Development and
`Manufacture of
`Protein Pharmaceuticals
`
`Edited by
`Steven L. Nail
`
`Purdue Universiry
`»~sf Lofayeue, Indiana
`
`and
`Michael J. Akers
`
`Baxter Plumnaceutical Solwions LLC
`Bloomington, Indiana
`
`Kluwer Academic I Plenum Publishers
`New York, Boston, Dordrecht, London, Moscow
`
`MYLAN INST. EXHIBIT 1024 PAGE 1
`
`

`

`~~ .................... ~~--------------------------------
`
`Library of Congress Cataloging-in-Publication Data
`
`Development and manufacture of protein pharmaceuticals/edited by Steven
`L. Nail and Michael J. Akers.
`p. em. -
`(Pharmaceutical biotechnology; v. 14)
`Includes bibliographical references and index.
`ISBN 0-306-46745-3
`l. Protein drugs.
`l. Chemistry. Pharmaceutical-methods. 2. Technology.
`[DNLM:
`Pharmaceutical- methods. 3. Peptides-pharmacology. 4.
`I. Nail, Steven L.
`Proteins- pharmacology. QV 744 0489 2002]
`Ill. Series.
`Michael J.
`RS431.P75 0 485 2002
`615' .19-dc21
`
`IL Akers.
`
`2002016154
`
`ISBN: 0-306-46745-3
`
`C> 2002 Kluwer Academic/Plenum Publishers
`233 Spring Street, New York. N.Y. 10013
`
`h!!p://www. wkap.nV
`
`10 9 8 7 6 5 4 3 2
`
`I
`
`A C.J.P. record for this book is available from the Library of Congress
`
`All rights reserved
`
`No part of this book may be reproduced. stored in a retrieval system. or transm itted in any fom1
`or by any means, electronic. mechanical. photocopying, microfilming. recording. or otherwise,
`without wriuen permission from the Publ isher, with the exception of any material supplied
`specifically for the purpose of being entered and executed on a computer system, for exclusive
`use by !he purchaser of the work
`
`Pri nted in the United States of America
`
`MYLAN INST. EXHIBIT 1024 PAGE 2
`
`

`

`2
`
`Formulation Development of Protein
`Dosage Forms
`
`Michael J. Akers, Vasu Vasudevan, and
`Mary Stickelmeyer
`
`1. INTRODUCTION
`
`A formulation scientist assigned the task of developing a stable, elegant, and
`manufacturable dosage form of a therapeutic protein drug has been given a
`significant challenge. Most proteins, as natural physiological molecules, are
`inherently unstable outside the human or animal body. Stability challenges
`in protein formulation development are typically enormous. The instability
`of these reactive and complex molecules must be considered not only in the
`formulation process, but also in development of the packaging system and
`the manufacturing process. These three areas are intimately and inseparably
`connected.
`Protein dosage forms are also sterile dosage forms. Sterile dosage forms
`must be essentially free* from microbial contamination (sterile), free from
`pyrogenic (including endotoxin) contamination, and free from particulate
`
`*The term "essentially free" is preferable over the more absolute term "free" when dealing with
`the subject of microbiological contamination. Except for products which can be terminally
`sterilized, which do not include proteins, there is no total and absolute assurance that each unit
`of product is, in fact, sterile.
`
`Michael J. Akers
`• Baxter Pharmaceutical Solutions LLC, Bloomington, Indiana 47402.
`Vasu Vasudevan and Mary Stickelmeyer
`• Lilly Research Laboratories, Indianapolis,
`Indiana 46285.
`Development and Manufacture of Protein Pharmaceuticals, edited by Nail and Akers. Kluwer Academic/
`Plenum Publishers, New York. 2002.
`
`47
`
`MYLAN INST. EXHIBIT 1024 PAGE 3
`
`

