Guidance for Industry
`
`Non-Penicillin Beta-Laetarn
`
`Drugs:
`A CGMP Framework for
`
`Preventing Cross-
`Contamination
`
`U.S. Department of Health and Human Services
`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 fiom:
`Oflice ofCommunications
`Division ofDrug Information, W051, Room 2201
`Cen torfor Drug Evaluation and Research
`Food and Drug Administration
`1 0903 New Hampshire A ve.
`Silver Spring, [MD 20993-0002
`Phone: 301—796—3400; Fax: 301—84 7—8714
`druginfo@fda.hhs.gov
`
`
`Minx/"am ' w. filo. Go V/Driivzst’73taidrmc‘erComolianr *eift’evitlafo.r"rfn formmionr/i'fltti-slrmcwdefault,him
`
`
`
`
`
`US. Department of Health and Human Services
`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.................................................................................................................... 1
`
`II. BACKGROUND ......................................................................................................................2
`
`III. RECOMMENDATIONS .........................................................................................................7
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`

`Guidance for Industry1
`
`Non-Penicillin Beta-Lactam Drugs:
`A CGMP Framework for Preventing Cross-Contamination
`
`This guidance represents the Food and Drug Administration's (FDA‘S) current thinking on this topic. It
`does not create or confer any rights for or on any person and does not operate to bind FDA or the public.
`You can use an alternative approach if the approach satisfies 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 FDA staff, call the appropriate
`
`number listed on the title page of this guidance.
`
`1.
`
`IN TROD UCTION
`
`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 pose health risks associated with cross-
`reactivity .
`
`Drug cross-contamination is the contamination of one drug with one or more different 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,
`eg, 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.
`
`1 This guidance was developed by the 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
`
`II.
`
`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). Drugs that are not in compliance with CGMPs are
`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, § 21 1.42(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 has clarified that separate buildings may
`not be necessary, provided that the section of the manufacturing facility dedicated to
`manufacturing penicillin is isolated (i.e., completely and comprehensively separated) from the
`areas of the facility in which non-penicillin products are manufactured.2 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 CGMP regulations specific to APls, the Agency has provided
`guidance to API manufacturers in the guidance for industry, ICH4 Q7, Gooleamtfizctu/‘ing
`
`2 Preamble to the final rule, “Current Good Manufacturing Practice, Processing, Packing, or Holding.” 43 FR 45014
`at 45038 (September 29, 1978).
`
`3 See “A Review of Procedures for the Detection of Residual Penicillins in Dnrgs” (Appendix 1, Procedures for
`Detecting and [Measuring Penicillin Contamination in Drugs, FDA By-Lincs No. 8 (November 1977)), availablc at
`Eitr I/l'wwwfda. maidmm‘aoads/AbontFDA/‘CentcrsOfficos/(31332111(Ti/£0958i3, )df. NB: This link works as of
`
`5/ 18/2012.
`
`4 International Conference on Harmonization.
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`Practice Guidancefor Active Pharmaceutical Ingredients (ICH Q7 guidance).5 Because some
`APIs are sensitizing compounds that may cause anaphylactic shock, preventing cross-
`contamination in APIs is 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,6
`
`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 known as 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 actixge ingredients in drugs for human use.7 Beta-lactam antibiotics include the following five
`classes :
`
`penicillins (e.g., ampicillin, oxacillin)
`cephalosporins (e.g., cephalexin, cefaclor)
`penems (e.g., imipenem, meropenem)
`carbacephems (e. g, loracarbet)
`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 lgE-mediated reactions to
`other beta-lactams, such as cephalosporins and penems.9
`
`5 We update guidance documents periodically. To make sure you have the most recent version of a guidance, check
`the Guidance Page at
`hti JI/I’WW'W Ma min/IDriias/Giiiri.ance(jom iianceRearuiaic-rvInformai.ion/Guidaraces/'defauit.lam.
`
`
`0 See section IV.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 known as the Orange Book (available 011 the Internet at
`imp:x’iwww.accessdata,t'dagnv/scripts/cder/ob/detanltcfm). The Orange Book is searchable by active ingredient
`and updated as newer drug products are added.
`
`8 Yao, JDC, and RC Mocllcring, Jr., Antibactcnal agcnts, in A/Ianual of Clinical Alicrobiologv, 9”1 edition, cditcd by
`PR Murray et a1, Washington DC, ASM Press, 2007.
