephalosporins.
`
`B. Beta-Lactam Antibiotics
`
`Beta-lactam antibiotics, including penicillins and the non-penicillin classes, share a basic
`chemical structure that includes a three-carbon, one-nitrogen cyclic amine structure knownas the
`beta-lactam ring. The side chain associated with the beta-lactam ring is a variable group attached
`to the core structure by a peptide bond;the side chain variability contributes to antibacterial
`activity. As of the date of this publication, FDA has approved over 34 beta-lactam compounds
`as active ingredients in drugs for human use.’ Beta-lactam antibiotics include the following five
`
`classes”:
`
`penicillins (¢.g., ampicillin, oxacillin)
`cephalosporins(e.g., cephalexin, cefaclor)
`penems (e.g., imipenem, meropenem)
`carbacephems(e.g., loracarbef)
`monobactams(e.g., aztreonam)
`
`Allergic reactions associated with penicillins and non-penicillin beta-lactams range from rashes
`to life-threatening anaphylaxis. Immunoglobulin E (IgE) antibodies mediate the immediate
`hypersensitivity reactions that are responsible for the symptoms of hay fever, asthma, hives, and
`anaphylactic shock. IgE-mediated hypersensitivity reactions are of primary concern because
`they may be associated with significant morbidity and mortality. There is evidence that patients
`with a history of hypersensitivity to penicillin may also experience IgE-mediated reactions to
`other beta-lactams, such as cephalosporins and penems.”
`
`* We update guidance documentsperiodically. To makesure you have the most recentversion of a guidance, check
`the Guidance Page at
`hin/Aeww fide cov/Drurs/GuidanceComplianceRceulatory informationGudancesdotankhim.
`
`
`® See section [V.D Containment (4.4) of the ICH Q7 guidance.
`
`7 Approved beta-lactam antibiotics are listed in FDA’s Approved Drug Products with Therapeutic Equivalence
`Evaluations, generally knownas the Orange Book (available onthe Internetat
`hitn /Aywowaccessdaia fda gov/scripte/cdsr/obdetauitctm). The Orange Bookis searchable by active ingredient
`and updated as newer drug products are added.
`
`* Yao, JDC, and RC Moellering,Jr., Antibacterial agents, in.\danual ofClinical Microbiology, 9" cdition, cdited by
`PR Murrayet al., Washington D.C., ASM Press, 2007.
`
`° Saxon, A, DC Adelman,A Patel, R Hajdu, and GB Calandra, 1988, Imipenemcross-reactivity with penicillin in
`humans, J Allergy Clin Immunol, 82:213-217; Saxon, A, GN Beall, AS Rohr, and DC Adelman, 1987, Immediate
`hypersensitivity reactions to beta-lactamantibiotics, Ann Intern Med, 107(2):204-215; Prescott, Jr., WA, DD
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`All non-penicillin beta-lactams also have the potential to sensitize individuals, and subsequent
`exposure to penicillin may result in severe allergic reactions in some patients. Although the
`frequency of hypersensitivity reactions due to cross-reactivity between beta-lactam classes can
`be lower than the risk within a class,'” the hazard posedis present’! and potentiallylife-
`threatening. The potential health hazard of non-penicillin beta-lactams therefore is similar to that
`of penicillins. Further similarities between non-penicillin beta-lactams and penicillins are as
`follows:
`
`e=Itis difficult to define the minimal dose below which allergic responsesare unlikely to
`occur in humans.
`e There is a lack of suitable animal or receptor testing models that are predictive of human
`sensitivity.
`e The threshold dose at which allergenic response could occur is extremely low and
`difficult to detect with current analytical methods. '*
`
`While beta-lactam antibiotics are similar to one another in many ways, they may differ in
`pharmacokinetics, antibacterial activity, and potential to cause seriousallergic reactions.
`Becauseallergy testing methods have not been well-validated,* it is clinically difficult to
`determine the occurrence and rate of cross-reactivity between beta-lactam antibiotics in humans.
`Therefore, undiagnosed or underreported cases of cross-reactivity likely exist. Some beta-lactam
`antibiotics have negligible potential for cross-reactivity with beta-lactams of other classes,
`whereas other beta-lactam compounds mayexhibit sensitizing activity as derivatives before the
`incorporation of side chains that confer antibacterial activity.
`
`Regardless of the rate of cross-reactivity between beta-lactam drugs or the mechanism ofaction
`by which such cross-reactivity may occur, the potential health risk to patients indicates that drug
`
`DePestel, JJ Ellis, and RE Regal, 2004, Incidence of carbapenem-associated allergic-type reactions amongpatients
`with versus patients without a reported penicillin allergy, Clin Infect Dis, 38:1102-1107.