`

`48
`
`Michael J. Akers et al.
`
`matter contamination (ready-to-use and reconstitutable solutions). Depend(cid:173)
`ing on the route of administration, sterile dosage forms must also be
`isotonic. For example, the intravenous route of administration can tolerate
`fairly wide
`ranges of "tonicity"
`(osmolality or osmolarity) or
`oncocity* whereas subcutaneous and intramuscular routes may require
`tighter control of product tonicity. Sterile products administered into spinal
`fluid or topically applied to the eye must be as close to isotonic as possible
`because of the potential of irreparable damage of spinal or corneal cells due
`to extremes in the osmolar concentrations of administered products. In
`addition, products administered by the injectable or ophthalmic topical
`routes should be as close to physiological pH (7.4) as possible to minimize
`pain and tissue irritation or damage.
`This chapter was written to provide the basic approaches and techniques
`used to design and develop dosage forms of proteins. To develop dosage
`forms means not only to generate a viable formulation, but also to identify a
`final packaging system, to design and scale up a quality manufacturing
`method, and to employ valid measurements to assure product quality. In
`addition, in this era of globalization, formulations must be developed that
`are acceptable from a regulatory standpoint throughout the world.
`Since protein stabilization has already been extensively discussed in
`many excellent references (Table I), we also intend to cover other issues
`essential in the complete formulation development of protein products yet
`not covered elsewhere, such as antimicrobial preservation, packaging
`components, container-closure integrity, clinical trial manufacturing, and
`development history reports.
`We have reviewed the literature and have selected the articles which
`provide both intensive analysis and extensive information on solving protein
`formulation and other product development problems. Advanced injectable
`(e.g., controlled release, implantable devices, gene delivery) and noninject(cid:173)
`able (e.g., pulmonary, oral, buccal) protein formulation research will not be
`covered in this chapter, but other references are available that deal with these
`advances (Baker, 1980; Davis et al., 1986; Senior and Radomsky, 2000;
`Hillery et al., 2001).
`
`2. WHY PROTEINS PRESENT UNIQUE CHALLENGES
`TO THE DEVELOPMENT SCIENTIST
`
`Many texts and articles already discuss the great difficulties scientists
`experience in protein dosage formulation because of the significant
`
`* Oncotic pressure = osmotic pressure exerted by colloids (e.g., plasma proteins) in a solution.
`
`MYLAN INST. EXHIBIT 1024 PAGE 4
`
`

`

`Formulation Development of Protein Dosage Forms
`
`49
`
`Table I
`Major Protein Formulation References
`
`Stability of Protein Pharmaceuticals, Parts A and B, T. J. Ahern and M. C. Manning, (eds.),
`Pharmaceutical Biotechnology, vols 2 and 3, Plenum Press, New York 1992
`Formulation concerns of protein drugs, T. Chen Drug Dev Ind. Pharm. 18:1311-1354 (1992)
`The formulation of proteins and peptides, M. J. Groves, M. H. Alkan, and A. J. Hickey, in:
`Pharmaceutical Biotechnology (M. E. Klegerman and M. J. Groves, eds.), Interpharm Press,
`Englewood, CO, 1992
`Protein stability and degradation mechanisms, B. L. Currie and M. J. Groves, in: Pharmaceutical
`Biotechnology (M. E. Klegerman and M. J. Groves eds.), Interpharm Press, Englewood, CO,
`1992
`Stability and Characterization of Protein and Peptide Drugs: Case Histories, Y. J. Wang and
`R. Pearlman, Plenum Press, New York, 1993
`Factors affecting short-term and long-term stabilities of proteins, T. Arakawa, S. J. Prestrelski,
`W. C. Kenney, and J. F. Carpenter, Adv. Drug Deliv. Rev. 10:1-28, 1993
`Formulation and Delivery of Proteins and Pep tides, Design and Development Strategies,
`J. L. Cleland and R. Langer, (eds.), ACS Symposium Series 567, American Chemical Society,
`Washington, DC, 1994
`Formulation, Characterization, and Stability of Protein Pharmaceuticals, R. Pearlman and
`Y. J. Wang, Plenum Press, New York, 1996
`
`instabilities of these molecules. Depending on the amino acid types and
`sequence, proteins are subject to various types of degradation mechanisms,
`including hydrolysis, oxidation, racemization, and interaction with a variety
`of solutes and surfaces. These mechanisms are especially critical, because
`pharmaceutical proteins are very pure and removed from their natural
`environments where they are most stable (Hanson and Rouan, 1992).
`Dealing with physical instability (e.g., denaturation, aggregation, and
`adsorption) often is more a problem with proteins than dealing with their
`chemical stabilization. Physical instability actually involves solubility
`problems with large molecules. Although proteins generally contain many
`polar groups capable of ionization and hydrogen bonding with water, they
`also can contain many hydrophobic amino acids that under various
`conditions will preferentially self-associate, leading to aggregation and
`decreased solubility. Therefore, the development scientist needs to know the
`structure of the protein and its conformation in solution in order to
`anticipate potential chemical and physical stability difficulties and then,
`using principles outlined in this chapter, develop formulation strategies
`which will overcome these instabilities. Protein formulations may also have
`significant potential for supporting microbial growth as compared to
`smaller molecules. The problems associated with protein microbial growth
`promotion properties are covered in Chapter 3. Table II summarizes some
`of the primary differences one must recognize in developing protein dosage
`forms compared to nonprotein dosage forms.
`
`MYLAN INST. EXHIBIT 1024 PAGE 5
`
`