`
`9 Saxon, A, DC Adelman, A Patel, R Hajdu, and GB Calandra, 1988, Imipenem cross-reactivity with penicillin in
`humans, J Allergy Clin Immunol, 822213-217; Saxon, A, GN Beall, AS Rohr, and DC Adelman, 1987, Immediate
`hypersensitivity reactions to beta-lactam antibiotics, 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,10 the hazard posed is present11 and potentially life-
`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:
`
`0
`
`It is difficult to define the minimal dose below which allergic responses are unlikely to
`occur in humans. 12
`
`0 There is a lack of suitable animal or receptor testing models that are predictive of human
`sensitivity. 13
`0 The threshold dose at which allergenic response could occur is extremely low and
`difficult to detect with current analytical methods.14
`
`While beta-lactam antibiotics are similar to one another in many ways, they may differ in
`pharmacokinetics, antibacterial activity, and potential to cause serious allergic reactions.
`Because allergy testing methods have not been well-validated,13 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 may exhibit 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 of action
`by which such cross-reactivity may occur, the potential health risk to patients indicates that drug
`
`DePestel, J] Ellis, and RE Regal, 2004, Incidence of carbapenem-associated allergic-type reactions among patients
`with versus patients without a reported penicillin allergy, Clin Infect Dis, 38:1102-1107.
`
`m Salkind, AR, PG Cuddy. and JW Foxwonh, 2001, ls this patient allergic to penicillin? An evidence-based analysis
`of the likelihood of penicillin allergy, JAMA, 285:2498-2505.
`
`M Khan, D. and R Solensky , 2010, Drug Allergy, J Allergy Clin Immunol. 125(2): 8131.
`
`12 Dayan, AD, 1993, Allergy to antimicrobial residues in food: assessment of the risk to man, Vet Microbiol,
`35:213-226; Blanca, M, J Garcia, JM Vega, A Miranda, MJ Carmena et al., 1996, Anaphylaxis to penicillins after
`non-therapeutic exposure: an immunological investigation, Clin Exp Allergy, 26:335-340.
`
`13 Olson, H, G Betton, D Robinson, K Thomas, A Monro et al., 2000, Concordance of the toxicity of
`pharmaceuticals in humans and in animals, Regul Toxicol Pharmacol, 32:56-67.
`
`14 Perez Pimiento, A, M Gomez Martinez, A Minguez Mena, A Trampa] Gonzalez, S de Paz Arranz, and M
`Rodriguez Mosquera, 1998, Aztrconam and ccftazidimc: evidence of in vivo cross-allergenicity, Allergy, 53:624-
`625; Shepard, GM, 1991, Allergy to B-lactam antibiotics, Immunol Allergy Clin North Am, 11(3):611-633.
`
`15 Bernstein, IL, IT Li, DI Bernstein et al., 2008, Allergy diagnostic testing: an updated practice parameter, Ann
`Allergy Asthma Imrnunol, 100281-8148.
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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 compounds such 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 compounds are 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 component rather than the inhibitor. Although there have
`been no case reports confirming anaphylactic reactions to a beta-lactamase inhibitor that is also a
`beta—lactam, these compounds are potentially sensitizing agents, and manufacturers should
`implement controls to reduce the risk 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
`crossfireactivity 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 Characteri stics 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-APA includes unbroken beta-lactam
`and thiazolidine rings. The beta-lactam ring is relatively unstable, and it commonly breaks 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 moiety is thought to be a common cause of
`penicillin urticaria] reaction,17 Degradation of 6-APA can also result in the formation of minor
`antigenic determinants, including penicilloic 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-APA is not a true antibiotic, it still carries with it a
`potential to induce allergenicity.
`
`16 Following publication of the draft version of this guidance (76 FR 14024), several commenters suggested that
`monobactams, specifically aztreonam, have a lower risk profile than other beta-lactam products and therefore should
`be exempted from the separation and control reconnnendations set forth in this guidance. We have reviewed
`relevant scientific and medical literature and determined that the relative risk of cross-reactivity associated with
`aztreonam, when compared to other beta-lactams, is a Inatter of scientific uncertainty. Accordingly, at this time,
`FDA does 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 altemative
`approach if the alternative approach satisfies the requirements of the applicable statutes and regulations,
`Manufacturers who wish to discuss an alternative separation and control strategy for a non-penicillin beta-lactam
`such as aztreonam with FDA are invited to do so through the application submission and review process.
`
`17 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 produce allergic reactions. Beta-lactam
`chemical manufacturing processes including, but not limited to, fermentation and synthesis, may
`create beta-lactam intermediates or derivatives with unknown health con sequences. 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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`III.
`
`RECOMlVIENDATIONS
`
`Because of 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 manufacturers to 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 recommends that 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 independent air
`handling system.
`
`As with 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 any other products are manufactured,
`including any other class 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
`
`LUTZ HEINEMANN, PHD
`BERND RICHTER, MD
`
`Nowadays, human insulin is used daily by millions of diabetic patients. The biolog-
`ical effect of human insulin is comparable to that of porcine insulin. However, after
`subcutaneous injection, pharmacological and clinical studies showed pharmacoki-
`netic and pharmacodynamic differences between human and animal insulins. Human
`insulin tends to have faster absorption and shorter duration of action compared with
`animal
`insulin. These differences are more pronounced and can be of clinical
`relevance with intermediate- and long-acting insulin preparations. Optimal meta-
`bolic control can be achieved with either human or highly purified animal insulin
`preparations, provided appropriate insulin replacement strategies are used.