`
`' Salkind, AR, PG Cuddy, and JW Foxworth, 2001, Is this paticnt allergic to penicillin? An evidence-based analysis
`of the likelihood of penicillin allergy, JAMA, 285:2498-2505.
`
`‘Khan, D. and R Solensky , 2010, Drug Allergy, J Allergy Clin Immunol. 125(2): $131.
`
`” Dayan, AD, 1993, Allergyto antimicrobial residues in food: assessmentof the risk to man, Vet Microbiol,
`35:213-226; Blanca, M, J Garcia, JM Vega, A Miranda, MJ Carmona etal., 1996, Anaphylaxis to penicillins after
`non-therapeutic exposure: an immunological investigation, Clin Exp Allergy, 26:335-340.
`
`8 Olson, H, G Betion, D Robinson, K Thomas, A Monro etal., 2000, Concordance of the toxicity of
`pharmaceuticals in humansand in animals, Regul Toxicol Pharmacol, 32:56-67.
`
`" Derez Pimiento, A, M Gomez. Martinez, A Minguez Mena, A Trampal Gonzalez, S de Paz. Arranz, and M
`Rodriguez Mosquera, 1998, Aztrconam and ceftazidime: evidence of in vivo cross-allergenicity, Allergy, 53:624-
`625; Shepard, GM, 1991, Allergy to B-lactamantibiotics, Immunol Allergy Clin North Am, 11(3):611-633.
`
`© Bernstein, IL, JT Li, DI Bernstein, et al., 2008, Allergy diagnostic testing: an updated practice parameter, Ann
`Allergy Asthma Immunol, 100:81-S148.
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`manufacturers should take steps to control for the risk of cross-contamination for all beta-lactam
`16
`products.
`
`C. Beta-Lactamase Inhibitors
`
`Beta-lactam compoundssuch as clavulanic acid, tazobactam, and sulbactam have weak
`antibacterial activity but are irreversible inhibitors of many beta-lactamases. These compounds,
`which are potential sensitizing agents, are typically used in combination with specific beta-
`lactam agents to preserve antibacterial activity (e.g., amoxicillin-clavulanate, piperacillin-
`tazobactam). Because these compoundsare almost always used in combination with specific
`beta-lactam agents, any clinical observations of hypersensitivity reactions likely would be
`attributed to the beta-lactam antibiotic componentrather than the inhibitor. Although there have
`been no case reports confirming anaphylactic reactions to a beta-lactamase inhibitorthat is also a
`beta-lactam, these compoundsare potentially sensitizing agents, and manufacturers should
`implement controls to reduce therisk of cross-contamination with beta-lactamase inhibitors as
`with all other beta-lactam products.
`
`D. Beta-Lactam Intermediates and Derivatives
`
`Some beta-lactam intermediate compounds and derivatives also possess similar sensitization and
`cross-reactivity properties. Beta-lactam intermediate compounds usually are API precursor
`materials that undergo molecular change or purification before use in the manufacture of beta-
`lactam antibiotic APIs. As a result of these changes, the intermediate compounds may develop
`antigenic characteristics that can produce allergic reactions. For example, 6-aminopenicillanic
`acid (6-APA) serves as the intermediate for the formation of all synthetic penicillins that are
`formed by attaching various side chains. The structure of 6-APAincludes unbroken beta-lactam
`and thiazolidine rings. The beta-lactam ringis relatively unstable, and it commonlybreaks open.
`In the case of 6-APA,this breakage leads to the formation of a penicilloyl moiety, which is the
`major antigenic determinant of penicillin. This motety is thought to be a common cause of
`penicillin urticarial reaction.’ Degradation of 6-APA can also result in the formation of minor
`antigenic determinants, including penicillcic acids, penaldic acid, and penicillamine.
`Anaphylactic reactions to penicillins usually are due to the presence of IgE antibodies to minor
`determinants in the body. Although 6-APAis not a trueantibiotic, it still carries with it a
`potential to induce allergenicity.
`
`'® Following publicationof the draft versionof this guidance (76 FR 14024), several commenters suggested that
`monobactams, specifically aztreonam, have a lowerrisk profile than other beta-lactam products and therefore should
`be exempted fromthe separation and control recommendations set forth in this guidance. We have reviewed
`relevant scientific and medical literature and determinedthat the relative risk of cross-reactivity associated with
`aztreonam, when compared to other beta-lactams, is a matter of scientific uncertainty. Accordingly, at this time,
`FDAdoes not recommend manufacturing controls that treat aztreonam differently from other beta-lactam products.