`

`50
`
`Michael J. Akers et al.
`
`Table II
`Protein versus Small Molecule Comparison from a Stability Standpointa
`
`Protein
`
`Small molecule
`
`Many potential reactive sites
`Many ionizable sites
`Buffer effect usually a unique
`acid/base catalysis
`Secondary/tertiary /q uarternary structure
`Disperse (colloidal) aqueous systems
`Temperature effects can be
`discontinuous (denaturation)
`Readily supports microbial growth
`
`Few reactive sites
`Few ionizable sites
`Buffer effect usually general
`acid/base catalysis
`Lacks "higher order" structure
`Single and continuous phase in solution
`Temperature effects are continuous
`
`a Courtesy, in part, of Dr. Lee Kirsch. University of Iowa. Iowa City. IA.
`
`3. GENERAL FORMULATION PRINCIPLES FOR PROTEINS
`
`Protein stability, both in the dry state and in solution, is the main
`reason why formulation science has such a presence in the commercial
`development of protein dosage forms. Proteins are complex in size and
`structure and, as macromolecules, contain a large number of functional
`groups. Generally, their biological activity in solution depends on a specific
`three-dimensional conformation. Almost every conceivable environmental
`factor (e.g., temperature, light, water, pH, presence of glass, rubber, or
`plastic, shear, presence of salts and other solutes, both macromolecules and
`low molecular weight compounds, detergents, or sanitizing agents, nature of
`the filling processes, freeze-thawing, freeze-drying) can effect conforma(cid:173)
`tional changes and lead to denaturation, aggregation, or adsorption to
`surfaces. The challenge to formulation scientists is to develop a stable
`formulation that can be consistently manufactured and is stable in a given
`packaging system over the shelf life of the product. A reasonable target
`expiration dating is 18 months to 2 years at ambient temperature or, failing
`this, at refrigerated conditions. Aqueous, ready-to-use solutions are
`preferable dosage forms for many reasons (convenience, cost, customer
`acceptance), but most proteins are not sufficiently stable in solution to allow
`practical expiration dating. Therefore, most protein dosage forms are solid
`forms in the commercial package with the solid form being produced by
`freeze-drying. Stability data should include not only the freeze-dried solid,
`but also solution stability after reconstitution with an appropriate vehicle.
`Most additives in protein formulations are needed for stability
`purposes. These include buffers to enhance stability against specific acid/
`base-catalyzed hydrolysis, antioxidants, chelating agents, and inert gases to
`
`MYLAN INST. EXHIBIT 1024 PAGE 6
`
`

`

`Formulation Development of Protein Dosage Forms
`
`51
`
`enhance stability against oxidative degradation, cryoprotectants/lyopro(cid:173)
`tectants to enhance stability during freeze-drying of the protein product,
`surface-active agents to minimize interfacial denaturation, and excipients
`(e.g., albumin) to minimize protein adsorption to inert surfaces such as
`glass. The best formulation strategy is to keep the formulation as simple as
`possible, to have a clear reason for including each additive, and, if
`possible, to use excipients that have previously been used in Food and
`Drug Administration (FDA)-approved formulations. Hundreds of articles
`have appeared in the literature just in the last 20 or so years that report
`the stabilizing effects of additives on various proteins. We will reference
`those we feel have the most relevance to the industrial formulation
`scientist.
`It is also important in this age of globalization that the formulation
`scientist develops a formulation that is acceptable worldwide. This is not an
`easy assignment, because there are many commonly used additives
`acceptable in one country, but not another. For example, disodium
`ethylenediaminetetraacetic acid (DSEDT A) is acceptable for use in inject(cid:173)
`able products in the United States and Europe, but is not acceptable in
`Japan. Levels of antimicrobial preservative agent(s) needed to pass the
`United States Pharmacopeia (USP) preservative efficacy test are much lower
`than levels required to pass the European Pharmacopoeia (EP) test.
`Other additives in protein products (not including controlled drug
`delivery systems) serve one or more of the following functions:
`
`• Agents for antimicrobial preservation
`• Agents for solubility enhancement
`• Bulking agents for freeze-dried products
`• Agents for achieving isotonicity
`
`Most proteins alone and in final product formulations support the
`growth of microorganisms. The microbial growth properties of proteins
`alone and in the final product formulation should be well known and steps
`should be taken to assure that the antimicrobial properties of the final
`formulation meet the appropriate acceptance criteria. For multiple-dose
`products, the addition of an antimicrobial preservative system is required to
`provide antimicrobial properties to the final product. Although including
`an antimicrobial preservative in a single-dose product has the advantage of
`providing additional assurance against introduction of microorganisms
`during manufacturing, this practice is generally frowned upon by regulatory
`agencies. Therefore, strict microbial control during manufacturing and the
`integrity of the packaging system all must be optimized in order to
`minimize the risk of inadvertent microbial contamination of the final
`product.
`
`MYLAN INST. EXHIBIT 1024 PAGE 7
`
`

