Formulation and Stability of Cytokine Therapeutics

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Formulation and Stability of Cytokine Therapeutics 1 1 ¨ ¨ TIINA LIPIAINEN, MARIKKI PELTONIEMI,1 SANJAY SARKHEL,1 TEIJO YRJONEN, HEIKKI VUORELA,1 ARTO URTTI,1,2 ANNE JUPPO1 1 2

University of Helsinki, Faculty of Pharmacy, Helsinki, Finland University of Eastern Finland, School of Pharmacy, Kuopio, Finland

Received 30 June 2014; revised 29 September 2014; accepted 13 October 2014 Published online in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.24243 ABSTRACT: Cytokines are messenger proteins that regulate the proliferation and differentiation of cells and control immune responses. Interferons, interleukins, and growth factors have applications in cancer, autoimmune, and viral disease treatment. The cytokines are susceptible to chemical and physical instability. This article reviews the structure and stability issues of clinically used cytokines, as well as formulation strategies for improved stability. Some general aspects for identifying most probable stability concerns, selecting excipients, and developing stable cytokine formulations are presented. The vast group of cytokines offers possibilities for new biopharmaceuticals. C 2014 Wiley The formulation approaches of the current cytokine products could facilitate development of new biopharmaceuticals.  Periodicals, Inc. and the American Pharmacists Association J Pharm Sci Keywords: biopharmaceuticals characterization; biosimilar; excipients; injectables; lyophilization; oxidation; protein aggregation; protein formulation; protein structure; solid state

INTRODUCTION Cytokines are small secreted proteins that enable receptormediated communication between cells and act as key modulators of immune responses by controlling growth, differentiation, and activation of various cell types. Hundreds of cytokines have been identified1 and some of these proteins are currently used in therapeutic products, mostly for treating cancer, autoimmune, and viral diseases (Table 1). The biological mechanisms and clinical use of cytokines have been well reviewed,1–5 but aspects of formulation and stability have been less widely studied. Here, we review cytokine therapeutics marketed in the United States and in Europe, aspects related to their structural instability, and the role of formulation excipients toward improving stability. There are decades between the market introduction of the first and the most recent cytokine products. Formulation strategies have evolved during that time and some of the first-generation products have been reformulated. The main difference to the older approaches is the current preference of albumin-free formulations. In addition, cytokines with conjugated polyethylene glycol (PEG) chains have been introduced to market. These pegylated proteins have greatly increased halflives compared with the unmodified cytokines, allowing more convenient administration regimes. Cytokines bind to the cell surface cytokine receptors with high affinity, making them high-potency molecules. Thus, cytokines are generally administered at low doses and they have a narrow therapeutic index. For this reason, structural stabilAbbreviations used: CHO, Chinese hamster ovary; G-CSF, granulocyte colony-stimulating factor; GM-CSF, granulocyte-macrophage colonystimulating factor; EDTA, ethylenediaminetetraacetic acid; EMA, European Medicines Agency; Fimea, Finnish Medicines Agency; HSA, human serum albumin; MAH, marketing authorization holder; PEG, polyethylene glycol; SDS, sodium dodecyl sulfate. ¨ Correspondence to: Tiina Lipiainen (Telephone: +358-294159346; Fax: +358294159138; E-mail: [email protected]) Journal of Pharmaceutical Sciences  C 2014 Wiley Periodicals, Inc. and the American Pharmacists Association

ity is a critical factor in ensuring biological function and efficacy. Low concentrations of active ingredient cause difficulties characteristic to cytokine products. Analysis of the cytokine protein structure and stability becomes challenging, especially if the product additionally contains albumin as a stabilizing excipient. Another important result is loss of protein because of adsorption on container or other surfaces. Most cytokines have a helical bundle fold, generally associated with marked hydrophobicity.6 Hydrophobicity can lead to problems related to solubility, tendency to aggregate and adsorption on surfaces, and consequent challenges in manufacturing and long-term storage. Physical and chemical instabilities may also lead to the formation of immunogenic degradation products. Oxidation is a major chemical degradation pathway for cytokines.7–13 Cytokine aggregates, especially those formed by oxidized proteins, have been linked to immunogenicity.9,10 Immunogenicity of therapeutic proteins is a topic of concern that has gained wide interest lately.14–17 Many of the cytokine products are intended for long-term use, and many patients eventually develop an antibody response to the protein drug.15 Antibodies formed against therapeutic proteins may cause serious adverse effects if they cross-react with endogenous proteins.18 Protein aggregates are a key risk factor for immunogenicity.16 Therefore, the hydrophobicity and aggregation tendency of cytokines is a matter of concern, and a major aim in formulation development is to inhibit unfolding and aggregation. In this review, we have covered the available information on structural characteristics (Table 2) and degradation pathways of interferon-alpha (IFN-"), interferonbeta (IFN-$), interferon-gamma (IFN-(), interleukin-2 (IL-2), IL-11, granulocyte colony-stimulating factor (GCSF), and granulocyte-macrophage colony-stimulating factor (GM-CSF). As a result, we attempt to identify common properties within these molecules and formulation strategies, and to provide a rationale for developing stable cytokine products. Lipi¨ainen et al., JOURNAL OF PHARMACEUTICAL SCIENCES

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Table 1. Cytokine IFN-"

IFN-$

IFN-(

IL-2

IL-11

Cytokine Products Approved for Clinical Use in the United States and Europe145–147 Trade Name (Company)

Active Ingredient

Production Host

Indication

Year of Authorization (Responsible Agency)

IntronA (US: Schering, EU: Merck Sharp and Dohmea )

IFN-"-2b

E. coli

Roferon-A (Roche)

IFN-"-2a

E. coli

PegIntron (EU: Mercka ,US: Schering) ViraferonPeg (Mercka ) Sylatron (Schering) Pegasys (Roche)

Peginterferon-"-2b

E. coli

Multiple myelomab Chronic myelogenous leukaemiab Chronic hepatitis B Carcinoid tumorb Hairy cell leukemia Follicular lymphoma Malignant melanoma Chronic hepatitis C Condylomata acuminatac AIDS-related Kaposi’s sarcomac Hairy cell leukemia AIDS-related Kaposi’s sarcomab Chronic myelogenous leukaemia Cutaneous T cell lymphomab Chronic hepatitis Bb Chronic hepatitis C Follicular lymphomab Renal cancerb Malignant melanomab Chronic hepatitis C

Peginterferon-"-2b

E. coli

Chronic hepatitis C

2000 (EMA)

Peginterferon-"-2b Peginterferon-"-2a

E. coli E. coli

Infergen (US: Intermune Pharms) Alferon N Injection (Interferon Sciences)

Interferon-alfacon-1

E. coli

Melanoma Chronic hepatitis B Chronic hepatitis C Chronic hepatitis C

2011 (FDA) 2002 (EMA) 2002 (FDA) 1997 (FDA)

IFN-"-n3

Human leucocytes

Condylomata acuminata

1989 (FDA)

Betaseron (US)/Betaferon (EU) (Bayer) Extavia (Novartis)

IFN-$-1b

E. coli

Multiple sclerosis

1993 (FDA) 1995 (EMA)

IFN-$-1a

CHO cells

Multiple sclerosis

Avonex (Biogen Idec)

IFN-$-1a

CHO cells

Multiple sclerosis

Rebif (US: Serono, EU: Merck Serono Europe) Actimmune (US) (Intermune/Vidara Ther.) /Imukin (EU) (Boehringer Ingelheim) Proleukin (EU: Novartis, US: Chiron) Ontak (Eisai) Neumega (Wyeth Pharms Inc.)

