formulation and stability of solutions

c08 IHBK072-Akers July 8, 2010 7:49 Trim: 10in× 7in

8 Formulation and stability of solutions

Ready-to-use solution dosage forms comprise the largest percentage of sterile dosage forms inthe marketplace. The solution formulation must be resistant both to physical and chemical degra-dation. Drugs in solution are subject to several major mechanisms of degradation–hydrolytic,oxidative, photolytic, and, for proteins, covalent and noncovalent aggregations, deamidation,cleavages, oxidation, and surface denaturation reactions.

Optimal formulations can minimize or prevent these degradation reactions. Typical addi-tives that help to stabilize injectable drugs in solution include surface-active agents, buffers,sugars, salts, antioxidants, chelating agents, competitive binders, and amino acids. Also, stor-ing solutions at colder temperatures (i.e., refrigerated or even frozen) can help to minimize drugdegradation. Drugs in solution also may have a tendency to form insoluble forms, therefore,physical stabilization is vitally important. This chapter focuses on formulation and stabilizationof sterile drugs in solution, particularly biopharmaceutical drugs with more complex structuresthat present greater or a wider variety of challenges (1,2). There are many other primary liter-ature resources for sterile solution drug formulation including an exhaustive updated reviewarticle on protein stability by Manning et al. (3). Proteins and other biopharmaceutical moleculesnot only readily degrade chemically, but also, and perhaps more readily, are prone to physicalinstabilities such as aggregation and precipitation.

OPTIMIZING HYDROLYTIC STABILITYHydrolysis is the reaction between water and the drug molecule resulting in the loss of potencyand stability. One of the first major studies to be conducted in early drug dosage form devel-opment is to determine the solubility and stability of the drug in solution as a function of pH.Therapeutic proteins, being structurally more complex with secondary and tertiary structuresand amino acids of differing properties being potentially exposed to an aqueous environment,experience a variety of potential degradation pathways over a broad pH range. While smallmolecules do not have this range of potential degradation pathways, many follow pH-stabilityprofiles like the one depicted for penicillin in Figure 8-1. It is common for weak electrolytesto have “V-shaped” degradation versus pH profile where the objective with such moleculesis to identify the pH range where drug stability is greatest. However, typically, the pH rangewhere stability is greatest also is where drug solubility is lowest, again clearly shown in Fig-ure 8-1. Solution pH and type of solvent used also significantly matters for minimizing proteinaggregation, an example of which is shown in Figure 8-2 for recombinant human granulocytecolony-stimulating factor (rhGCSF).

Proteins and some small molecules may degrade in solution by more than one mecha-nism and each degradation mechanism has a different pH-stability profile. Tissue plasminogenactivator undergoes dimer formation, loss of clot lysis or peptidolytic activity, each of whichhave slightly different pH-stability profiles. Glucagon in solution will degrade by hydrolysis,oxidation, and aggregation; the same is true for growth hormone. Insulin degrades by hydrol-ysis (deamidation) and formation of higher molecular weight forms as do many other proteinmolecules.

Hydrolysis or deamidation occurs with peptides and proteins containing susceptibleasparagines (Asn) and glutamine (Gln) amino acids, the only two amino acids that are primaryamines. The side chain amide linkage in a Gln and Asn residue may undergo deamidation toform free carboxylic acid. Deamidation can be promoted by a variety of factors including highpH, temperature and ionic strength (1).

Minimizing hydrolytic stability of drugs, particularly peptides and proteins, can be accom-plished through one or more of the following approaches:

1. Optimization of amino acid sequence; that is, engineering protein structures toremove unstable amino acids or insert amino acid that sterically hinder Asn or Gln

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FORMULATION AND STABILITY OF SOLUTIONS 97

Rate of Degradationof Penicillin

Solubilityof Salt

pH

Lo

g k

1 (H

R−1

) at

35°

C

So

lub

ility at 35°C, g

/L

1

0

2 3 4 5 6 7 8 96

8

10

12

14

16

18

20

−1

−2

−3

Figure 8-1 Effect of pH on solubility and stability of procaine penicillin G. Source: From Ref. 4.

deamidation, as long as this does not affect protein activity, potency, toxicity, or any otherquality attribute.

2. Formulate at optimal solution pH. For example, human epidermal growth factor 1–48demonstrates some interesting pH-dependent stability in that at pH <6, succinimide forma-tion at Asp11 is favored while at pH >6, deamidation of Asn1 is favored (6). The optimal pH,therefore, is right at pH of 6. Generally, deamidation occurs above pH 5.0 with the optimalpH range to minimize deamidation being between 3.0 and 5.0.

3. Store at low temperatures although this will always create difficulties in complying withthe requirement during distribution and long-term storage of the product. However, withthe advent of cold storage distribution businesses, this is less of a problem than in previousyears.

4. Optimize the effects of ionic strength using empirical approaches to determine the effects ofadded electrolytes.

120

100

80

60

40

20

00 1 2 3

Incubation Time (days)

Mo

no

mer

(%

of

tota

l pro

tein

)

4 5

pH 7 PBS

pH 7 PBS 0.5M Sucrose

pH 6.1 PBS

pH 3.5 HCl

pH 3.5 HCl 150mM NaCl

Figure 8-2 Aggregation profiles of recombinant human granulocyte colony-stimulating factor (rhGCSF) as afunction of pH and type of solution. Source: From Ref. 5.