`

`52
`
`Michael J. Akers et al.
`
`Table III
`Common Stability and Compatibility Problems with Proteins and Possible Solutions
`
`Stability problem
`
`Hydrolysis, deamidation
`(e.g., asparagine deamidation)
`Oxidation (e.g., methionine
`oxidation)
`
`fJ- Elimination
`Transpeptidation
`Racemization
`Disulfide exchange
`Denaturation during freeze-drying
`Aggregation, precipitation
`
`Adsorption to surfaces
`
`Possible solutions
`
`pH control, buffers, low ionic strength
`
`Antioxidants, chelating agents,
`low pH, oxygen-free processing
`and packaging
`Low pH, chelating agents
`pH control, lower concentration
`pH control, buffers
`Thiol scavengers (e.g., cysteine)
`Cryo-, lyoprotectants
`pH control, surface-active agents, minimize
`mechanical stress
`Surface-active agents, albumin, presaturation
`
`The formulation scientist must be aware of the potential for adverse
`effects of low-level impurities in formulation components and packaging
`materials on physical and chemical stability of proteins. Impurities such as
`peroxides from surface-active agents and other polymeric agents, aldehydes
`from polymer synthesis and degradation, and extractables from rubber
`closures must be known and controlled to avoid both short-term and long(cid:173)
`term adverse effects on product quality.
`Table III summarizes common stability and/or compatibility issues
`with protein dosage forms and suggested approaches for solving these
`issues. These approaches will be covered in more detail later in this
`chapter.
`
`4. WHY PACKAGING, PROCESSING, AND FORMULATION
`ARE INTERRELATED
`
`Most publications that deal with protein formulation do not cover
`aspects of the manufacturing process or packaging. Yet the three are
`interrelated. A formulation is not stable unless the product can be
`manufactured consistently at a large scale and packaged in a container/
`closure system that can maintain sterility and stability for a relatively long
`period of time. Packaging of proteins is especially challenging because of
`the inherent interactive nature of proteins with inert surfaces such as glass,
`rubber, and plastic. For many proteins, adsorption at these surfaces
`
`MYLAN INST. EXHIBIT 1024 PAGE 8
`
`

`

`Formulation Development of Protein Dosage Forms
`
`53
`
`sometimes results in the surface denaturation and subsequent aggregation
`of the protein (Cleland et at., 1993). This includes interfacial denaturation
`at the air-water interface (e.g., the headspace in a vial containing a
`protein solution). Minimizing foaming caused by agitation during
`manufacture, as well as during use of the product, may be critical in
`order to avoid significant loss of protein activity or generation of visible
`particulate matter.
`It is well known now that processing of protein formulations can affect
`protein stability. Examples include adverse effects of freezing and/or drying
`that occur during the lyophilization process, mixing/agitation processes, the
`filtration process, complicated manufacturing procedures requiring longer
`filling or hold times, and the movement of intermediate product from one
`location or site to another. In all these examples, the protein formulation
`must be designed to resist changes in potency, purity, and other physical(cid:173)
`chemical characteristics of the protein itself and the finished formulation.
`The bottom line message here is simple: A formulation scientist
`developing a protein (or, for that matter, any) dosage form must consider
`the formulation, process, and package together, not focus on one aspect
`exclusive of others. The smart formulation scientist, in fact, not only will
`consider all aspects of the formula, process, and package, but also will develop
`close interactions with packaging engineers, polymer scientists, manufactur(cid:173)
`ing experts, and other experts in areas outside of the formulation scientist's
`direct expertise. ("None of us is as smart as all ofus"-Satchel Paige.)
`
`5. COMMERCIALLY AVAILABLE PROTEIN DOSAGE FORMS
`
`Table IV summarizes U.S. marketed protein dosage forms approved by
`the FDA through 2000. The table contains information from the Physicians'
`Desk Reference (2001) on the dosage form, route of administration, and
`types and quantities of additives. Although preferential interaction experi(cid:173)
`ments (e.g., Arakawa and Timasheff, 1982) can predict which solutes can
`serve as protein stabilizers, the majority of protein formulation research and
`development requires a great amount of trial and error to finalize the type
`and amount of formulation components. Prior "art," in the sense of
`knowing what has worked before and, particularly for injectable formula(cid:173)
`tions, which additives have a history of safety and regulatory acceptance,
`greatly assists the protein formulation scientist in developing stable, elegant,
`and manufacturable dosage forms. Characterization of protein structure, as
`well as collecting preformulation data as described in Chapter 1, will
`provide supporting data for stabilizers and other additives that are most
`
`MYLAN INST. EXHIBIT 1024 PAGE 9
`
`