IFN-$-1a

CHO cells

Multiple sclerosis

2008 (EMA) 2009 (FDA) 1996 (FDA) 1997 (EMA) 1998 (EMA) 2002 (FDA)

IFN-(-1b

E. coli

Chronic granulomatous disease Osteopetrosis

1994 (Fimea) 1999 (FDA)

Aldesleukin (rhIL-2)

E. coli

Renal cell carcinoma Melanomac

1992 (FDA) 1992 (Fimea)

Denileukin diftitox Oprelvekin (rhIL-11)

E. coli E. coli

T-cell lymphoma Chemotherapy-induced thrombocytopenia

1999 (FDA) 1997 (FDA)

1986 (FDA) 2000 (EMA)

1986 (FDA) 2001 (Fimea, Finnish Medicines Agency)

2000 (EMA) 2001 (FDA)

Continued

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Table 1. Cytokine G-CSF

GM-CSF

3

Continued Trade Name (Company)

Active Ingredient

Production Host

Indication

Year of Authorization (Responsible Agency)

Neupogen (Amgen)

Filgrastim (recombinant methionyl hG-CSF)

E. coli

Neutropenia Hematopoietic stem cell transplantation Cancer

1991 (FDA) 1991 (Fimea)

Biograstim (MAH: AbZ-Pharma; Batch release: Merckle Biotec) Ratiograstim (MAH: Ratiopharm; Batch release: Merckle Biotec) Tevagrastim (MAH: TEVA Generics; Batch release: Teva Pharma) Filgrastim Hexal (MAH:Hexal; Batch release: Sandoz) Zarzio (Sandoz) Nivestim (Hospira) Grastofil (Apotex Europe) Granocyte (ChugaiSanofi-Aventis)

Filgrastim Biosimilar, reference drug Neupogen

E. coli

Neutropenia Hematopoietic stem cell transplantation Cancer

2008 (EMA)

Lenograstim (rhG-CSF)

CHO cells

Neulasta (Amgen)

Pegfilgrastim (pegylated r-metHuG-CSF) Lipegfilgrastim (glycol-pegylated r-metHuG-CSF)

E. coli

Sargramostim (rhGM-CSF)

Lonquex (MAH: Teva; Batch release TEVA and Merckle Biotec) Leukine (Berlex/Sanofi)

2008 (EMA)

2008 (EMA)

2009 (EMA)

Neutropenia

2009 (EMA) 2010 (EMA) 2013 (EMA)

Neutropenia Hematopoietic stem cell transplantation Cancer Neutropenia Cancer

1994 (Fimea)

E. coli

Neutropenia

2013 (EMA)

S. cerevisiae

Bone marrow transplantation Cancer

1991 (FDA)

2002 (FDA) 2002 (EMA)

a

Marketing authorization transferred from Schering-Plough Europe in 2011–2012. Indication approved by EMA or Fimea only. Indication approved by FDA only. MAH, marketing authorization holder. b c

Pharmaceutical Protein Formulations As for proteins in general, structural integrity of a cytokine is critical for its biological function. Changes in the native structure often lead to loss of activity, as the cytokine no longer binds its receptor in a proper manner, and may cause harmful immunogenic reactions. Proteins are prone to alterations in their tertiary fold (physical instabilities, e.g., unfolding and aggregation) and/or modifications in their primary structures (chemical degradation, e.g., oxidation, deamidation, disulfide scrambling, and proteolysis).19–22 The risk of such changes is pronounced when the environmental conditions are different from the physiological conditions where the protein has evolved to function. Furthermore, the product protein concentration generally differs markedly from physiological conditions, bringing about additional stress to the protein. Product manufacturing and processing steps including purification, filtration, and filling, DOI 10.1002/jps.24243

as well as transportation, storage, and administration expose the protein to various stress factors.23 Shear forces, contacts with various interfaces, and temperature variations increase the risk of protein degradation.24 The magnitude of this risk can be greatly mitigated by efficient formulation.25–30 When formulating a pharmaceutical protein, the choice of excipients, ionic strength, and pH need to be optimized for a stable and safe product. The storage conditions and shelf-life of a product depends on the stability of the formulation. Product shelf-lives may reflect business decisions rather than absolute protein stability, though. Stability studies to extend product shelf-life can be time-consuming and expensive and might not give any further value for the company. Pharmaceutical protein formulation development is often based on previous experience and on the process of trial-anderror.21 One of the first steps of development is the choice of an ideal buffer system to maintain optimal pH. Commonly Lipi¨ainen et al., JOURNAL OF PHARMACEUTICAL SCIENCES

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165–166

165

166

143 (x2)

133

178 174

127

IFN-"

IFN-"2

IFN-$

IFN-(

IL-2

IL-11 G-CSF

GM-CSF

177 174 (rhG-CSFc ) 175 (r-methG-CSFd ) 127

132

140

166 (IFN-$-1a) 165 (IFN-$-1b)

166 (IFN-"-con) 165–166 (IFN-"-n3) 165

Product

15–23

19.0 ∼20

15–18

30–50

∼23

19–21

17.5–22.0

Natural

19.0 ∼20 (rhG-CSF) 18.8 (r-metHuG-CSF) 19.5, 16.8, and 15.5

15.3

16.5 (x2)

19.4 (IFN-"-con) 16.0–27.0 (IFN-"-n3) 19.2 (IFN-"-2a) IFN-"-2b 22.5 (IFN-$-1a) 18.5 (IFN-$-1b)

Product

Molecular Weight (kDa)

Yes (six sites)

No Yes (one site)

No Yes (rhG-CSF) No (r-methG-CSF) Yes

b

No

No

Yes/Noa (two sites) Yes (one site)

Yes (IFN-$-1a) No (IFN-$-1b)

No

No

Product

Yes

Yes (one site)

Most subtypes not (one site)

Natural

Glycosylation

IFN-( has two glycosylation sites on each monomer, and of these 0, 1, or 2 may be occupied by a carbohydrate chain. 3D structure has not been verified. c Lenograstim. d Filgrastim.

a

Natural

Number of Amino Acids

Summary of Cytokine Protein Characteristics

Cytokine

Table 2.

Two

One (+ one free Cys. In rhIL-2, Cys is substituted by Ser.) 0 Two (+ one free Cys)