`As with any non-binding recommendations offered in guidance to industry, manufacturers can use an altcrnative
`approachif the alternative approachsatisfies the requirements of the applicable statutes and regulations.
`Manufacturcrs who wish to discuss an alternative separation and control strategy for a non-penicillin beta-lactam
`such as aztreonam with FDAare invited to do so through the application submission and review process.
`
`" Middleton’s Allergy: Principles and Practice,7th ed. (electronic) (2009). Chapter 68: Drug Allergy.
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`Contains Nonbinding Recommendations
`
`Derivatives are unintended by-products that occur during the manufacturing process(i.e., an
`impurity or degradant). Like intermediates, beta-lactam derivatives could have sensitizing
`properties and may develop antigenic properties that can produceallergic reactions. Beta-lactam
`chemical manufacturing processes including, but not limited to, fermentation and synthesis, may
`create beta-lactam intermediates or derivatives with unknown health consequences. Although
`the health risk of sensitization and cross-reaction is difficult to predetermine for beta-lactam
`intermediates and derivatives and is not always well-defined, manufacturing controls intended to
`reduce the risk of cross-contamination should be considered for operations that produce beta-
`lactam intermediates or derivatives.
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`TH.
`
`RECOMMENDATIONS
`
`Becauseof the potential health risks associated with cross-reactivity (cross-sensitivity) of beta-
`lactams, manufacturers should assess and establish stringent controls (including appropriate
`facility design provisions assuring separation) to prevent cross-contamination. Just as FDA
`considers the separation of production facilities for penicillins to be current good manufacturing
`practice, FDA expects manufacturersto treat sensitizing non-penicillin beta-lactam-based
`products similarly. Specifically, FDA recommends that manufacturers establish appropriate
`separation and control systems designed to prevent two types of contamination: (1) the
`contamination of a non-penicillin beta-lactam by any other non-penicillin beta-lactam, and (2)
`the contamination of any other type of product by a non-penicillin beta-lactam. Accordingly,
`FDA recommendsthat the area in which any class of sensitizing beta-lactam is manufactured be
`separated from areas in which any other products are manufactured, and have an independentair
`handling system.
`
`Aswith penicillin, the section of a facility dedicated to manufacturing a sensitizing non-
`penicillin beta-lactam should be isolated (i.e., completely and comprehensively separated) from
`areas in the facility in which other products are manufactured. This control applies to each of the
`five classes of sensitizing beta-lactams; the area in which any class of sensitizing beta-lactam is
`manufactured should be separated from areas in which anyother products are manufactured,
`including any otherclass of sensitizing beta-lactam. Manufacturing that is restricted to a specific
`class of beta-lactam compound (e.g., the cephalosporin family of products) generally would not
`mandate separate facilities and air handling systems, and could permit production campaigning
`and cleaning as sufficient control.
`
`Finally, as discussed above, beta-lactam intermediates and derivatives may induce allergic
`reactions and therefore pose risks of cross-contamination. Accordingly, firms that manufacture
`beta-lactam intermediates or receive them for further processing, as well as firms whose
`manufacturing processes result in beta-lactam derivatives, should evaluate their manufacturing
`operations for the possibility of cross-contamination and implement appropriate controls to
`reduce or mitigate the potential for cross-contamination. As with penicillin and non-penicillin
`beta-lactam drugs, such controls could include, but are not limited to, isolation and separation of
`intermediate and derivative materials, facilities, equipment, and personnel.
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`Clinical Pharmacology of
`Human Insulin
`
`Lutz HEINEMANN, PHD
`BERND RICHTER, MD
`
`Nowadays, humaninsulin is used daily by millions of diabetic patients. The biolog-
`ical effect of humaninsulin is comparable to that of porcine insulin. However,after
`subcutaneous injection, pharmacological and clinical studies showed pharmacoki-
`netic and pharmacodynamic differences berween human and animalinsulins. Human
`insulin tends to have faster absorption and shorter duration of action compared with
`animal
`insulin. These differences are more pronounced and can be ofclinical
`relevance with intermediate- and long-acting insulin preparations. Optimal meta-
`bolic control can be achieved with either human orhighly purified animal insulin
`preparations, provided appropriate insulin replacementstrategies are used.