`

`~
`~
`~
`::r
`~ c;-
`
`> i'I"
`
`:-
`I:l
`~
`;;!
`0
`
`Sodium citrate
`Polyethylene glycol (2 mg/ml)
`
`Albumin (10 mg/ml)
`Calcium chloride (3 mM)
`Tri-n-butyl phosphate (5 ppm)
`Polysorbate 80 (25 ppm)
`Glycine (0.05 M)
`PEG (1500 ppm)
`
`Incompatible with preservatives
`Phosphoric acid
`Polysorbate 80 (0.011 %)
`L-Arginine
`
`Human serum albumin (1%)
`Sodium citrate
`
`Polysorbate 80 (0.001 %)
`Sodium chloride
`Sodium phosphate
`
`Excipientsd
`
`IV
`
`IV
`
`IV
`
`sterile WFI)
`(recons. with
`
`Freeze-dried
`
`sterile WFI)
`(recons. with
`
`Freeze-dried
`
`D5Wor NS)
`(recons. with
`
`Freeze-dried
`
`Nabi
`
`Autoplex T
`
`Anti-inhibitor coagulant
`
`Genetics Institute
`
`Recombinate Baxter
`Miles
`Koate-HP
`
`Antihemophilic factor
`
`Genentech
`
`Activase
`
`Alteplase
`
`embolism)
`(Acute Ml; pulmonary
`
`IV infusion
`
`Solution
`
`Genzyme
`
`Cerdase
`
`(Gaucher disease)
`
`Alglucerase
`
`IV
`
`Solution
`
`Lilly
`Centocor
`
`of blood clots)
`(Anti platelet prevention
`
`ReoPro
`
`Abciximab
`
`administrationC
`
`Route of
`
`Physical formb
`
`Manufacturer
`
`Trade name
`
`Generic nameD
`
`Sodium chloride
`Calcium chloride
`
`Inhalation
`
`Aerosol
`
`Genentech
`
`Pulmozyme
`
`(Cystic fibrosis)
`DNAse
`
`Dornase alpha
`
`[Heparin])
`clotting factors
`(Vitamin k dependent
`complex
`
`~
`
`Current commercial protein forms
`
`Table IV
`
`MYLAN INST. EXHIBIT 1024 PAGE 10
`
`

`

`VI
`VI
`
`(continued)
`
`'"
`0 8
`"rj
`~
`0 '" I»
`0
`::s
`ff,
`0
`....
`"C
`-.
`0
`::s ....
`E! CD
`'0
`~
`CD
`0
`0 ::s
`~ :t,
`8
`
`0
`"rj
`
`Benzyl alcohol (0,9%) (diluent)
`Mannitol
`Sodium phosphate
`
`Benzyl alcohol (0,9%) (diluent)
`Sodium chloride (diluent)
`Sodium phosphate
`
`Lactose
`Phenol (0,2%) (diluent)
`Benzyl alcohol (2%) (diluent)
`
`Thimerosal
`aluminum
`
`5.40 mcg adsorbed on 0,5 mg
`
`Sodium phosphates
`Sodium chloride
`Thimerosal
`aluminum
`
`1M
`
`1M
`
`1M
`
`1M
`
`bact, WFl)
`(recons, with
`
`Freeze-dried
`
`speical diluent)
`(recons, with
`
`Freeze-dried
`
`special diluent)
`(recons, with
`
`Serono
`
`Profasi
`
`Organon
`
`Pregnyl
`
`Freeze-dried
`
`Wyeth-Ayerst
`
`A,P,L.
`
`Suspension
`
`Recombinax Merck
`
`gonadotropin
`
`Human chorionic
`
`gonadotropin
`
`Human chorionic
`
`20 mcg adsorbed on 0,5 mg
`
`1M
`
`Solution
`
`GlaxoSK
`
`Engerix-B
`
`Hepatitis B vaccine
`
`Polysorbate 80 (0,004%)
`Mannitol
`Sodium acetate
`
`Human serum albumin (0,25%)
`Sodium citrate
`Sodium chloride
`
`Sodium hydroxide to pH 5,25
`Citric acid
`
`IV or SC
`
`Solution
`
`Amgen
`
`Neupogen
`
`(Granulocyte CSF)
`
`Filgrastim
`
`IV or SC
`
`Solution
`
`Ortho Biotech
`Amgen
`
`Procrit
`
`Epogen
`
`(Anemia)
`
`Erythropoietin
`
`IV
`
`Solution
`
`Cor
`
`Integrilin
`
`Eptifibatide
`
`aggregation)
`inhibits platelet
`(Acute coronary syndrome
`
`MYLAN INST. EXHIBIT 1024 PAGE 11
`
`