One (+ one free Cys. In IFN-$-1b, Cys is substituted by Ser.) 0

Two

Two

Number of Disulfide Bridges

Helical bundle

Helical bundle Helical bundle

Dimer of two helical bundles Helical bundle

Helical bundleb Helical bundle

Helical bundle, also $-sheet content

∼6 ∼6 ∼9 ∼10

∼11 ∼6 ∼5

∼7

Overall Fold

Isoelectric Point

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used buffers include phosphate (pKa = 7.21), acetate (pKa = 4.76), citrate (pKa = 6.39), histidine (pKa = 6.0), and Tris(hydroxymethyl)aminomethane (pKa = 8.10) in pH ranges of 4–7.21 Pain perception associated with injection may vary with buffer choice. Subcutaneous injection of a citrate-buffered solution was found more painful than a histidine-containing solution, for example.31 Often a liquid formulation is desired. In case long-term stability cannot be achieved by liquid formulation, the formulation may be dried by lyophilization. In the dry solid state, degradation reactions are generally slowed down, facilitating storage at higher temperatures. This is because of greatly reduced water content and molecular mobility compared with solutions. However, the drying process and storage in the dry state cause stress to the protein. Therefore, additional stability-enhancing excipients are usually required. As a general rule, minimum number of excipients necessary should be used, but specific needs relating to inhibition of protein degradation and administration route requirements often necessitate the inclusion of many excipients. The most commonly used excipients include protein stabilizers (e.g., sugars, polyols, polymers, and amino acids), albumin, surfactants (e.g., polysorbates and poloxamers), bulking agents for dry formulations (e.g., mannitol and glycine), antioxidants (e.g., methionine), chelating agents [ethylenediaminetetraacetic acid (EDTA) and citric acid], tonicity modifiers (e.g., NaCl and sucrose), and preservatives for multidose products (e.g., benzyl alcohol, phenol, metacresol, and chlorbutanol).21,26,27,29,30 Formulations of therapeutic cytokine products currently on the market are presented in Table 2. The roles of specific excipients in the formulations and stability of each cytokine are discussed in the following sections. Interferon Alpha Interferon-alpha was the first recombinant cytokine approved for therapeutic use (IntronA and Roferon-A in 1986). It has antiviral, antiproliferative, and immunoregulatory properties, mechanisms of which are still not completely understood. Several reviews have covered this topic of interest.32–34 The diverse biological activities of IFN-" render therapeutic applications in cancer and viral disease treatment (Table 1). Interferon-alpha is a family of multiple protein species.34 Fourteen genes encode for different interferon alphas harboring post-translational modifications. They are composed of 165 or 166 amino acids and contain two conserved disulfide bonds. Some IFN-"s are glycosylated and their molecular weights vary in the range of 17.5–22 kDa. Alferon, a product manufactured from pooled human leukocytes, contains a mixture of interferon subtypes (at least 14) as active ingredient labeled as IFN-"-n3.35 The product is claimed to have reduced immunogenicity compared with the other interferon products, probably because of the presence of natural glycosylation and multiple subtypes instead of only one.35 However, there are safety concerns related to natural source materials because of the risk of blood-borne diseases in plasma-derived products.3 All other products contain recombinant proteins, either IFN-"2 or interferon alfacon-1. The latter is a non-naturally occurring protein, with a synthetic consensus sequence derived by scanning several natural IFN-" subtype sequences and selecting the most frequently observed amino acid in each corresponding position. Interferon alfacon-1 possibly has higher activity than the natural interferons.36 DOI 10.1002/jps.24243

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Interferon-alpha-2 is the most widely used and the best characterized IFN-". It contains 165 amino acids and occurs naturally in three subtypes (2a, 2b, and 2c). IFN-"-2a is the active ingredient in products marketed by Roche (Roferon-A and Pegasys), whereas the Schering/Merck products (IntronA, PegIntron ViraferonPeg, and Sylatron) contain IFN-"-2b as the active component. These two variants only differ by one amino acid: position 23 is occupied by lysine in IFN-"-2a and by arginine in IFN-"-2b. The variants have similar structure and biological activity. In both, the three-dimensional structure is formed by a cluster of five "-helices.37,38 Although topographically similar to other helical bundle proteins, the fifth helix (also present in IFN-$) is a unique feature, distinguishing the IFN-fold. Klaus et al.37 have suggested that IFN-"-2a might have more stability in the monomeric form than IFN-"-2b when stressed at high concentration and low ionic strength. Two pegylated forms of IFN-" that are less prone to proteolytic degradation and renal clearance have been developed to improve half-life and efficacy. This allows reduced dosing frequency, from three times a week to once a week. However, pegylation has also raised concerns. PEG-linked proteins with molecular weights less than 70 kDa have resulted in renal accumulation in rats because the PEG molecules cannot be degraded in the lysosomes.39 Furthermore, there has been discussion about the immunogenicity of the PEG moiety itself; however, the conclusions are controversial.40,41 Pegasys contains IFN-"-2a conjugated with a branched 40 kDa PEG moiety, characterized as a mixture of nine positional isomers.42 Pegylated IFN-"-2b is the active ingredient in the identical formulations PegIntron and ViraferonPeg. PegIntron comprises 14 different monopegylated variants (positional isomers), as well as dipegylated and unpegylated (<5%) forms of IFN-"-2a.43 The PEG moiety in PegIntron/ViraferonPeg is a linear 12 kDa polymer. The average molecular weight of the conjugated protein is about half lower as compared with Pegasys (31 kDa vs. 63 kDa). The biological activities and serum half-lives of these two modified interferons are not identical. Sylatron is approved for melanoma treatment by US FDA with a formulation identical to PegIntron except for a higher Peg-IFN-"-2a concentration. The natural human IFN-"2 is glycosylated (O-linked sugar chain at Thr-106) with variation in the sugar composition leading to size heterogeneity of 19–21 kDa.44 However, the therapeutically used recombinant IFN-"s are nonglycosylated and less hydrophilic than the natural protein. The lack of glycosylation does not cause a dramatic reduction in solubility, though, as is the case for recombinant IFN-$.44 The proteins are described water soluble in the manufacturers’ documentation. Interestingly, and somewhat unexpectedly (compared with IFN-$), the glycosylation results in slightly decreased thermal stability of IFN-"-2b (melting temperature decreases from about 65◦ C–66◦ C to 64◦ C).45 Interferon-alpha is acid labile and undergoes structural changes and unfolding below pH 4.46,47 IFN-"-2b is reported to have maximal conformational stability at pH 7, where it may be self-associated as dimers or even higher-order oligomers.46,48 Self-association might favor formation of aggregates and for this reason ways to suppress it have been evaluated. Conjugation to PEG moieties or albumin, or lowering the pH to 4, and increasing ionic strength (e.g., 400 mM NaCl) could change the self-association behavior.48 The marketed products containing IFN-"-2b, IFN-alfacon, or IFN-"-n3 are formulated in pH 7 phosphate buffer. Apparently, the possible self-association does Lipi¨ainen et al., JOURNAL OF PHARMACEUTICAL SCIENCES

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not adversely affect biological activity of IFN-". Different from the other IFN-" products, the IFN-"-2a formulations have acetate buffer and lower pH (Roferon pH 547,49 and Pegasys pH 6). The acetate-containing products are only available as liquid products. Dry product development would require change of buffer system, as acetate is volatile and partially sublimes during lyophilization.25 Loss of acetate results in altered buffer composition and pH shift in the lyophilized product. Interferon-alpha-2a adsorbs to borosilicate glass surfaces, causing loss of protein and possible induction of denaturation especially at low protein concentrations.7 Potential surface adsorption has also been reported for dilute Pegasys samples.42 Polysorbate 80 inhibits adsorption by competing with the protein for the hydrophobic surfaces and interfaces,7,50 and it is included in nearly all IFN-" formulations (at 0.05–0.2 mg/mL). Polysorbate-free IFN-" formulations contain albumin that is known to compete with proteins for surface adsorption sites.51 In fact, polysorbate and albumin seem to be interchangeable in aqueous IFN-" solutions.52 Infergen is a rare cytokine product without surfactant or albumin. This is noteworthy because the IFN concentration in Infergen is low (30 :g/mL) and loss of protein because of surface adsorption is expected. Perhaps the modified sequence of IFN-alfacon results in different surface adsorption tendency compared with the natural sequence IFN-"s. In addition to glass surfaces, contact with chlorobutyl stoppers and the process of sealing the ampoules by heating may cause degradation. This was reported for IFN-"-2b in phosphate buffer, where adsorption and chemical instability (appearance of degradation impurities in HPLC) was observed.7 Improved stability was achieved in citrate or sodium-citrate phosphate buffer, or by adding EDTA or polysorbate 80 in the formulation. EDTA and citric acid act as chelators that bind metal ions.28 Redox-active transition metal ions such as Fe, Cu, Mn, and Cr have the potential to initiate chemical degradation, oxidation in particular.28 These metal ions are often found in pharmaceutical preparations, originated from impurities in excipients (especially in sugars and polymers), or from storage and processing containers (e.g., stainless steel manufacturing vessels).22 Phosphate is not generally named as a metal-chelating excipient for protein formulations, but it might prevent oxidative damage of proteins by binding iron.53 Interferon-alpha contains amino acid residues that are susceptible to oxidation and deamidation reactions. Formation of methionine sulfoxide was suggested as one of the degradation products induced by IFN-"-2b contact with chlorobutyl stoppers.7 Various methionine sulfoxide forms were also observed in another IFN-"-2b stability study; however, deamidation products could not be detected.54 Furthermore, an oxidized form was identified in bulk IFN-"-2b solution, where methionine sulfoxide was present at about 3%. However, it was concluded not to impair the biological activity.8 Even though the oxidation products might not directly affect activity, oxidation is clearly a concern. Oxidized IFN-"-2b is prone to aggregate and in particular, metal-catalyzed oxidation might result in the formation of highly immunogenic aggregates.9 To mitigate the risk of oxidation and immunogenicity from aggregates, EDTA has been included in the IntronA solution formulation. It is the only cytokine formulation with EDTA. Surprisingly, no metal chelators are included in the other IFN-" products and no literature is available on IFN-"-2a oxidation. Lipi¨ainen et al., JOURNAL OF PHARMACEUTICAL SCIENCES