`
`The development of manufacturing
`
`techniques for human insulin has
`madeit possible to weat IDDM pa-
`tients with a hormonethat has an amino
`acid sequence identical
`to endogenous
`insulin. After characterization of the bi-
`ological activity of human insulin in vitro
`and in animal studies, a series of efficacy
`and safety crials with human insulin in
`humanswas performed (1,2), In thefirst
`years, several studies compared the po-
`tency of human insulin and animal insu-
`lin preparations with regard to their
`pharmacological properties. Later, such
`studies were performed to compare hu-
`man insulin preparations manufactured
`using different methods G,4).
`It is surprising how muchofthe
`literature on human insulin,
`including
`proceedings of commercially sponsored
`symposia as well as papers and reports
`
`published in books and supplements to
`well-known journals, was printed 10
`years ago, all non-peer-reviewed, com-
`pared with the numberoforiginal papers
`published on human insulin that have
`passed a peer-review system. This is dis-
`turbing, because pharmacological differ-
`ences between humaninsulin and ani-
`mal
`insulin might have practical
`implications for the daily therapy of mil-
`lions of patients.
`In this paper, we will review the
`properties of humaninsulin preparations
`available today for clinical practice. Fur-
`thermore, we will describe the pharma-
`cological differences between human insu-
`lin and highly purified (Gnonocomponent)
`insulin preparations of animal origin. We
`attempt to give a balanced overview ofthe
`results of all studies, comparing various
`pharmacological aspects of human insulin
`
`From the Department of Nutrition and Metabolic Diseases (WHO Collaborating Center for
`Diabetes), Heinrich-Heine-University of Diisseldorf, Diisseldorf, Germany.
`Address correspondence and reprint requests to Lutz Heinemann, PhD, Department of
`Nutrition and Metabolic Diseases, Heinrich-Heine-University of Dtisseldorf, P.O. Box 10 10
`07, Moorenstr. 5, 40001 Dusseldorf, Germany.
`IDDM,insulin-dependent diabetes mellitus; NIDDM, non-insulin- dependent diabetes mel-
`litus.
`
`and animal insulin. As a result, it was nec-
`essary to quote papers that were notpeer-
`reviewed.
`A major emphasis of this review
`is the presentation of the time-action
`profiles of the most widely used human
`insulin preparations. A mere discussion
`of differences between human insulin
`and animal insulins would be somewhat
`out of date, because, in many countries,
`humaninsulin is already used by most
`patients.
`
`STRUCTURE, PRODUCTION,
`PURITY, AND POTENCY OF
`HUMANINSULIN
`
`Structure
`The structure of animal insulin has mi-
`nor but potentially importantdifferences
`from humaninsulin: Porcine insulin dif-
`fers by one amino acid (alanine instead
`of threonine at the carboxy-terminal of
`the B-chain, i.e., position B30), and beef
`insulin differs by two additional alter-
`ations of the sequence of the A-chain
`(threonine and isoleucine on positions
`A8 and AlO are alanine and valine).
`Thus, there is nearly a complete homol-
`ogy berween humaninsulin and porcine
`insulin in the amino acid sequence.
`None of the differences between
`human insulin and animal
`insulins is
`thoughtto be atsites crucial to the bind-
`ing or action of insulin. Therefore,
`it
`could be expectedthat the receptor bind-
`ing and cellular interactions of human
`insulin would not differ significantly
`from those of pork or beef insulin (2).
`The amino acid on position B30 is near
`one of the parts of the insulin molecule
`thought
`to be involved in the self-
`association of two insulin molecules into
`dimers. Thus,
`the self-association ten-
`dency could be different berween human
`insulin and porcine insulin (5).
`The physicochemical properties
`of human, pork, and beefinsulins differ
`somewhat because of their different
`amino acid sequence. Threonine adds
`
`90
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`Heinemann and Richter
`
`one extra hydroxyl group to the human
`insulin molecule. This increases its hy-
`drophilic properties and decreases the
`lipophilic properties, as compared wich
`that of porcine insulin. Thus, the solu-
`bility of humaninsulin in aqueous solu-
`tions is higher than thatof porcine insu-
`lin.
`
`Production
`One wayto mass produce humaninsulin
`was to exchange alanine in position B30
`of porcine insulin with threonine, using
`an enzymartic-chemical method (semi-
`synthetic rechnique) (6). During thelast
`decades, biosynthetic production of hu-
`man insulin was made possible through
`advances in genetic engineering, espe-
`cially in recombinant DNA technology
`(7,8). Methods used to produce human
`insulin have changed considerably dur-
`ing the last decade. At
`the end of the
`1980s, the semi-synthetic production of
`humaninsulin was essentially stopped
`and replaced by biosynthetic production.