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98 STERILE DRUG PRODUCTS: FORMULATION, PACKAGING, MANUFACTURING, AND QUALITY

0.12

0.10

0.08

Pro

tein

Co

nce

ntr

atio

n (

mg

/mL

)

0.06

0.04

0.02

0.00

0 1 2Time (hrs)

(A) (B)

3 4

0.6

Pro

tein

Co

nce

ntr

atio

n (

mg

/mL

)

0 1 2

Time (hrs)3 4

0.5

0.4

0.3

0.2

0.1

0.0

0.6

Pro

tein

Co

nce

ntr

atio

n (

mg

/mL

)

0 1 2

Time (hrs)

(C)

3 4

0.5

0.4

0.3

0.2

0.1

0.0

Figure 8-3 Protein concentration in the supernatant after isothermal incubation of rBoNTA(Hc) at 50◦C at pH 5,6, and 8. (A) rBoNTA(Hc) in 20 mM Na-succinate, pH 5 at 0.1 mg/mL protein concentration in buffer alone (circles)and with 150 mM NaCl (triangles), at 0.55 mg/mL protein concentration in buffer alone (downward triangles), andwith 150 mM NaCl (squares); (B) rBoNTA(Hc) in 20 mM histidine, pH 6 at 0.1 mg/mL protein concentration inbuffer alone (circles), with 150 mM NaCl (triangles), at 0.55 mg/mL protein concentration in buffer alone (downwardtriangles), and with 150 mM NaCl (square).(C) rBoNTA(Hc) in 20 mM K-phosphate, pH 8 at 0.1 mg/mL proteinconcentration in buffer alone (circles), and with 75 mM NaCl (triangles), at 0.55 mg/mL protein concentration inbuffer alone (downward triangles), and with 75 mM NaCl (square). Source: From Ref. 7.

Buffers and Hydrolytic StabilityBuffers prevent small changes in solution pH. Even pH changes of 0.1 can affect drug solubilityand stability. Buffers are composed of salts of ionic compounds. The most common buffersused in sterile product formulations are acetate, citrate, and phosphate. Recent buffer systems,especially effective for monoclonal antibody formulations, are amino acid-based buffers. Buffersystems acceptable for use in sterile solutions are listed in Table 8-1, both ionic compounds andamino acids.

The proper selection of buffer type and concentration is determined by performing solu-bility and stability studies as a function of pH and buffer species, an example of which is shownin Figure 8-3 for pH stability of recombinant botulinum serotype A vaccine in three differentbuffers with varying sodium chloride levels at three different pHs (7). However, a pH rangethat is a good compromise between solubility and stability can be selected and that pH rangemaintained with the proper selection of the appropriate buffer component.

Generally, deamidation is much slower at acidic pH than at neutral or alkaline pH. Forexample, ACTH deamidation in the pH range of 7 to 11 was catalyzed by increasing bufferconcentrations, whereas no buffer catalysis occurred at pH 5 to 6.5 (8).

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Table 8-1A Most Common Buffers Used in Sterile Drug Solutions

Buffer system pKa Typical buffer pH range

Lactic acid/lactate 3.1 2.0–4.0Tartaric acid/tartrate 3.0, 4.2 2.0–5.3Glutamic acid/glutamate 2.1, 4.3, 9.7 2.0–5.3Malic acid/malate 3.4, 5.1 2.5–5.0Citric acid/citrate 3.1, 4.8, 5.2 2.5–6.0Gluconic acid/gluconate 3.6 2.6–4.6Benzoic acid/benzoate 4.2 3.2–5.2Succinic acid/succinate 4.2, 5.6 3.2–6.6Acetic acid/acetate 4.8 3.5–5.7Histidine 1.8, 6.1, 9.2 5.5–7.4Phosphoric acid/phosphate 2.1, 7.2, 12.7 6.0–8.2Glycine/glycinate 2.4, 9.8 6.5–7.5Tromethamine (TRIS, THAM) 8.1 7.1–9.1Diethanolamine 8.0 8.0–10.0Carbonic acid/carbonate 6.4, 10.3 5.0–11.0

Table 8-1B Dissociation Constants of Amino Acids Used as Buffers inSterile Drug Solutions, Especially Monoclonal Antibody Products

Amino acid � -Carboxylic acid � -Amino group Side chain

Alanine 2.35 9.87 –Arginine 2.01 9.04 12.48Aspartic acid 2.10 9.82 3.86Cysteine 2.05 10.25 8.00Glycine 2.35 9.78 –Histidine 1.77 9.18 6.10Lysine 2.18 8.95 10.53

One of the great challenges in scale-up and technology transfer from laboratory scale toproduction scale batch sizes is the adjustment of pH. Despite the presence of a buffer, target pHoften is not met following addition of all components. Buffers are typically not used to adjustpH of production batches; rather dilute solutions of strong acids (e.g., hydrochloric, acetic orphosphoric acids) and strong bases (e.g., sodium hydroxide) are used. Careful pH adjustmentwith these dilute acids and/or bases is very important, because if target pH is missed, additionaluse of these strong acids and bases may alter buffer capacity and ionic strength of the finalformulation.

General acid and/or general base buffer catalysis can accelerate the hydrolytic degrada-tion. An example is given in Figure 8-4 where the inactivation rate of an experimental drugwas affected by both type and concentration of buffer component (9). The deamidation rate of asmall peptide using different buffers found that the peptide was most unstable in a phosphatebuffer and most stable in Tris buffer (10). Buffer type and concentration will affect aggregation ofbasic fibroblast growth factor depending on pH (11). At pH 5, aggregation increased as citratebuffer concentration increased. Citrate buffer at pH 3.7 caused aggregation, whereas acetatebuffer at pH 3.8 did not. At pH 5.5 to 5.7, phosphate, acetate, and citrate buffers all showedsimilar aggregation rates.

Histidine has been found to be an excellent buffer component for monoclonal antibodies(e.g., Synagis R©, Herceptin R©, Xolai R©, Raptiva R©) maximally stable in the pH 6 range. The pKa ofhistidine is 6.0 that makes it an ideal buffer at pH of 6.0. Histidine is the only amino acid withpH 7.4 within its buffering range, therefore, it has found importance in parenteral formulationsrequiring buffering in the physiological pH range (12).

High concentrations of monoclonal antibodies (≥50 mg/mL) have the ability to self-buffer(13). IgG2 was found to be more stable at pH 5 after accelerated stability studies as a self-bufferedformulation than in formulations containing conventional buffers such as acetate, glutamate,and succinate.

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0.15

0.14

GlycineCitrateTartrateMalate

0.13

0.12

0.11

0.090 0.01 0.02 0.03 0.04

Buffer Concentration (M)

k (1

/day

)

0.05 0.06

0.1

Figure 8-4 Rate of hydrolysis of GW280430as function of buffer type and concentration.Source: From Ref. 9.