All liquid IFN-" formulations contain sodium chloride as tonicity modifier, whereas the two powder formulations have glycine and sucrose for that purpose (Table 3). Sucrose also acts as a lyoprotectant, providing stability to the dried PEGIFN product. In the dry powder formulation of IntronA, the bulking agent glycine is combined with albumin, which also has stability-enhancing roles as lyoprotectant and cryoprotectant. The multidose products comprise m-cresol, benzyl alcohol, or phenol as antimicrobial preservatives. Infergen represents one of the simplest formulations of all cytokine products: sodium chloride in phosphate buffer. The pegylated IFN products have shelf-lives of 3 years: the longest among all cytokine formulations. Interestingly, a liquid formulation PegIntron was not introduced to the market because of depegylation on storage in this form.55 In contrast, Pegasys is formulated as a solution and it showed stability problems because of PEG-IFN oxidation.56 Degradation of PEG-IFN via oxidation was inhibited by addition of benzyl alcohol,56 although benzyl alcohol is not commonly known to possess antioxidant properties. Interferon Beta Interferon beta belongs to the same category of Type I interferons, binds to the same receptor complex, and activates similar, though not identical, antiviral, antiproliferative, and immunomodulatory effects as IFN-".34 Products containing rhIFN-$ as active substance are used to treat patients with multiple sclerosis. Unlike IFN-", human IFN-$ is only encoded by one gene. IFN-$ is a glycoprotein composed of 166 amino acid residues with a molecular weight of about 23 kDa.57 It has one disulfide bridge, one free cysteine residue, and the tertiary structure is composed of five "-helices, the overall fold being similar to that of IFN-".57 The recombinant protein in Avonex and Rebif, IFN$-1a, is produced in Chinese hamster ovary (CHO) cells. It has an identical sequence and similar glycosylation pattern as the natural protein. On the contrary, the active ingredient in Betaseron, Betaferon, and Extavia, IFN-$-1b, is not glycosylated and has a lower molecular weight of 19 kDa. Furthermore, the sequence of IFN-$-1b has been modified by deleting a methionine residue (Met-1) and substituting cysteine (Cys-17) to serine, for enhanced stability. The free cysteine residue in the native protein is buried but close to the surface, and will readily participate in disulfide scrambling in an event of unfolding, leading to heavy aggregation.57 Despite the high costs of protein production in mammalian cell lines (in this case CHO cells) compared with bacterial fermentation, it has clear advantages with IFN-$. The N-linked carbohydrate complex attached at Asn-80 shows some variation in its composition, but it plays a key role as its removal dramatically reduces solubility of the protein.58 Importantly, Runkel et al.59 reported that the specific activity of IFN-$-1b is 10 times lower compared with IFN-$-1a. The reduction in activity is caused by the lack of glycosylation and the resulting decrease in solubility, not by the sequence modifications.59 They accounted for 60% of IFN-$-1b in Betaseron being lost because of aggregation when exposed to physiological pH. There is a high number of hydrophobic side chains located on the protein surface close to the glycosylation site and the carbohydrate complex exhibits an unusually high degree of order. Therefore, it is believed that the carbohydrates shield the side chains from solvent exposure and stabilize the folded state of the protein.60 DOI 10.1002/jps.24243

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Alferon

Infergen

Pegasys

Sylatron

PegIntron/ ViraferonPeg

Roferon-A

IntronA

IFN-α–2b (23–192 µg/mL) Dibasic sodium phosphate anhydrous (1.8 mg/mL) Monobasic sodium phosphate monohydrate (1.3 mg/mL) Edetate disodium (0.1 mg/mL) Sodium chloride (7.5 mg/mL) m -Cresol (1.5 mg/mL) Polysorbate 80 (0.1 mg/mL) Water for injections (ad 0.5–3.0 mL) Lyophilized powder c IFN-α-2b (38–192 µg) Glycine (20 mg) Sodium phosphate dibasic (2.3 mg) Sodium phosphate monobasic (0.55 mg) Human albumin (1.0 mg) Solvent (1.0 mL): water for injections IFN-α-2a (22 –110 µg/mL) Ammonium acetate (0.77 mg/mL) Sodium chloride (7.2 mg/mL) Benzyl alcohol (10 mg/mL) Polysorbate 80 (0.2 mg/mL) (Acetic acid, Sodium hydroxide) Water for injections (ad 0.5 mL) Lyophilized powder: Peg-IFN-α-2b (74–222 µg) Dibasic sodium phosphate anhydrous (1.11 mg) Monobasic sodium phosphate dihydrate (1.11 mg) Sucrose (59.2 mg) Polysorbate 80 (0.074 mg) Solvent (0.7 mL): water for injections Lyophilized powder: Peg-IFN-α-2b (296 –888 µg) Excipients identical to PegIntron. Peg-IFN-α-2a (135–360 µg/mL) Sodium chloride (8.0 mg/mL) Polysorbate 80 (0.05 mg/mL) Benzyl alcohol (10 mg/mL) Sodium acetate trihydrate (2.6 2 mg/mL) Acetic acid (0.05 mg/mL) Water for injections (ad 0.5 or 1.0 mL), pH 6 ± 0.5 Interferon alfacon-1 (30 µg/mL) Sodium chloride (5.9 mg/mL) Sodium phosphate (3.8 mg/mL) Water for injections (ad 0.3 or 0.5 mL), pH 7.0 ± 0.2 IFN-α-n3 (25 µg/mL) Phenol (3.3 mg/mL) Human albumin (1.0 mg/mL) Sodium chloride (8.0 mg/mL) Sodium phosphate dibasic (1.7 mg/mL) Potassium phosphate monobasic (0.20 mg/mL) Potassium chloride (0.20 mg/mL) Water for injections (ad 1.0 mL), pH 7.4

Formulation (Shaded Background for Liquid Products)

Vial (single use)

Vial

SC Three times a week or daily

IL Two time s a week

Vial, prefilled syringe, prefilled pen/autoinjector (single use )

Vial (single use)

SC Once a week

SC Once a week

Vial Prefilled pen (single use)

Prefilled syringe (single use)

SC Daily or three times a week

SC Once a week

Vial (single use )c

Vial (s ingle use b and multidose) Penb (multidose)

Container

IM, SC, IV, IL Daily or three times a week

IM, SC, IV, ILa Daily or three times a week

Route and Frequency of Administration

Cytokine Product Formulations, Shelf Lives and Special Precautions for Storage145–147

Trade name

Table 3.