`

`~
`~
`!>l
`::r
`n·
`~
`
`I:) :-
`~
`'"
`"
`....
`> ~
`
`Polysorbate 80 (0.01 %)
`Sodium chloride
`Glycine
`
`Zinc oxide
`Sodium acetate
`Sodium chloride
`Methylparaben (0.1 %)
`
`Sodium phosphate
`Phenol (0.065%)
`m-Cresol (0.16%)
`Protamine
`
`m-Cresol (0.25%)
`Glycerin
`Sodium citrate 10 mM
`Polysorbate (0.4%)
`Phenol (0.5%)
`Sodium chloride
`
`Benzyl alcohol (0.9%) (diluent)
`Sodium phosphates
`Mannitol
`
`Glycerin (diluent)
`Meta cresol (0.3%) (diluent)
`Sodium phosphate
`Glycine
`Mannitol
`
`Excipientsd
`
`~
`Ul
`
`IV or 1M
`
`0.9% sodium chloride)
`accompanying
`
`(recons. with
`Freeze-dried
`
`Nabi
`
`WinRho SDF
`
`(Gamma globulin /gG)
`
`Immune globulin
`
`SC
`
`SC
`
`SC
`
`SC
`
`Suspension
`
`Squibb-Novo
`Lilly
`
`Humulin L
`
`Human insulin
`
`Suspension
`
`Solution
`
`Squibb-Novo
`Lilly
`
`Squibb-Novo
`Lilly
`
`Humulin N
`
`Human insulin
`
`Humulin R
`
`Human insulin
`
`Solution
`
`Nutropin NQ Genentech
`
`SC
`
`(recons. with bact. WFI)
`Freeze-dried
`
`Genentech
`
`Nutropin
`
`SC
`
`administration"
`
`Route of
`
`special diluent)
`(recons. with
`
`Freeze-dried
`
`Lilly
`
`Humatrope
`
`hormone
`
`Human growth
`
`Physical formb
`
`Manufacturer
`
`Trade name
`
`Generic name"
`
`(continued)
`Table IV
`
`MYLAN INST. EXHIBIT 1024 PAGE 12
`
`

`

`~
`
`(continued)
`
`Incompatible with preservatives
`
`dilute in 50 mL D5W
`
`After reconstitution,
`
`IV Infusion
`
`sterile WFl)
`(recons. with
`
`Freeze-dried
`
`Chiron
`
`Proleukin
`
`carcinoma)
`(Metastatic renal cell
`
`Interleukin-2
`
`0
`'l'j
`~
`0 '"
`I»
`0
`g. =
`... 0
`'tj
`.....,
`0
`a
`8 n
`~
`0 n
`=
`0
`::to
`~
`0 8
`
`'"
`8
`
`'"0
`
`'l'j
`
`Sodium citrate
`Polysorbate 20 (0.01 %)
`Sodium succinate
`Mannitol
`
`Sodium chloride
`Mannitol
`Human serum albumin
`
`Parabens
`Human serum albumin
`Sodium phosphates
`Glycine
`
`Benzyl alcohol (0.9%)
`Human serum albumin
`Sodium phosphates
`Glycine
`
`Sodium phosphates
`Potassium chloride
`Sodium chloride
`Human albumin (0.1%)
`Phenol (0.33%)
`
`Human serum albumin
`Phenol (0.3%)
`Sodium chloride
`
`SC
`
`SC
`
`Intralesion
`
`1M or SC
`
`Intralesion
`
`1M or SC
`
`Solution
`
`Genentech
`
`Actimmune
`
`Interferon y-I B
`
`Freeze-dried
`
`Chiron
`Berlex
`
`Betaseron
`
`(Multiple Sclerosis)
`
`Interferon P-IB
`
`Solution
`
`Schering
`
`Intron A
`
`(Hairy cell leukemia)
`
`Interferon 1X-26
`
`bact WFI)
`(recons. with
`
`Freeze-dried
`
`Schering
`
`Intron A
`
`(Hairy eel/leukemia)
`
`Interferon 1X-2b
`
`Intralesional
`
`Solution
`
`Interferon
`
`Alferon N
`
`(Genital warts)
`
`Interferon lX-n3
`
`1M or SC
`
`RTU Solution
`Freeze-dried and
`
`Roche
`
`Roferon-A
`
`(Hairy cell leukemia)
`
`Interferon 1X-2a
`
`MYLAN INST. EXHIBIT 1024 PAGE 13
`
`