Ionic StrengthIonic strength is a measure of the intensity of the electrical field in a solution. Ionic strengthdepends on the total concentration of ions in solution and the valence of each ion. The ionicstrength of a 0.1 M solution of sodium chloride is 0.1. The ionic strength of a 0.1 M solutionof sodium sulfate is 0.3, because sulfate ions have a valence of 2 added to the valence of1 for the sodium ions. Ionic strength may have an effect on drug stability in solution. TheDebye–Huckel theory predicts that increased ionic strength would be expected to decrease therate of degradation of oppositely charged reactants and increase the rate of degradation ofsimilarly charged reactants. For example, increasing ionic strength will increase the stabilityof recombinant alpha-1-antitrypsin (14). Conversely, increasing ionic strength will increase therate of deamidation of human growth hormone (hGH) (15), bovine somatotropin (BST) (16) andlead to opalescence and higher viscosity of a monoclonal antibody (17).

OPTIMIZING OXIDATIVE STABILITYDrugs containing such functional groups as phenols, catechols, and thioethers will be subjectto oxidative degradation. Epinephrine, phenylephrine, dobutamine, dopamine, morphine, Ter-ramycin, ascorbic acid, and many others are examples of small molecule drugs that will oxidizein solution. Proteins containing amino acids methionine, cysteine, cystine, histidine, trypto-phan, and tyrosine are susceptible to oxidative and/or photolytic degradation depending onthe conformation of the protein and resultant exposure of these sensitive amino acids to thesolvent and environmental conditions. Environment conditions that catalyze oxidative degra-dation include the presence of dissolved oxygen in solution, light exposure, high temperature,low solution pH, metal ions (ppm, even ppb levels), and impurities such as peroxide. Oxidationof sulfhydryl-containing amino acids (e.g., methionine and cysteine) will lead to disulfide bondformation and loss of biological activity. The free-thiol group that is present in a cysteine residueof any native biologically active protein may oxidize not only to produce an incorrect disulfidebridge, but also can result in other degradation reactions such as alkylation, addition to doublebonds, and complexation with heavy metals.

Human growth hormone, chymotrypsin, lysozyme, parathyroid hormone, human granu-locyte colony-stimulating factor, insulin-like growth factor I, acidic and basic fibroblast growthfactors, relaxin, the monoclonal antibody OKT3, interleukin 1�, and glucagon are a few of theexamples of proteins that may undergo oxidative degradation.

For protection against oxidation, choice of an effective antioxidant is one of the severalprecautions that must be practiced in formulation development and final product manufacture.Indeed, minimizing drug oxidative degradation requires a combination of several approaches,

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Table 8-2 Various Approaches Used To Minimize Oxidative Drug Degradation

� Preparation and storage at low temperatures� Use of chelating agents to eliminate metal catalysis� Increasing ionic strength� Elimination of peroxide and metallic contaminants in formulation additives� Protection from light� Awareness of possible interaction of light exposure and phosphate buffer in forming free radicals� Replacing oxygen with nitrogen or argon during manufacturing� Removing oxygen from the headspace of the final container� Formulation established at the lowest pH possible while still maintaining adequate solubility and overall

stability� Use of a container/closure system that allows no oxygen transmission through the package during

distribution and storage� Assuring that phenolic or other oxidizing cleaning agent residues are minimal in the production environment

not only formulation, but also hermetic packaging, oxygen-free processing, and all other pre-cautions listed in Table 8-2.

Formulators should be aware of the potential for polysorbate 80 to adversely affect theoxidative stability of proteins. Polysorbate 80 is a commonly used surface-active agent in pro-tein formulations to minimize surface aggregation problems. However, it has the tendency toproduce peroxides that can oxidize methionine and cysteine residues. This phenomenon wasreported in studies involving formulation development of Neupogen R© (18) and recombinanthuman ciliary neurotrophic factor (19).

AntioxidantsThere are several choices of antioxidants that can be used in sterile formulations. Those usedmost frequently are ascorbic acid, salts of sulfurous acid (sodium bisulfite, sodium metabisulfiteor sodium thiosulfate), and thiols such as thioglycerol and thioglycolic acid. Dithiothreitol,reduced glutathione, acetylcysteine, mercaptoethanol, and thioethanolamine are thiols whichusually oxidize too readily to be of practical use in pharmaceutical formulations requiringlong-term storage.

Precautions must be applied when considering certain antioxidants in certain drug formu-lations. Here is one example. Ascorbate in the presence of Fe3+ and oxygen actually induces theoxidation of methionine in small-model peptides (20). Ascorbate is a powerful electron donor inthat it is readily oxidized to dehydroascorbate. It also generates highly reactive oxygen speciessuch as hydrogen peroxide and peroxyl radicals. These, in turn, will accelerate the oxidationof methionine. Phosphate buffer accelerated the degradation of methionine in the presence ofascorbic acid. The addition of EDTA did not enhance stability even though ferric ion and othertransition metals were components in the formulation, either purposely added or as trace com-ponents of the buffer and peptide. This pro-oxidant effect of ascorbate methionine oxidationwas concentration dependent and occurred most readily at pH 6 to 7.

Chelating AgentsChelating agents are used in formulations to aid in inhibiting free radical formation and resultantoxidation of active ingredients caused by trace metal ions such as copper, iron, calcium, man-ganese, and zinc. There are several examples of commercial formulations (Nebcin R©, Decadron-LA R©, Versed R©, Cleocin R©, and others) where a chelating is all that is needed, that is, no antioxi-dant in the formulation, to protect the active ingredient against metal-catalyzed oxidation. Themost common chelating agent used is disodium ethylenediaminetetraacetic acid (DSEDTA),typically at very low concentrations, for example, ≤0.04%. DSEDTA tends to dissolve slowlyand is usually among the first of formulation ingredients to be dissolved during compoundingbefore adding other ingredients, including the active. Citrate buffer can also serve as a chelatingagent although not as effective as DSEDTA.