Storage at 2°C – 8°C Do not freeze Do not shake

Storage at 2°C – 8°C Do not freeze. Do not shake Protect from light

3 years (vials and syringes) or 2 years (pens) at 2°C – 8°C. 24 h at room temperature Do not freeze. Do not shake. Protect from light

Storage at 25°C 24 h after reconstitution (2°C – 8°C) Do not freeze. Do not shake

3 years (2°C – 8°C or 25°C)e 24 h after reconstitution (2°C – 8°C) Do not freeze Do not shake (pens)

2 years (2°C –8°C) 28 days at or below 25°C Do not freeze Protect from light

3 years d (2°C – 8°C) 24 h after reconstitution (2°C –8°C) Do not freeze

Continued

Single dose vials:18 months (2 °C – 8°C) 7 days at or below 25°C Multidose vials:2 years 28 days after opening (2°C – 8°C) 7 days at or below 25°C Multidose pens:15 months 27 days after opening (2°C – 8°C) Do not freeze

Shelf Life and Precautions

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Neumega

Ontak

Proleukin

Actimmune/ Imukin

Rebif

Avonex

Betaferon/ Betaseron/ Extavia f

Lyophilized powder: IFN-β-1b (300 µg) Human albumin (15 mg ) Mannitol (15 mg) Solvent (1.2 mL): Sodium chloride (5.4 mg/mL) Water for injections Lyophilized powder: IFN-β-1a (33 µg) HSA (16.5 mg) Sodium phosphate dibasic (6.3 mg) Sodium phosphate monobasic (1.3 mg) Sodium chloride (6.4 mg) Solvent (1.0 mL): water for injections, pH 7.3 (after reconstitution) IFN-β-1a (60 µg/mL) Sodium acetate trihydrate (1.6 mg/mL) Acetic acid, glacial (0.5 mg/mL) Arginine hydrochloride (32 mg/mL) Polysorbate 20 (0.05 mg/mL) Water for injections (ad 0.5 mL), pH 4.8 US: IFN-β-1a (44 or 88 µg/mL) HSA (4 or 8 mg/mL) Mannitol (55 mg/mL) Sodium acetate (0.8 mg/mL) Water for injections (ad 0.2 or 0.5 mL) EU: IFN-β-1a (44 or 88 µg/mL) Mannitol (45 or 55 mg/mL) Poloxamer 188 (0.5 mg/mL) L-methionine (0.1 mg/mL) Benzyl alcohol (5 mg/mL) Sodium acetate Acetic acid and NaOH for pH adjustment Water for injections (ad 0.2– 1.5 mL), pH 4 ± 0.5 IFN-γ-1b (200 µg/mL) Mannitol (40 mg/mL) Sodium succinate (0.72 mg/mL) Polysorbate 20 (0.1 mg/mL) Water for injection (ad 1.0 mL) Lyophilized powder Aldesleukin (rhIL-2) (1.3 mg) Mannitol (60 mg) SDS (0.22 mg) Sodium phosphate monobasic (0.20 mg) S odium phosphate dibasic (1.1 mg) Solvent (1.2 mL):water for injections, pH 7.5 ± 0.3(after reconstitution) Denileukin diftitox (150 µg/mL) Citric acid ( 20 mM) EDTA (0.05 mM) Polysorbate 20 (<1%) Water for injection (ad 2.0 mL), pH 6.9 –7.2 Lyophilized powder Oprelvekin (rhIL-11) (5.0 mg) Glycine (23 mg) Dibasic sodium phosphate heptahydrate (1.6 mg) Monobasic sodium phosphate monohydrate (0.55 mg) Solvent (1.0 mL): water for injections (ad 1.0 mL), pH 7.0

Formulation (Shaded Background for Liquid Products)

Continued

Trade name

Table 3.

Vial (single use)

IV Daily during short treatment cycles separated by rest periods.

Vial (single use)

Vial (single use)

SC or IV Daily during short treatment cycles separated by rest periods.

SC Daily

Vial (single use)

Prefilled syringe (single use), Prefilled pen (single use) , Prefilled cartridge for electronic injection device or manual pen-injector device (multidose)

Storage at 2°C –8°C 3 h after reconstitution Do not freeze. Protect from light. Do not shake reconstituted solution.

Store frozen (at or below –10°C)

2 years (2°C– 8°C) 24 h after reconstitution (2°C– 8°C) 48 h after dilution (2°C– 8°C) Do not freeze Protect from light Do not dilute with NaCl solution

3 years (2°C– 8°C) 12 h below 25°C Do not freeze Do not shake

18 months (2°C– 8°C) g 4 days below 25°C Multidose products: 28 days after opening Do not freeze Protect from light

Storage at 2°C– 8°C 30 days below 25°C Do not freeze

Prefilled syringe, autoinjector (single use)

Continued

Vial (single use)

3 years (2°C– 8°C) 7 days at 15°C– 30°C Do not freeze Protect from light

2 years (EU: below 25°C, US: at 2°C–8°C, 30 days at 25°C) 6 h after reconstitution (2°C– 8°C) Do not freeze

Prefilled syringe. Prefilled pen/autoinjector (single use)

2 years (below 25°C) 3 h after reconstitution (2°C– 8°C) Do not freeze

Shelf Life and Precautions

Vial (single use)

Container

SC Three times a week

SC Three times a week

IM Once a week

SC Every other day

Route and Frequency of Administration

8 REVIEW

DOI 10.1002/jps.24243

DOI 10.1002/jps.24243

SC or IV Daily

SC or IV Daily

Vial (single use)

Vial (single use)

Prefilled syringe (single use)

Prefilled syringe (single use)

SC Once after each chemotherapy cycle

SC Once after each chemotherapy cycle

P refilled syringe (single use)

Prefilled syringe (single use)

Prefilled syringe (single use)

Vial Prefilled syringe (single use)

Container

SC or IV Daily

SC or IV Daily

SC or IV Daily

SC or IV Daily

Route and Frequency of Administration

(2°C –8°C) Do not freeze 6 h (without preservative) 20 days at 2°C –8°C (0.9% benzyl alcohol) after reconstitution Do not shake

(2°C –8°C) Do not freeze Do not shake 20 days (2 °C –8°C) after vial entering

2 years (below 30°C) 24 h after reconstitution (2°C –8°C) Do not freeze

2 years (2°C –8°C) 3 days below 25°C Do not freeze Protect from light

3 years (2°C –8°C) 72 h at room temperature Do not freeze. One -time freezing does not cause harm. Do not shake. Protect from light .

Same as Neupogen

36 months (2°C –8°C). 72 h at RT. 24 h after dilution. One-time freezing not harmful. Protect from light. Do not dilute with saline

Same as Neupogen

30 months (2°C –8°C) 24 h at room temperature. 24 h after diluti on. Do not shake. One -time freezing does not cause harm. Do not dilute with NaCl solution.

Shelf Life and Precautions

IL, intralesional; IM, intramuscular; IV, intravenous; RT, room temperature; SC, subcutaneous; WFI, water for injections. a Recommended routes of administration are different in the US and Europe. FDA label states that solution for injection is not recommended for IV administration, instead the lyophilized product should be used for this purpose. In contrast, the European product information documents instruct to administer the liquid product via IV or SC routes. b Only available in Europe. c Only available in the US. d Lyophilized Intron A product currently only authorized by FDA. In a European public assessment report (EPAR) summary, a powder product containing HSA was stated to have a shelf life of 3 years (2◦ C–8◦ C). e Available as single-use vials and prefilled pens. The European product information document recommends storage of both product types at 2◦ C–8◦ C (document revised 5/2010), but an FDA label (revised 6/2013) states that vials should be stored at room temperature (at 25◦ C, excursions permitted to 15◦ C–30◦ C). f Brand name is Betaseron in the US and Betaferon in Europe. Extavia is marketed by Novartis but manufactured by Bayer and has identical composition and pharmaceutical form as Betaseron/Betaferon. g The original Rebif formulation in Europe contained HSA and had a shelf-life of 24 months. h Neupogen Novum, 0.5 mL prefilled syringe filgrastim product licensed in Europe, has an additional strength of 0.96 mg/mL.