`In the beginning of the biosynthetic pro-
`duction of human insulin, the A and B
`chains were produced separately and had
`to be combined. At present, biosynthetic
`humaninsulin is produced with a perfect
`three-dimensional structure;
`that is, all
`foldings and disulfide bridges of the in-
`sulin precursor produced by the bacteria
`or yeast cells are identical to endogenous
`insulin. The correct spherical structureis
`important for the insulin-insulin recep-
`tor interaction, and hencefor the biolog-
`ical action of insulin. Porcine insulin has
`a slightly different
`three- dimensional
`structure when compared with human
`insulin (9).
`
`Purity
`To ascertain a low immunogenicity of
`humaninsulin preparations, impurities
`had to be avoided. The semi-synthetic
`humaninsulin production could take ad-
`vantage of the well-established produc-
`tion and purification methods for por-
`cine insulin, which was used as the
`original substrate. Possible contamina-
`tions with proinsulinlike or glucagonlike
`
`substances, pancreatic polypeptide, so-
`matostatin, and vasoactive intestinal pep-
`tides were avoided by using monocom-
`ponent porcine insulin. Conramination
`by enzymes or waste products, as a result
`of the enzymatic-chemical exchange of
`one amino acid during the secondary
`production step, also could be avoided
`(10). In contrast, the insulin production
`methods that use recombinant DNA
`technology have a higher propensity for
`contamination of the insulin product
`with various bacterial or yeast cell poly-
`peptides. The first biosynthetic human
`insulin production using bacteria had
`more obstacles in achieving purity, at-
`triburable to the face that
`the A-and
`B-chains had to be extracted separately,
`and the two chains had to be combined
`with an intact insulin molecule. Thus,
`proteins and other substances of bacte-
`rial origin, as well as waste products of
`the insulin recombination, had to be
`eliminated. Later, purification methods
`were developed to obtain insulin prepa-
`rations free of any potentially harmful
`contamination by Escherichia coli-derived
`peptides (11-13). Antibodies to such
`peptides could not be detected in 10
`patients treated with humaninsulin for 6
`mo (12). Some of the problems of the
`recombinant DNA technique were cir-
`cumvented when it became possible to
`produce homologous proinsulin by E.
`coli (13). Thus, only the C-peptide-like
`sequence had to be cleaved to achieve
`human insulin. Humaninsulin produced
`biosynthetically from yeast cells with a
`different insulin precursor (not identical
`to human proinsulin) was even easier to
`clear from impurities because the precur-
`sor is secreted into the medium, and after
`cleavage of C-peptide, the intact mole-
`cule can be obtained (14,15). Because of
`the sophisticated purification tech-
`niques, it can be assumed that advanced
`humaninsulin preparations are pure and
`free of any significant contamination
`(16). In regular insulin preparations, in-
`sulin molecules self-associate co dimers
`and large oligomers. In addition, a small
`amountof covalently aggregated dimers
`
`and other insulin-transformation prod-
`ucts is formed in commercial
`insulin.
`These transformation products prevail in
`the blood of insulin-treated diabetic pa-
`tients because they have a slower meta-
`bolic clearance relative to insulin mono-
`mers (17-19). Human insulin was
`reported as more susceptible to the pro-
`duction of such products than beef insu-
`lin (19), These transformation products
`are claimed to be highly immunogenic.
`In addition, degradation of the injected
`insulin occurs in the subcutaneous de-
`pot, resulting in degradation products
`that also might have immunogenicactiv-
`ity (20).
`It has to be emphasized that even
`with a hormoneidentical to the human
`insulin, there are still major differences
`compared with the naturally occurring
`hormone. The route of insulin adminis-
`tration is different, and the insulin prep-
`arations contain additives like antisep-
`tics, stabilizers, and, with NPH-insulins
`(Isophane), xenomorphousproteins like
`protamine.
`
`Potency
`In the first study that reports theeffects
`of short-acting human insulin produced
`by recombinant DNA technology in
`healthy men, the plasma glucose decre-
`mentafter subcutaneousinjection of hu-
`maninsulin was similar to that of highly
`purified porcine insulin (21,22). The po-
`tency of semi-synthetic humaninsulin or
`biosynthetic humaninsulin also was re-
`ported to be similar to that of animal
`insulin after intravenousinsulin infusion
`at various doses or after subcutaneous
`injection in diabetic patients (2).
`In the rabbit