EDTA should not be used in formulations of metalloproteins such as insulin or hemoglobinor fibrolase as the chelating agent will attack the metal that is part of the stable conformation of

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the protein. EDTA will accelerate the oxidative degradation of methionine in human insulin-likegrowth factor I solutions (21). Oxidation of methionine 59 was catalyzed by light and ferric ionsin combination with EDTA. It was suggested that EDTA actually enables ferric ions to be activeby stabilizing the transfer of electrons from ferric ions to ferrous ions. Methionine in this proteinis radicalized by light and then oxidized to methionine sulfoxide. Light may also trigger thegeneration of hydroxyl radicals by decomposition of water that may oxidize the methionine.Thus, the formulator must not indiscriminately include EDTA in protein formulations withoutcarefully determining that its presence aids in oxidative stabilization of the protein.

To illustrate how several multiple approaches can be applied to minimize oxidativedegradation, parathyroid hormone was used as a model protein to investigate stabilizationof methionine, tryptophan, and histidine amino acids from oxidative degradation (22). Success-ful approaches included using polysorbate free from peroxide contamination; mannitol alsohelped protect against peroxide-induced oxidation, EDTA to complex heavy metals originatingfrom stainless steel surfaces, and free-radical scavenger stabilizers such as Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) and pyridoxine.

Inert GasesInert gases are frequently used in production of sterile dosage forms. The most commonly usedinert gas is nitrogen. Other inert gases used, although not often primarily because of expense,include argon and helium. Argon, however, has been shown to be more efficient in displacingoxygen because it is heavier than air and will more readily stay in the vial compared to nitrogen.The normal use of inert gases in sterile product manufacturing involves solution and headspacesaturation. Addition to water and compounding solutions prior to aseptic filtration saturatesthe solution and minimizes the level of dissolved oxygen. However, oxygen is never completelydisplaced with an inert gas when the solution is sparged. Many manufacturers use a dualneedle that permits simultaneous filling of a liquid and purging of gas at the same time. Inertgas introduced into the headspace of a filled vial right before the vial is stoppered with a rubberclosure theoretically displaces oxygen in the headspace. Again, a dual needle can be used to fillsolution and purge gas into the final container at the same time.

The inert gas must be high quality grade and must be sterilized, usually with a 0.22-�mhydrophobic membrane filter. The integrity of the gas filter is tested before and after use bydiffusion flow methods.

Packaging and OxidationAll the appropriate formulation and processing procedures can be in place for stabilizing pro-tein solutions against oxidation, but if the packaging system is inadequate from an integritystandpoint, the product will readily degrade. Most injectable products are packaged in glassvials or syringes with rubber closures or plungers. The rubber-glass interface and the oxygentransmission coefficient of the rubber closure will dictate the quality of the container/closuresystem (chap. 30).

Oxygen transmission coefficients are determined for a particular rubber closure formu-lation by the rubber closure manufacturer. Rubber formulations having the lowest oxygentransmission coefficients are the synthetic butyl and halobutyl types. The formulator shoulddetermine from the rubber manufacturer how the halobutyl rubber is cured (shaped, molded)since common curing agents are zinc oxide, aluminum, and peroxide, which potentially canleach out of the rubber formulation with time and catalyze oxidative degradation.

Many drugs (catecholamines, cephalosporins, aminoglycosides, some steroids, iron-containing molecules, and many others) are sensitive to light. Effective packaging is the primary(in most cases only) way to protect drugs from light degradation. Good light protective sec-ondary packaging, use of amber-colored primary packaging (although more expensive anddifficult to inspect for particulate matter), and maintaining product storage in the dark are theways that sensitive drugs are protected from light degradation. There is no practical formulationapproach to stabilize light-sensitive drugs; good packaging is the key to protect against lightdegradation.

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OTHER STABILIZERS TO MINIMIZE DRUG DEGRADATIONThe literature contains many examples of excipient stabilization phenomena with injectabledrugs. The following are only a few examples. Sugars and polyols, such as ethylene glycol,glycerol, glucose, and dextran, at high concentrations, can inhibit the metal-catalyzed oxidationof human relaxin (23). All but dextran act as chelating agents in complexing transition metalions, whereas dextran, which has a higher binding affinity to metal ions and undergoes depoly-merization in a metal-catalyzed oxidation, protects relaxin by a radical scavenging mechanism.

Mannitol has been shown to inhibit the iron-catalyzed oxidation of Met-containing pep-tides (22,24). Mannitol is the most commonly used excipient in freeze-dried formulations oftenserving a dual role as a bulking agent and a stabilizer.

Fibroblast growth factors, both acidic and basic, possess nearly identical three-dimensionalstructures of 12 antiparallel �-strands arranged with approximate threefold-internal symme-try (25). Acidic fibroblast growth factor was found that its tendency to aggregate in solutionwas inhibited by a variety of polyanionic additives such as inositol hexasulfate or sulfate�-cyclodextrin and by a number of commonly used excipients such as sucrose, dextrose, tre-halose, glycerol, and glycine. In all cases, these interactions between acidic fibroblast growthfactor and various excipients resulted in an increase in the protein’s Tm, the midpoint of thetemperature of the transition from the folded to unfolded protein. Basic fibroblast growth factorhas a major degradation pathway that involves not only aggregation and precipitation, but alsoa succinimide replacement of aspartate at position 15 of the protein sequence (26). Adjustingsolution pH from 5 to 6.5 and storage at low temperatures will help to avoid this reaction.

A variety of co-solvents can stabilize proteins in solution because the co-solvent is prefer-entially excluded from surface interaction with the protein (27). Co-solvents behaving this wayinclude glycerol and sorbitol. Polyethylene glycol (PEG) also is preferentially excluded from theprotein, yet will still denature or destabilize proteins in solution.

OPTIMIZING PHYSICAL STABILITYPhysical stability problems are rare with small molecules except with sparingly solublemolecules that are borderline soluble in the formulation vehicle. However, proteins, because oftheir unique ability to adopt higher order secondary and tertiary three-dimension structures,tend to undergo a number of physical changes, independent of chemical modifications. Phys-ical instability of proteins is sometimes a greater cause for concern and is more difficult tocontrol compared to chemical instability. Many proteins, particularly when exposed to stressfulconditions, for example, extremes in temperature, will unfold such that the hydrophobic por-tions become exposed to the aqueous environment. Such exposure will promote aggregation orself-association, possibly leading to physical instability and loss of biological activity since theinteraction with the receptor site requires folded structures with correct conformation. The rela-tionship of the different pathways of physical destabilization of proteins is shown in Figure 8-5.