`
`T he development of manufacturing
`
`techniques for human insulin has
`made it possible to treat IDDM pa-
`tients with a hormone that has an amino
`
`to endogenous
`acid sequence identical
`insulin. After characterization of the bi-
`
`ological activity of human insulin in Vitro
`and in animal studies, a series of efficacy
`and safety trials with human insulin in
`humans was performed (1,2). In the first
`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 dilIerent methods (3,4).
`It is surprising how much of the
`literature on human insulin,
`including
`proceedings of commercially sponsored
`syrnposia 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 number of original papers
`published on human insulin that have
`passed a peer-review system. This is dis-
`turbing, because pharmacological differ-
`ences between human insulin and ani-
`
`insulin might have practical
`mal
`implications for the daily therapy of mil-
`lions of patients.
`In this paper, we will review the
`properties of human insulin preparations
`available today for clinical practice. Fur~
`thermore, we will describe the pharma-
`cological dilIerences between human insu-
`lin and highly purified (monocomponent)
`insulin preparations of animal origin. We
`attempt to give a balanced overview of the
`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 Dijsseldorl, Dusseldorf, Germany.
`Address correspondence and reprint requests to Lutz Heinemann, PhD, Department of
`Nutrition and Metabolic Diseases, Heinrich-Heine-University of Dilsseldorf, PO. Box 10 10
`07, Moorenstr. 5, 40001 Diisseldorf, 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 not peer-
`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,
`human insulin is already used by most
`patients.
`
`STRUCTURE, PRODUCTION,
`PURITY, AND POTENCY OF
`HUMAN INSULIN
`
`Structure
`The structure of animal insulin has mi-
`
`nor but potentially important differences
`from human insulin: Porcine insulin dif-
`
`fers by one amino acid (alanine instead
`of threonine at the carboxy-terminal of
`the B-chain, i,e., position 1330), and beef
`insulin differs by two additional alter-
`ations of the sequence of the A—chain
`(threonine and isoleucine on positions
`A8 and A10 are alanine and valine).
`Thus, there is nearly a complete homol-
`ogy between human insulin and porcine
`insulin in the amino acid sequence.
`None of the differences between
`human insulin and animal
`insulins is
`
`thought to be at sites crucial [O the bind-
`ing or action of insulin. Therefore,
`it
`could be expected that 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 between human
`insulin and porcine insulin (5).
`The physicochemical properties
`of human, pork, and beef insulins differ
`somewhat because of their different
`
`amino acid sequence. Threonine adds
`
`90
`
`P.1
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`Heinemann and Richter
`
`one extra hydroxyl group to the human
`insulin molecule. This increges ILS hy-
`drophilic properties and decreases the
`lipophilic properties, x compared with
`that of porcine insulin. Thus, the solu-
`bility of human insulin in aqueous solu-
`tions is higher than that of porcine insu-
`lin.
`
`Production
`
`One way to mass produce human insulin
`was to exchange alanine in position B30
`of porcine insulin with threonine, using
`an enzymatic-chemical method (semi-
`synthetic technique) (6). During the last
`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
`19805, the semi—synthetic production of
`human insulin 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
`human insulin 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 structure is
`important for the insulin-insulin recep-
`tor interaction, and hence for the biolog-
`ical action of insulin. Porcine insulin has
`
`three- dimensional
`a slightly different
`structure when compared with human
`insulin (9).
`
`Purity
`To ascertain a low immunogenicity of
`human insulin preparations, impurities
`had to be avoided. The semi-synthetic
`human insulin 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 vgoactive intestinal pep-
`tides were avoided by using monocom-
`ponent porcine insulin. Contamination
`by enzymes or mte 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-
`tributable to the fact
`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 (I l—13). Antibodies to such
`peptides could not be detected in 10
`patients treated with human insulin 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. Human insulin 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
`human insulin preparations are pure and
`free of any significant contamination
`(16). In regular insulin preparations, in-
`sulin molecules self-associate to dimers
`
`and large oligomers. In addition, a small
`amount of 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-
`tiens 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 immunogenic activ-
`ity (20).
`It has to be emphasized that even
`with a hormone identical 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), xenomorphous proteins like
`protamine.
`
`Potency
`In the first study that repors the effects
`of short- acting human insulin produced
`by recombinant DNA technology in
`healthy men, the plasma glucose decre—
`ment after subcutaneous injection of hu-
`man insulin was similar to that of highly
`purified porcine insul