Leukine

Granocyte

Lonquex

Neulasta

Nivestim and Grastofil

Filgrastim Hexal/ Zarzio

Biograstim/ Ratiograstim/ Tevagrastim

Neupogen

Filgrastim (r-methG-CSF) (300 or 600 µg/mL)h Acetate (0.59 mg/mL) Sorbitol (50 mg/mL) Polysorbate 80 (0.04 mg/mL) Sodium (0,035 mg/mL) Water for injections (ad 0.5–1.6 mL) Filgrastim (600 µg/mL) Biosimilar, same composition except slight differences in polysorbate content and in pH value compared to Neupogen Filgrastim (600 and 960 µg/mL) Quantitatively identical composition to Neupogen, except that buffer system is glutamate instead of acetate Filgrastim 600 and 960 µg/mL Biosimilars, same composition as Neupogen Pegfilgrastim (pegylated r-methG-CSF) (10 mg/mL protein) Acetate (0.58 mg/mL) Sorbitol (US: 50 mg/mL; EU: 10 mg/mL) Polysorbate 20 (US: 0.03 mg/mL) Sodium (0.03 mg/mL) Water for injections (ad 0.6 mL), pH 4 Lipegfilgrastim (10 mg/mL protein) Acetic acid Sodium hydroxide Polysorbate 20 Sorbitol (30 mg) Water for injections (ad 0.6 mL) Lyophilized powder Lenograstim (rhG-CSF) (105 µg or 263 µg) Arginine (10 mg) Phenylalanine (10 mg) Methionine (1.0 mg) Mannitol (25 mg) Polysorbate 20 (0.1 mg) Hydrochloric acid Solvent (1.0 mL): water for injections, pH 6.5 after reconstitution Sargramostim (rhGM-CSF) (500 µg/mL) Mannitol (40 mg/mL) Sucrose (10 mg/mL) Tromethamine (1.2 mg/mL) Benzyl alcohol (11 mg/mL) Water for injections (ad 1.0 mL), pH 6.7–7.7 Lyophilized powder Sargramostim (rhGM-CSF) (250 µg) Mannitol (40 mg) Sucrose (10 mg) Tromethamine (1.2 mg) Solvent (1.0 mL): WFI or 0.9% benzyl alcohol pH 7.1–7.7

Formulation (Shaded Background for Liquid Products)

Continued

Trade name

Table 3.

REVIEW

9

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In contrast, the corresponding sites in IFN-" (not close to its Olinked carbohydrate chain) are occupied by charged residues.60 Even though IFN-$-1a is much less hydrophobic than IFN$-1b, aggregation and surface adsorption seem to be concerns with both proteins. This is reflected by the inclusion of human serum albumin (HSA) in all original formulations. Both Betaseron (300 :g IFN-$-1b) and Avonex (30 :g IFN-$-1a) contain a high amount of albumin in the lyophilized formulations (15 mg HSA in both formulations, compared with, e.g., IntronA with 1 mg HSA). Albumin also serves other roles in lyophilized formulations (bulking agent, lyoprotectant, and cryoprotectant). The liquid Rebif (US) formulation has a lower content of albumin (Table 3). Recombinant production of albumin can alleviate the safety concerns related to plasma-derived albumin; however, analytical challenges posed by the presence of two proteins in the formulation remain. Thus, albumin-free formulations are more desirable, and reformulation cases where albumin has been replaced by surfactants are described in the following paragraphs. Betaseron was the first IFN-$ product to be approved. The powder formulation consists of IFN-$-1b, albumin, and mannitol; reconstitution is carried out in sodium chloride solution. It has 10-fold higher amount of active ingredient compared with other IFN-$ products. In a related case, an albumin-free formulation has been developed for a hydrophobic cytokine that resembles IFN-$-1b.61 In a study by Hawe and Friess,61 major stability concerns of solubility and surface adsorption could be mitigated without the addition of albumin. The solution pH was adjusted between 3.5–5.0 and low ionic strength, with the inclusion of polysorbate 20 or glycine in the formulations. Long-term stability (6 months) was afforded by a liquid formulation containing 4.5% mannitol and 0.02% polysorbate (pH 3.5–4.0) or a lyophilized formulation with 4% mannitol, 1% sucrose, and 0% or 0.005% polysorbate.61 Conditions of low ionic strength are achieved in Betaseron by including sodium chloride (tonicity modifier) in the reconstitution solution. Lyophilized Avonex formulation in sodium phosphate buffer contains IFN-$-1a, albumin, and sodium chloride. It has a much higher pH (7.3 for reconstituted solution) than the other IFN$-1a formulations (pH 3.5–4.8). IFN-$-1a has been stated to be more stable at physiological pH than IFN-$-1b59 ; however, IFN-$-1a has also been reported to form potentially immunogenic aggregates when formulated in sodium phosphate buffer and sodium chloride at pH 7.2.62 Phosphate-buffered saline can undergo large pH changes during freezing because of precipitation of the less-soluble buffer component (dibasic sodium phosphate), such as a decrease from pH 7.0 to 4.3–5.8 depending on the excipients present.63 Shift in formulation pH can deactivate proteins and for this reason phosphate buffer is not generally recommended for lyophilized protein formulations. However, in the case of Avonex, a pH decrease might be an advantage for protein stability (no data to support this hypothesis). Another interesting fact about Avonex formulation is the inclusion of sodium chloride. It is generally not recommended for lyophilized protein formulations to contain sodium chloride because of exposure to highly supersaturated salt solution during freezing.64 It is known that the unglycosylated IFN-$ aggregates at high ionic strength.61 Furthermore, the low eutectic melting temperature of sodium chloride water solution (−21◦ C) is not favorable for freeze-drying, because the product temperature must be kept low during the primary drying step, leading to a less efficient drying process.30,65 Lipi¨ainen et al., JOURNAL OF PHARMACEUTICAL SCIENCES

A liquid Avonex formulation without albumin is currently marketed along with the lyophilized product.66 In this formulation, phosphate buffer has been replaced by acetate (pH 4.8). Reduced pH prevents IFN-$-1a aggregation.62 In addition, arginine and polysorbate 20 have been introduced and sodium chloride excluded from the formulation. The surfactant reduces adsorption and aggregation propensity, similar to the role played by albumin in the previous formulation. Arginine is a basic amino acid that can be used to preserve protein activity, especially when alternatives to the use of serum albumin are sought.67 Its importance as a protein formulation excipient is rapidly growing.50 It is known to increase protein solubility and stability via multiple molecular mechanisms, such as by disrupting protein–protein interactions and thus preventing aggregation (see reviews for more detailed discussion on protein– arginine interactions).27,50,68 In Avonex formulation, arginine hydrochloride also ensures formulation isotonicity along with the buffer components. Rebif is also available as two formulations. The original formulation (the one marketed in the US) is composed of albumin and mannitol in acetate buffer (pH 3.5–4.5). A new formulation, developed in conjunction with the introduction of a genetically engineered clone that supports enhanced expression and yield, was introduced in the European market in 2003.69 This formulation consists of mannitol, poloxamer 188, methionine, and benzyl alcohol in sodium acetate buffer (pH 3.5–4.5). The surfactant, poloxamer, serves to mimic the role of albumin in the formulation. However, the albumin-free, poloxamer-containing formulation has a shorter shelf life (18 months) as compared with the original albumin-containing product (24 months in EU). The difference in promised shelf life does not necessarily correlate with product stability but may be a mere company decision. Another Rebif formulation with poloxamer, lysine, and sodium acetate also showed promise in the prescreening phase of the development process.69 It is interesting to note the proposition of Jaber et al.69 that albumin protects IFN-$ against oxidation by acting as a scavenger, a property not commonly attributed to albumin. IFN-$-1a is vulnerable to oxidation and the oxidized form has a strong tendency to aggregate.10 Furthermore, oxidized and aggregated IFN-$-1a is potentially highly immunogenic.70 Antioxidant methionine is thus added to the formulation. However, the liquid formulation Avonex does not contain any excipient with known antioxidant properties. Uribeta is a biosimilar IFN-$ product marketed in Central and South America, with the same composition as Betaseron except that mannitol is replaced by glucose.71 Zheng et al.71 reported formation of potentially harmful glycated forms of both IFN-$ and HSA in Uribeta even when stored at +4◦ C and the extent of glycation increased when stored at room temperature. These results highlight the risk of using reducing sugars such as glucose in protein formulations. Interferon Gamma Interferon gamma is a proinflammatory cytokine, with powerful immunomodulatory properties and some antiviral activity.72 This cytokine is important in tumor surveillance and in immune responses against various types of infections, including intracellular bacterial infections.73,74 IFN-( is the only type II interferon; it has a unique receptor and differs structurally from other interferons.74 The biologically active form is a dimer composed of two identical 143 amino acid polypeptides. Each DOI 10.1002/jps.24243