Native State Molten Globule State Unfolded (Denatured) State

Soluble Aggregates Misfolded State

Insoluble Aggregatesor Precipitates

Figure 8-5 Schematic pathway of physical degradation of a protein. Source: From Ref. 28.

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DenaturationProtein denaturation occurs when native higher order structure is disrupted. Denaturation canlead to unfolding and the unfolded polypeptide chain may undergo further reactions. Such inac-tivation could be association with surfaces and/or interaction with other protein molecules lead-ing to aggregation and precipitation. Denaturation may be reversible or irreversible. Reversibledenaturation results from high temperature exposure, or in purposeful experimental conditionswhen the protein is exposed to chaotropic agents (e.g., urea, guanidine hydrochloride). Whenthe denaturing condition is removed, the protein will regain its native state and maintain itsactivity. Reversible denaturation can be decreased by the use of additives such as salts that bindto nonspecific binding sites on the proteins (29–31). Preferential hydration of proteins in thepresence of a glycerol-water mixed solvent system is a prerequisite for stabilizing the nativestructure of several globular proteins (32).

Irreversible denaturation means that the protein, once unfolded, will not regain its nativeform and activity. Aggregation phenomena lead to irreversible denaturation.

Protein AggregationAggregation of peptides and proteins is caused mainly by hydrophobic interactions that eventu-ally lead to denaturation. Sources of hydrophobic conditions include exposures to air–liquid andsolid–liquid interfaces, light, temperature fluctuations, impurities, and foreign particles. Whenthe hydrophobic region of a partially or fully unfolded protein is exposed to water, a thermo-dynamically unfavorable situation is created. The normally “buried” hydrophobic interior isnow exposed to a hydrophilic aqueous environment. Consequently, the decrease in entropyfrom structuring water molecules around the hydrophobic region forces the denatured proteinto aggregate, mainly through the exposed hydrophobic regions. Thus, solubility of the proteinmay also be compromised. In some cases self-association of protein subunits, either native ormisfolded, may occur under certain conditions and this may lead to precipitation and loss inactivity (Fig. 8-6). Irreversible aggregation can be minimized, even prevented, through expertformulation approaches involving stabilizers such as surfactants, polyols, or sugars.

Factors that affect protein aggregation in solution generally include protein concentration,pH, temperature, other excipients, and mechanical stress. Some factors (e.g., temperature) canbe easily controlled during compounding, manufacturing, storage, and use. Other factors, (e.g.,mechanical stress, temperature excursions during shipping and distribution, inherent instabilityof the active ingredient) cannot be so easily controlled. Formulation studies will dictate appro-priate choice(s) of pH and excipients that will not induce aggregation and/or, in fact, will aid inthe prevention of aggregation. A new class of alkyl saccharide excipients originally intended todramatically enhance transmucosal absorption of peptide and protein drugs was found to behighly effective in preventing protein aggregation (33). These alkyl saccharide excipients stabi-lize and reduce aggregation of peptides or proteins in therapeutically useful formulations, andthey may provide solutions for aggregation-related manufacturing or formulation problemsand/or unwanted immunogenicity. Examples of proteins stabilized by these excipients (0.125%concentrations) include insulin and growth hormone.

Figure 8-6 Example of aggregated protein.

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The desire to identify stable solution preparations of insulin for use in novel deliverysystems such as continuous infusion pumps, led to the development of test methodologyfor assessing the impact of various additives on physical stability. Insulin (and many otherproteins) physical stability typically is evaluated using thermomechanical procedures involvingagitation or rotation of protein solutions at elevated temperature. Turbidity resulting fromaggregation is usually determined as a function of time by visual inspection or light scatteringanalysis. Alternatively, reductions in the soluble protein content due to precipitation can bequantified by HPLC assay as a function of time. Relative stability is defined by the lengthof time a preparation remains on the test without showing a change in either parameter. Itshould be noted that the greatest difficulty in applying such testing strategies is interpretingthe experimental data and correlating it in a practical way to “real life” conditions that theformulation may actually experience. Nevertheless, regulatory agencies may request data fromsuch testing to support dating periods or other product claims. Physical stress testing, however,is more appropriately used as a development screening tool to identify the capability of variousadditives to prevent aggregation.

Analytical methods used for determining protein aggregation are listed on pages 177–178(chapter 11 under “Answers for Case Study 10”).

Foreign Particles, Protein Aggregation, and ImmunogenicityThe reality of protein aggregation has raised the concerns about such aggregates, even atsubvisible levels, leading to an immune response resulting from antibody-mediated neutraliza-tion of the protein’s activity or alterations in bioavailability (34,35). Among many causes forprotein aggregation are protein particles resulting either from the protein alone or resultingfrom heterogenous nucleation on foreign micro- or nanoparticles originating from the manu-facturing process (mixing tanks, process tubing, filter systems, filling machines) and from thecontainer/closure system (36). Silicone oil, used as a lubricant for rubber closures on vials andrubber plungers in prefilled syringes also can induce protein aggregation (37).

Large protein aggregates are subvisible particles (smaller than 10 micrometers) that are notcurrently monitored and quantified by compendial subvisible particulate matter measurementsystems. Carpenter, et al. (34) have questioned this current practice and have proposed that(i) scientists from industry and academia work together to define the quantitative capabilitiesof particle counting instruments for particles as small as 0.1 �m, (ii) develop new particlecounting instruments for more reliable measurement of particles at sizes approaching 0.1 �m,and (iii) more studies be conducted and published on the impact of protein aggregation onimmunogenicity including the role of protein class, amount of aggregate, size of aggregates,and protein conformation in aggregates.

Also, the reader is referred to the end of chapter 29 where there is some discussion aboutthe huge variety of biopharmaceutical commercial product package insert language regardingacceptability of visible particulate matter and use of different types of transfer and in-line filters.