`

`I:> :-
`~
`;;!
`0
`:0;-
`~
`~
`
`'" !!.
`::r
`s:: o·
`
`~
`
`Soduim chloride
`Sodium phosphates
`Polysorbate 80 (0.1 %)
`
`D-mannitol, Polysorbate 80
`containing CMC sodium,
`Reconstituted with diluent
`D-mannitol
`DL-Lactic/glycolic acids
`Gelatin
`
`Acetic acid
`Benzyl alcohol
`Sodium chloride
`
`Phosphate buffer to pH 7
`Glycine
`
`IV
`
`Solution
`
`Ortho Biotech
`
`Orthoclone
`
`OKT3
`
`(Immuno suppressant)
`
`Muromonab-CD3
`
`1M
`
`SC
`
`Freeze-dried
`
`Lupron Depot TAP Pharma
`
`Leuprolide Acetate
`
`Solution
`
`TAP Pharma
`
`Lupron
`
`Leuprolide Acetate
`
`sterile WFl)
`(recons. with
`
`platelet transfusions)
`reducing need for
`thrombocytopenia;
`(Prevention of severe
`
`SC
`
`Genetics Institute Freeze-dried
`
`Neumega
`
`Interleukin-II
`
`Excipientsd
`
`administrationC
`
`Route of
`
`Physical formb
`
`Manufacturer
`
`Trade name
`
`Generic nameQ
`
`(continued)
`Table IV
`
`MYLAN INST. EXHIBIT 1024 PAGE 14
`
`

`

`~
`
`8
`en '"
`0
`0
`::s
`9.
`0
`:?
`-,
`0
`n ::s ...
`8
`~ 't:I
`n
`0
`::s
`0
`:to
`~
`8
`
`en
`
`0
`"'!j
`~
`
`0
`"'!j
`
`phosphate buffer
`
`pH adjusted with sodium
`Mannitol
`
`Incompatible with preservatives
`TRIS
`Sucrose
`Mannitol
`
`Polysorbate (0.07%)
`Sodium citrate
`Sodium chloride
`
`"CMC, carboxymethylcellulose.
`'IV, intravenous; SC, subcutaneous; 1M, intramuscular.
`hrecons., reconstituted; D5W, dextrose 5% in water; NS, 0.9% sodium chloride; WFI, water for injection; bact., bacterial; RTU, ready to use.
`"MI, myocardial infarction; CSF, colony stimulating factor.
`
`Human serum albumin
`Sodium chloride
`Mannitol
`
`Infusion
`coronary
`
`IV, Intra-
`
`SC
`
`IV Infusion
`
`sterile WFl)
`(recons. with.
`Freeze-dried
`
`chloride
`0.9% sodium
`accompanying
`
`(recons. with
`Freeze-dried
`
`sterile WFl)
`
`(recons. with
`Freeze-dried
`
`IV
`
`pH 6.5
`Solution
`
`Abbott
`
`Abbokinase
`
`Urokinase
`
`hormone)
`gland to release growth
`(Stimulates pituitary
`
`Geref Ampuls Serono
`
`Sermorelin acetate
`
`transplant)
`CSF for bone marrow
`(Granulocyte macrophage
`
`Immunex
`
`Leukine
`
`Sargramostim
`
`Genentech
`
`Rituxan
`
`Rituximab
`
`non-Hodgkins lymphoma)
`(Treatment of B-cell
`
`MYLAN INST. EXHIBIT 1024 PAGE 15
`
`

`

`60
`
`Michael J. Akers et at.
`
`likely to be effective. For example, pH-solubility/stability studies will
`provide direction on what type of buffers to use, if any. Having knowledge
`of the structural conformation of a protein in order to predict which amino
`acids in the protein sequence may be particularly vulnerable to degradation
`because of exposure to the environment may give the formulation scientist
`some direction on the stabilizers required. The final selection of excipients,
`unfortunately, must be a result of much empirical evaluation. However,
`information such as that given in Table IV summarizes what others have
`done with their protein products, and thus can give significant guidance to
`formulation scientists facing the development and stabilization of new
`peptide and protein dosage forms.
`Note that protein dosage forms primarily are divided into three types
`
`1. Ready-to-use solutions
`2. Freeze-dried powders that are reconstituted into solutions immedi(cid:173)
`ately before administration
`3. Ready-to-use suspensions
`
`Proteins are commonly formulated at very low doses (very dilute
`solutions), although there are examples of relatively high dose protein
`products, such as formulations of immunoglobulin G (lgG) at 50 mg/ml. In
`general, dilute solutions are less physically stable than more concentrated
`solutions (Hanson and Rouan, 1992) and adsorption to surfaces will result
`in a higher fractional loss of protein. However, in the case of the Neutral
`Protamine Hagedorn (NPH) formulation of insulin, the rate of formation of
`higher molecular weight polymers increases as a function of concentration
`(Brange et at., 1992b). Also, for interleukin 1f3 (IL-1fJ), aggregation/
`precipitation was shown to demonstrate biphasic kinetics (slower rate
`followed by a more rapid rate) at temperatures lower than 55°C and to be
`dependent on concentration. When the concentration was increased from
`lOO to 500 mg/ml, the slower rate was observed to be suppressed and a more
`rapid degradation was observed (Gu et at., 1991). In general, however, there
`are surprisingly few literature reports of protein stability as a function of
`concentration.
`
`6. CHEMICAL STABILIZATION
`
`6.1. pH, Hydrolysis, and Buffers
`
`The effect of solution pH on stability is probably the most important
`factor to study in early protein dosage form development. Figure 1
`
`MYLAN INST. EXHIBIT 1024 PAGE 16
`
`