REVIEW

monomer is composed of six "-helices and although the overall fold of the dimer does not resemble that of other interferons, five of the six helices have a folding topology similar to the type I IFN fold.75 IFN-( does not have any disulfide bonds and the dimer structure is stabilized by intertwining of the helices along the subunit interface.75 This kind of intimate linkage is unusual among globular proteins. Glycosylation is not essential for the biological activity of IFN-(. There are two glycosylation sites in each monomer and of the two sites either both, one or none, are occupied by an N-linked carbohydrate.72 Depending on the degree of glycosylation, a monomer has a molecular weight of 17, 20, or 25 kDa. The size heterogeneity is further increased by natural variation in the length of the carboxy-terminal. The mature dimers vary in the molecular weight range of 30–50 kDa.72 IFN-( activity is lost because of structural changes and dissociation of the dimer, when exposed to acidic conditions below pH 4.5, temperature above 50◦ C or high pressure.76,77 The monomers are prone to aggregate at certain conditions, such as combination of low pH and high ionic strength.76 Recombinant IFN-(-1b is approved for the treatment of chronic granulomatous disease, an inherited leukocyte disorder, and for osteopetrosis, a rare bone disease. The recombinant protein differs from the natural IFN-( in lacking glycosylation, having a shorter sequence of 140 residues with a methionine residue instead of a blocked pyroglutamate in the amino-terminus.73 The two marketed products, Actimmune and Imukin, are liquid products with identical compositions of IFN(-1b, mannitol, and polysorbate 20 in sodium succinate buffer. The formulation pH is 5.0 and a low ionic strength (5 mM sodium succinate) is critical for the cytokine stability. A study reported that acidification of a phosphate-buffered IFN-( formulation to pH 5.0 caused significant structural loss at high ionic strength.63 Feasibility of freeze-drying the product formulation has been investigated. A stable formulation could not be obtained by direct lyophilization as the monosodium form of succinic acid crystallized upon freezing and resulted in a pH drop and loss of activity.78 Replacing succinate with glycolate buffer in the marketed formulation retained activity after lyophilization and during storage in the solid state. Interestingly, Lam et al.,78 observed substantial deamidation in both succinate and glycolate formulations, but concluded that the chemical degradation had no effect on biological activity. There are no multiple-dose products available in the market, possibly because benzyl alcohol causes structural changes and aggregation of IFN-(-1b.79

Interleukin-2 Interleukin-2 stimulates T cells, natural killer cells, and antibody production by B cells. It is a key cytokine for immune response activation and also for immune tolerance and memory induction.80,81 Clinically, IL-2 is interesting because of its ability to enhance antitumor responses. It is a hydrophobic protein composed of 133 amino acids, that contains one disulfide bridge and one free cysteine residue, as well as a site for O-linked glycosylation.82 Because of variation in carbohydrate content, IL-2 is heterogeneous in size (15–18 kDa) and charge, but this does not seem to affect bioactivity.82 The overall structure is formed by a bundle of "-helices.83,84

DOI 10.1002/jps.24243

11

Aldesleukin (rhIL-2) is used for the treatment of renal cell carcinoma and melanoma. The recombinant protein differs from wild-type IL-2; it does not contain N-terminal alanine, it has serine in lieu of cysteine at position 125 and it is not glycosylated.85 Recombinant IL-2 is very hydrophobic and prone to aggregate. The aggregates have been held accountable for antidrug antibody formation.15 In addition, oxidation of IL-2 has been reported. EDTA and methionine are able to protect IL2 from oxidation.11 Glycine increases activity retention during storage, whereas polysorbate 80 may cause IL-2 degradation.11 Proleukin is a lyophilized formulation consisting of mannitol as stabilizer and bulking agent, sodium dodecyl sulfate (SDS; 0.02%) and sodium phosphate buffer, pH 7.5. SDS is an ionic surfactant that binds to proteins and solubilizes them. It is not commonly used in therapeutic protein products because the strong binding interactions often lead to denaturation of proteins. In fact, SDS is included in the Proleukin formulation to promote IL-2 aggregation.86 In case of aldesleukin, in vivo efficacy is improved when the cytokine is administered as aggregates, because monomers are rapidly cleared from the body.86 Proleukin contains noncovalently bound SDS/IL-2 microaggregates (27 interleukin molecules per aggregate, on average) that possibly have micellar or micelle-like structures.85,86 Thus, SDS does not have a conventional surfactant role in this formulation. Proleukin pH 7.5 is noteworthy as IL-2 is structurally more stable at pH 4 than at pH 7.87 Additionally, lyophilization with various buffers at pH 7 lead to extensive unfolding and aggregation of rIL-2. However, structural integrity was not compromised when freeze dried from solutions at pH 5 or below.88 Sucrose and other excipients were able to stabilize the structure at pH 7, but mannitol did not improve structure or activity retention.88 Furthermore, decreasing the pH to 4 lead to inhibition of surface adsorption in a liquid formulation of a similar IL-2 mutant (position 125 occupied by alanine instead of serine).11 Ontak, another product containing IL-2, is used to treat T cell lymphoma. The active ingredient, denileukin diftitox, is a recombinant fusion protein where fragments of diphtheria toxin are combined with IL-2.89 The cytokine serves to target the active ingredient to the cancerous T cells, allowing the toxin to be released in the diseased cells. Ontak is a liquid formulation with EDTA and polysorbate 20 in citric acid, pH 6.9–7.2, with recommended storage as frozen. Interleukin-11 Interleukin-11 is a thrombopoietic growth factor that stimulates megakaryocytopoiesis and platelet production, among multiple other biological activities.90–94 Recombinant IL-11 (oprelvekin; in Neumega) is used for the prevention of severe thrombocytopenia and reduction of the need for platelet transfusions after chemotherapy.95 Compared with the natural IL-11, the recombinant IL-11 (oprelvekin) lacks the amino-terminal proline and has 177 amino acids, one less than the natural protein.95 More than 50% of the polypeptide chain adopts "-helical conformation and a four-helix bundle tertiary fold resembling that of G-CSF has been suggested.91 However, the tertiary structure of IL-11 has not been validated. The helix-bundle structure is striking as IL11 has an exceptionally high content of proline residues (12%)