ADSORPTIONProteins exhibit a certain degree of surface activity; that is, they adsorb to surfaces due to theirinnate nature of being amphiphilic polyelectrolytes. Consequently biological activity may beeither reduced or totally lost if such adsorption occurs during manufacturing, storage, or use ofthe final product. Insulin has been the most studied protein with respect to surface adsorption.Potential problems may be encountered while delivering insulin because of its ability to adsorbonto the surfaces of delivery pumps, glass containers, and to the inside of the intravenous bags.Insulin adsorption usually is finite once binding sites are covered and such adsorption is usuallynot clinically significant.

Adsorption to surfaces depends on protein–protein interactions, time, temperature, pH,and ionic strength of the medium and the nature of the surface (38). Interactions that determinethe overall adsorption process between a protein and a surface include redistribution of chargedgroups in the interfacial layer, changes in the hydration of the sorbent and the protein surface,and structural rearrangements in the protein molecule. Surface denaturation which commonlytakes place at the liquid–solid and liquid–air interface to involve conformational changes suchas loss of �-helices to �-sheets and certain random structures (39). These structural changes,

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Table 8-3 Possible Strategies to Overcome Protein Adsorption

� Increase protein concentration during filtration and/or use extra volume to saturate the filter with proteinsolution

� Modify (e.g., siliconize) the surface of the glass containers, providing a resistant barrier to protein-surfaceinteraction

� Decrease the rate of mixing when it is known that shear will affect protein adsorption� Add excipients such as surfactants that have higher surface activity� Add macromolecules such as albumin and gelatin (must be synthetic) to complete for binding sites on the

surface.

determined by the nature of the interfaces, are similar to those observed with aggregationcaused by heat, high pressure, or chemical denaturants. In the case of proteins, sources suchas the polymer of the membrane filter, the administration set, agitation that occurs during thepurification process as well as the method of manufacture are known or at least suspectedto cause surface denaturation. Strategies often used to overcome protein denaturation due toadsorption are presented in Table 8-3.

SURFACTANTSSurface-active agents (surfactants) exert their effect at surfaces of solid–solid, solid–liquid,liquid–liquid, and liquid–air because of their chemical composition containing both hydrophilicand hydrophobic groups (see chap. 6). Surfactants effectively compete against proteins for theseinterfacial hydrophobic locations, thus helping to minimize protein adsorption and potentialaggregation.

Generally, ionic surfactants can denature proteins. However, nonionic surfactants usuallydo not denature proteins even at relatively high concentrations (1% w/v) (40). Most parenterallyacceptable nonionic surfactants come from either the polysorbate (sorbitol-polyethylene oxidepolymers) or polyether (polyethylene oxide-polypropylene oxide block co-polymers) groups.Polysorbate 20 and 80 and sodium dodecyl sulfate are effective and acceptable surfactant sta-bilizers in marketed protein formulations (Table 6-6). The chemical structure of polysorbates,factors affecting micelle formation and degradation pathways of polysorbates 20 and 80 are thesubject of a review article by Kerwin (41). Effectiveness of polysorbate stabilization is dependenton the structure of polysorbate (monomer or micelle) and polysorbate–protein ratio (42). Othersurfactants that have been used in protein formulations for clinical studies and/or found in thepatent literature include Pluronic F68, and other polyoxyethylene ethers (e.g., the “Brij” class).

The choice of surfactant and the final concentration optimal for stabilization is quitedependent on a variety of factors including other formulation ingredients, for example, sugars,protein concentration, headspace in the container, the type of container, and test methodology.

Recombinant hGH will aggregate readily under mechanical and thermal stress. Aggre-gation from mechanical stress can be substantially reduced in the presence of surfactants (43).Mechanical stress may cause proteins to be more exposed to air–water interfaces where denat-uration is more likely to occur than in the bulk phase of water. Surfactants will preferentiallycompete with proteins for accumulation at the air–water interface and keep the protein fromundergoing interfacial denaturation resulting from mechanical stress. Pluronic F68 and Brij35 will stabilize hGH at their critical micelle concentrations (0.1% and 0.013%, respectively),whereas stabilization with polysorbate 80 requires a concentration of 0.1%, higher than the crit-ical micelle concentration value for polysorbate 80 of 0.0013%. The reasons for these differencesin stabilizing concentrations are not clear, but simply reflect differences in interactions betweendifferent surfactants and proteins. It is interesting to note that these surfactants do not stabilizehGH from aggregation due to high temperature stress.

Surface-active agents, particularly polysorbate 80, protect proteins against surface-induced denaturation during freezing (44). A strong correlation exists between freeze denatu-ration (quick freezing of the protein) and surface denaturation (shaking the protein in solution).Proteins that tend to denature under these conditions are protected by the addition of polysor-bate 80 (0.1%). Other surfactants—Brij 35, Lubrol-px, Triton X-10, and even the ionic surfactant,

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Figure 8-7 Effect of Tween 80 concentration on particle formation in solutions of recombinant human hemoglobinas a function of shear stress. Source: From Ref. 46.

sodium dodecyl sulfate—also protected the protein from denaturation although these surfac-tants have not yet been approved for use in injectable formulations. The authors pointed out thatsurfactants may be needed to protect proteins from denaturation during the freezing step only,and that other stabilizers, for example, sucrose, may be needed to further protect the proteinduring freeze drying.

Surfactants were ineffective in preventing BST aggregation and precipitation in solutionat elevated temperature1, whereas other stabilizers such as sucrose were more effective (45).Tween 80 was more effective in reducing the amount of measurable particles due to aggregationof recombinant human hemoglobin (Fig. 8-7).

1 While polysorbate 80 was not effective in stabilizing BST at elevated temperature, it was effective when theapplied stress was agitation. Also, the authors noted that polysorbate 80 destabilization of BST was not observedat ambient or refrigerated temperatures as other decomposition pathways, for example, deamidation, becamemore predominant at lower temperatures.

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Peroxides are known contaminants of nonionic surfactants (19). Peroxide levels fromdifferent sources of polysorbate 80 ranged from less than 1 mEq/kg to more than 27 mEq/kg.Peroxide levels increased upon storage at ambient temperatures probably due to headspace oxy-gen and/or the container/closure interface allowing ingress of air. Peroxides in polysorbate canresult in oxidative degradation of proteins. Improvements have been made in the manufacturingof polysorbate, for example, certified peroxide-free polysorbates are now readily available.