`

`Formulation Development of Protein Dosage Forms
`
`61
`
`schematically depicts expected stability problems of proteins as a function of
`pH. pH-stability studies are conducted very early to understand relative
`protein stability over a pH range, typically from about pH 3 to about pH 10.
`The relationship of stability and solubility to pH usually follows a pattern of
`higher solubility resulting in lower chemical stability and lower solubility
`resulting in lower physical stability. Protein solubility is usually at a minimum
`at its isoelectric point. Insulin, for example, has an isoelectric point of 5.4, and
`at this pH it is quite insoluble in water ( < 0.1 mg/ml). Adjusting the solution
`pH to less than 4 or greater than 7 greatly increases insulin solubility ( > 30
`mg/ml, depending on zinc concentration and species source of insulin), but
`also increases the rate of deamidation at these pH ranges (Brange, 1992). An
`example of the effect of pH on deamidation and polymerization of insulin is
`shown in Fig. 2 (Brange and Langkjaer, 1993). In dosage form development,
`the scientist must first determine what pH range provides acceptable
`solubility of the protein for proper dosage, then determine whether this pH
`range also provides acceptable stability. There is usually a trade-off between
`solubility and stability and it is up to the scientist to identify what pH is
`optimal for both. When an acceptable trade-off does not exist for a solution
`formulation, a freeze-dried formulation is usually indicated.
`Hydrolysis or deamidation occurs with proteins containing susceptible
`Asn and GIn amino acids, the only two amino acids that are primary
`amides. The side-chain amide linkage in a GIn or an Asn residue has been
`shown to undergo deamidation to form free carboxylic acid. Deamidation
`
`Un-ionized Terminal
`Carboxylic Acid
`
`Un-ionized Side Chain
`Carboxylic Acids
`
`Selectively Oxidized
`Under Acidic Conditions
`
`Peptide cleavage
`
`Cleavage of Dissacharide
`to Reactive Species
`
`Disaccharide browning
`
`Un-ionized Protein
`Amides
`
`Base-Catalyzed
`
`Ionized Cysteine
`
`Acid
`
`Neutral
`
`Base
`
`pH
`
`Figure 1. Protein reactions as a function of pH. Figure courtesy of Dr. Lee Kirsch.
`
`MYLAN INST. EXHIBIT 1024 PAGE 17
`
`

`

`62
`
`Michael J. Akers et al.
`
`A
`%
`Hydrolysis
`100,----r---.--.---.---~--r_--.__,--_,
`
`Monodesamido
`~ Didesamido
`EEl Splil produCI
`
`u w u ~ ~ M ~ ro U M
`pH
`
`80
`
`60
`
`40
`
`20
`
`o
`
`B
`
`%
`
`Polymerization
`20'---~--~--.--.---r--.---.---r--'
`
`5
`
`u w u ~ ~ M ~ ro U M
`pH
`
`Figure 2. Chemical transformation of insulin during storage of rhombohedral insulin crystals
`(bovine insulin crystals: 0.7% NaCI, 0.2% phenol) as a function of pH during storage at 25°C
`for 12 months. (A) Formation of the hydrolysis products mono and didesamido insulins and the
`insulin split product (AB-A9). (B) Formation of covalent dimers and oligomers. Reprinted with
`permission from Brange and Langkjaer (1993). Copyright 1993 Plenum Press.
`
`MYLAN INST. EXHIBIT 1024 PAGE 18
`
`

`

`Formulation Development of Protein Dosage Forms
`
`63
`
`can be promoted by a variety of factors, including extremes in pH,
`temperature, and ionic strength.