Electron paramagnetic resonance (EPR) spectroscopy has been used to determine thebinding stoichiometry of the surfactant to the protein and, thus, what potentially is the optimalamount of surfactant to use to stabilize the protein against surface denaturation and otherphysical instability reactions (47).

CYCLODEXTRINSCyclodextrins are cyclic (�-1,4)-linked oligosaccharides of �-D-glucopyranose containing a rel-atively hydrophobic central cavity and hydrophilic outer surface. Cyclodextrins come in a widevariety of structural derivatives, the most common being �-, �-, and � -cyclodextrins, whichconsist of six, seven, and eight glucopyranose units, respectively. Two parenteral cyclodex-trins are EncapsinTM, a hydroxylpropyl-�-cyclodextrin, and CaptisolTM, a sulfobutylether-�-cyclodextrin. They have been used widely for increasing the solubility stability, and bioavail-ability of small drug molecules (see chap. 6). Peptides and proteins can also be stabilized incyclodextrin complexes. �-cyclodextrins at a 25-fold excess stabilized leucine enkephalin againstenzymatic degradation in sheep nasal mucosa (48). Hydroxypropyl-�-cyclodextrin at a 1% con-centration was shown to enhance the reconstituted solution stability of keratinocyte growthfactor (49) and several other proteins in solution (50). Glucagon will form inclusion complexeswith � -cyclodextrin in acidic solution that results in enhancement of glucagon’s physical andchemical stability (51).

ALBUMINSerum albumin is a widely used stabilizer in protein formulations for minimizing proteinadsorption to glass and other surfaces (Table 8-4). Albumin preferentially competes with otherproteins for binding sites on surfaces, but why this is so is not clear.

Because albumin is a natural protein, concerns have been raised about potential con-tamination of albumin with human prion protein that is thought to be the infectious agent inbovine spongiform encephalopathy (BSE). Indeed, the use of animal-source excipients (and thisincludes not only albumin, but also glycerol and polysorbate 80) is no longer practiced. Thedevelopment of synthetic (e.g., recombinant HSA) versions of these materials has eliminatedconcerns over potential disease transmission.

OTHER PHYSICAL COMPLEXING/STABILIZING AGENTSPEG is a common co-solvent for solubilizing small nonproteinaceous molecules and may min-imize the aggregation of several peptides and proteins (52). PEG modification of proteins forsustained-release purposes has seen wide application. The concentration of PEG needs to be

Table 8-4 Some Examples of Commercial Protein Dosage FormsContaining Human Serum Albumin

Generic Brand R© % HSA in product

Alglucerase Ceredase 1.0Erythropoietin Epogen 0.25Interferon Alpha-2a Roferon-A 0.5Interferon Alpha-2b Intron-A 0.1 (after reconstitution)Urokinase Abbokinase 5.0 (after reconstitution)Alpha-1-Proteinase Aralast 0.5 (after reconstitution)Antihemophilic factor Recombinate 1.0 (after reconstitution)Botulism toxin Myobloc 0.05Streptokinase Streptase 2.0 (after reconstitution)Hyaluronidase Halozyme 0.1

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fairly low (< 1%, w/v) to serve as a stabilizer; otherwise at higher concentrations (>10% w/v)it can cause precipitation (53).

Poly(vinylpyrrolidone) (PVP) also is like PEG in that at low concentrations it canstabilize proteins, whereas at high concentrations it may help lead to protein aggregation andprecipitation. PVP at low concentrations (≤2.0%) effectively stabilizes human IgM monoclonalantibody against heat-induced aggregation, whereas PVP concentrations ≥5.0% will causeaggregation (54).

Fibroblast growth factors, acidic and basic, are prone to acid and thermal inactivation andcan be stabilized by a number of heparin and heparin-like molecules (25). Human keratinocytegrowth factor, also prone to aggregation at high temperature, is stabilized by heparin, sulfatedpolysaccharides, anionic polymers, and citrate ion (55).

OPTIMIZING MICROBIOLOGICAL ACTIVITY

Antimicrobial PreservativesMany products (perhaps around 25%) are commercially available as multiple-dose formulations.If a sterile product is intended for multiple dosing, then it must contain an effective antimicrobialpreservative (AP) agent. AP agents are formulated with the active pharmaceutical ingredient ifthe product is a ready-to-use solution or is part of the diluent used to reconstitute freeze-driedproducts intended for subsequent multiple dosing. While rare, there are examples of AP agentsformulated within the freeze-dried product and not part of the diluent.

Of 145 peptide and protein drug products listed in 2006 Physicians’ Desk Reference,36 contained preservatives (56). Most vaccine products used to contain AP agents, especiallythimerosal, but by 2006, only 8 vaccine products still were formulated as multidose products.

The most common APs used in multiple-dose formulations are phenol, meta-cresol, andbenzyl alcohol. Less common, especially for new formulations, but still used APs include methyland propylparaben. Some, although very few, vaccines still contain APs with phenoxyethanolbeing the most common. Thimerosal used to be commonly used for vaccine products, but nottoday. Examples of use of these preservatives are listed in chapter 6 (Table 6-7).

Use of antimicrobial agents requires passing a preservative efficacy test (PET) (USPchap. <51> provides the directions for conducting this test). Unfortunately, the United StatesPharmacopeia (USP) and the British and/or European Pharmacopeial (BP/EP) tests for PETare different in their requirements. Table 8-5 summarizes the differences between the tests. TheUSP basically requires a bacteriostatic preservative system while the BP/EP requires a bacteri-ocidal system. For example, the USP requires a 3-log reduction in the bacterial challenge by the14th day after inoculation, while criteria A of the BP/EP test requires the same 3-log reductionwithin 24 hours. This great difference in compendial requirements for preservative efficacy hascaused many problems in the formulation of protein dosage forms for various markets. Oneunpublished example involved a new protein product where the scientist developing the for-mulation was unaware of the different compendial requirements. The focus was minimizinginstability of the new protein in the presence of the AP and used a minimal amount of AP inthe formulation. The phase 1 clinical study was scheduled for a European clinic so the EP PETwas performed. The formulation failed miserably and the product had to be reformulated withstart of the clinical study delayed by almost a year.

Passing the BP/EP PET requires the use of relatively high amounts of phenol or cresolor other AP that may have an impact on the stability of the formulation and could result insorption of the preservative into the rubber closure. The formulator must keep in mind that

Table 8-5 Comparison of USP and EP Preservative Efficacy Tests

Test USP <51> EP <Chapter 5.1.3>

Bacterial 1-log reduction within 7 days 2-log reduction within 6 hoursChallenge 3-log reduction with 14 days 3-log reduction within 24 hoursFungal challenge No increase after 28 days 2-log reduction with 7 daysOverall requirement Bacteriostatic Bacteriocidal

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140

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00.7 0.9 1.1 1.3

Benzyl Alcohol (%)

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Loss in ellipticity at 280 nm 280 of rhIFN-γ as function of benzyl alcohol concentrationin 16 mM acetate buffer at pH 5.0 ( ) and 16 mM succinate buffer at pH 5.0 ( ).

Figure 8-8 Effect of benzyl alcohol on recombinant human interferon gamma aggregation. Note: Tobler, et al.J Pharm Sci, June, 2004 used hydrogen-deuterium isotope exchange detected by MS to detect tertiary structurechanges that involve only a limited part of this protein still causing irreversible loss of activity. Benzyl alcoholcauses protein to unfold forming very large aggregates. Source: From Refs. 62 & 64.

increasing the concentration of APs may have a negative impact on protein physical stability(precipitation, aggregation, etc). Increasing AP levels will increase the hydrophobicity of theformulation and could affect the aqueous solubility of the protein. Increasing AP concentrationsalso increases the potential for toxicological hazards.

It is well known that APs not only protect insulin formulations against inadvertent con-tamination, but also may have a significant effect on protein stability. For example, phenolicpreservatives have a profound effect on the conformation of insulin in solution (57) and theassembly of the specific type of LysPro insulin hexamer (58). Furthermore, phenol and/orm-cresol in insulin solutions will have a tendency to be adsorbed by and permeate rubberclosures (59). Therefore, rubber formulations must be designed to minimize these potentialproblems.

APs are known to interact with proteins and can cause stability problems such as aggre-gation. For example, phenolic compounds will cause aggregation of hGH (60). Phenol willproduce a significant decrease in the �-helix content of insulinotropin resulting in aggregationof �-sheet structures (61). Benzyl alcohol, above certain concentrations and depending on otherformulation factors, will interact with recombinant human interferon-� causing aggregationof the protein (Fig. 8-8) (62). Other examples are granulocyte-stimulating factor and recombi-nant interleukin-1R (56). These examples point out the need for the formulation scientist tounderstand the importance of potential effects of preservative type, concentration, and otherformulation additives on the interaction with proteins in solution while balancing the needs forantimicrobial efficacy.

In determining the appropriate AP agent or agents, insulin was studied as the proteinto be preserved and combining insulin with different types of AP agents either alone or incombination (63). These formulations were challenged with the five USP PET organisms andD values2 determined. The D-value determination allows a single-quantitative estimate of theAP effectiveness of a certain agent or combination of agents in a specific formulation against aspecific microorganism. The preservative combination of 0.2% phenol and 0.3% m-cresol gavethe lowest D-value (fastest time required for a 1-log reduction in the initial inoculum of S. aureusand, thus, was the most effective AP system in this particular insulin formulation.

There are instances where a manufacturer, because of concerns regarding aseptic process-ing and sterility assurance of the product throughout its shelf-life, will add an AP agent in

2 D value = Time required for a 1-log reduction in the microbial population due to the effect of the antimicrobialpreservative system. The smaller the D value, the greater the effect of the preservative on the microorganism inquestion. Covered in chapter 18.

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the protein formulation even though it is intended only for a single-dose injection. This is avery controversial practice. Regulatory agencies worldwide object to this approach if, in theiropinion, the use of APs in a single-dose injectable product is practiced in order to “cover up”for inadequate aseptic manufacturing practices and controls.

Many countries require PET be performed for routine stability protocols and for specialstability studies. Also there may be requests from agencies to do PET on containers that havebeen used (i.e., penetrated; partial volume withdrawn) to demonstrate that the product can stillkill microorganisms. In mid-1995, the Australian Drug Evaluation Committee (ADEC) passedresolutions that in light of safety concerns with contamination and cross-contamination, the useof injectable products in multi-dose packages is discouraged. In order to support the use of amultidose product and the shelf-life once a package has been reconstituted or opened for use,AP efficacy data are required for approval.

OSMOLALITY (TONICITY) AGENTSSalts or nonelectrolytes (e.g., glycerin) are added to protein formulations in order to achievean isotonic solution. Nonelectrolytes often are preferred over salts as tonicity adjusters becauseof the potential problems salts cause in precipitating proteins. Generally, solutions containingproteins administered IV, IM, or SC do not have to be precisely isotonic because of immediateeffects from dilution by the blood. Intrathecal and epidural injections into the cerebrospinal fluidrequire very precise specifications for the product to be isotonic and at physiological pH. This isbecause extremes in osmolality and/or pH can damage or destroy cells and cerebrospinal cellscannot be reproduced or replaced.

SIMPLE EXERCISEFor each of these commercial sterile solution formulations, name the purpose of each excipient.

Nebcin R© (Lilly)Tobramycin 80 mgSodium bisulfite 5 mgDisodium EDTA 0.1 mgPhenol 5 mg

Valium Injection (Roche)Diazepam 5 mgPropylene glycol 40%Ethanol 10%Benzoic acid/Sodium benzoate 5%Benzyl alcohol 1.5%

Nutropin AQ R© (Genentech)Somatropin 10 mgSodium chloride 17.4 mgPhenol 5 mgPolysorbate 20.4 mgSodium citrate 10 mM

Rebif R© (Serono)Interferon beta-1 a 44 mcgHuman albumin 4 mgMannitol 27.3 mgSodium acetate 0.4 mg

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