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and lacks structure-stabilizing disulfide bridges.91 The cytokine also has an unusually high amount of leucine (23%), which has been suggested to account for considerable hydrophobicity.94 However, Czupryn et al.91 have hypothesized that the hydrophobic side chains are located on the helical interfaces and contribute to stabilize the bundle structure. Recombinant IL11 has also been described highly cationic, rich in arginine and other basic amino acids.96 In addition, the lack of glycosylation of the natural protein implicates that this secreted protein is adequately soluble. The cytokine has a methionine on its surface (Met58) that is critical for bioactivity.91 Oxidation of the methionine is therefore a potential concern of stability. Neumega is a lyophilized powder composed of rhIL-11 (5 mg), glycine, and sodium phosphate buffer (pH 7 after reconstitution). The lack of surfactant in the formulation is noteworthy. The protein concentration in Neumega is high for a cytokine formulation, which may provide an explanation for the lack of surfactant. Only a small proportion of the total protein is lost because of adsorption when the concentration is high. Polysorbates may be undesirable because peroxide impurities, often present in the raw materials, could impair the bioactivity by oxidizing the critical methionine residue. Glycine crystallizes during the freezing step of lyophilization and generally it needs to be combined with amorphous stabilizing agents that provide protection to the protein remaining in the amorphous phase.97 In Neumega, no such amorphous excipient is required. This may also be explained by the high protein concentration that can provide stability during lyophilization.30 Powders with low concentrations of rhIL-11 have been challenging to formulate because of adsorption on glass surfaces leading to significant activity loss.94 A formulation containing 1 :g/mL rhIL-11, 0.5% HSA, 0.1% trehalose, and 0.02% polysorbate 20 in potassium phosphate buffer (pH 7.4) inhibited surface adsorption and loss of bioactivity during long-term (6 months) storage.94 Both HSA and the surfactant were necessary for full recovery of activity; however, oxidation products were not analyzed. In another study, storage stability of rhIL-11 (3.9 mg/mL) was optimized by using mixtures of disaccharides (2.5% or 5% sucrose or trehalose) and hydroxyethyl starch (2.5% or 5%) in Tris buffer pH 7.0.98 The disaccharides inhibited unfolding during drying, whereas the polymer raised the glass transition temperature of the dried formulation. This resulted in improved storage stability compared with either disaccharide alone and allowed a faster and more economical drying process. Granulocyte Colony-Stimulating Factor Granulocyte colony-stimulating factor is a hematopoietic growth factor that stimulates the proliferation and differentiation of blood progenitor cells and activation of neutrophils.99 G-CSF products are blockbuster drugs, widely used to treat or prevent neutropenia, including cancer patients receiving chemotherapy. They are also used to mobilize blood progenitor cells for transplantation. There are four different recombinant forms of G-CSF in clinical use: lenograstim, filgrastim, pegfilgrastim, and lipegfilgrastim. Lenograstim (rhG-CSF; the active ingredient in Granocyte) is recombinant G-CSF produced in mammalian (CHO) cells. It has the same amino acid composition of 174 residues and glycosylation site (O-linked sugar chain at Thr-133) as the natural human G-CSF.100 Fil-

Lipi¨ainen et al., JOURNAL OF PHARMACEUTICAL SCIENCES

grastim (r-met-hG-CSF; the active ingredient in Neupogen and biosimilars) differs from the natural protein in lacking glycosylation and having one additional amino acid, the unprocessed N-terminal methionine. The recombinant G-CSFs are 19–20 kDa in size, contain two disulfide bonds, and one free cysteine residue, and possess similar biological activity as the endogenous protein.100,101 The three-dimensional structure is characterized as a four-helix bundle, having structural similarity with human growth hormone (hGH), GM-CSF, IFN-$, IL-2, and IL-4.102 Pegfilgrastim (Neulasta) and lipegfilgrastim (Lonquex) are pegylated forms of filgrastim, with increased elimination halflives. This facilitates a less frequent dosage regimen. Pegfilgrastim has a 20-kDa PEG molecule directly conjugated to the protein. The conjugation is different in lipegfilgrastim where the PEG chain is attached by a carbohydrate linker. The linker consists of glycine, N-acetylneuraminic acid, and N-acetylgalactosamine and it is attached to the unused O-glycosylation site, resulting in improved selectivity of pegylation.103,104 Recombinant met-G-CSF rapidly aggregates at neutral pH.105 The protein is however stable at acidic environment because of repulsive electrostatic protein–protein interactions at pH 3.5106 and even at pH 2.1.107 High ionic strength neutralizes the charge–charge repulsions and induces aggregation even at low pH.106 Sucrose, on the contrary, inhibits aggregation via nonspecific thermodynamic stabilization.105 Benzyl alcohol accelerates rhG-CSF aggregation; the effect is reduced by sucrose or reduction of pH to 3.5.108 Glycosylation contributes to GCSF stability by rendering the protein less sensitive to heat denaturation and aggregation, even at neutral pH.109 Filgrastim products are formulated at pH 4,12 whereas lenograstim formulation has a pH of 6.5 (after reconstitution). All methionine residues except for the additional N-terminal residue are critical for G-CSF biological activity. They are all susceptible to oxidization by peroxides.110 They are the only amino acid residues in G-CSF vulnerable to oxidation.12 Methionine oxidation is an important degradation pathway for filgrastim, possibly even the shelf-life-limiting factor.13 Free methionine is an effective antioxidant to prevent oxidation of rhG-CSF.111 Interestingly, liquid filgrastim products do not contain antioxidants, whereas the lenograstim dry powder formulation does have methionine. It could be possible that the amino-terminal methionine residue in filgrastim (not critical to activity), serves an antioxidant function. Chemical instability arising from deamidation was found to be the main degradation pathway for both Neupogen and a biosimilar product.112 The free cysteine residue could be replaced by serine without affecting activity or stability, to avoid intermolecular disulfide scrambling.101 This approach has been used to improve rIL2 stability. In G-CSF, the free sulfhydryl group appears to be well buried inside the protein and is rather unreactive. Therefore, cysteine substitution has not been utilized in the current products. The free cysteine residue could still account for dimer formation observed in stability studies. Because aggregation can be effectively inhibited at low pH and ionic strength, oxidation, and deamidation are the major pathways that need to be prevented. Polysorbate concentrations in the filgrastim products are relatively low (0.04 mg/mL); this reduces the risk of peroxide-induced

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oxidation. Light and elevated temperature accelerates peroxide formation in polysorbates and often polysorbates contain residual peroxides as impurities.113 Of the four types of G-CSF products, the pegylated proteins are similar, but the rest have significant differences affecting formulation strategies. All filgrastim products are liquid formulations containing buffer, sorbitol as stabilizer/tonicity modifier, and polysorbate 80. The originator product Neupogen and most of the biosimilars are formulated with acetate buffer but in Filgrastim Hexal/Zarzio glutamate buffer system has been used. In stability studies comparing Filgrastim Hexal/Zarzio to Neupogen, the biosimilar product formulation showed lower levels of oxidation and deamidation. The stability of the products was concluded to be comparable, though.112,114 It is noteworthy that Filgrastim Hexal and Zarzio have a longer shelf-life of 36 months, whereas for Neupogen and the other acetatecontaining products it is 30 months. However, the different shelf lives may reflect business decisions rather than protein stability. One-time freezing of the product does not adversely affect product stability that could be attributed to the low crystallization tendency of sorbitol upon freezing.97 In contrast, mannitolcontaining formulations such as Granocyte and Rebif must be discarded if accidentally frozen. The pegylated filgrastim products differ from the nonpegylated versions in having a much higher protein concentration. The Neulasta and Lonquex products contain 10 mg/mL protein, whereas the concentration range is 0.3–0.9 mg/mL in the filgrastim products. The formulations are otherwise similar to Neupogen (acetate buffer and sorbitol). The surfactant concentration required is lower compared with Neupogen, probably because of the reduced aggregation tendency because of pegylation.115 Polysorbate 20 is used instead of polysorbate 80 because of its availability from vegetable-derived source.115 It is difficult to rationalize the different sorbitol concentrations in the Neulasta product marketed in US (50 mg/mL) and in Europe (10 mg/mL). Higher sorbitol concentration reduces the amount of degradation products.115 The Granocyte formulation clearly differs from all the above described products. It is a lyophilized powder product with a lower cytokine concentration enabled by the higher activity of lenograstim compared with filgrastim.116 Lenograstim is less sensitive to degradation at neutral pH compared with filgrastim; therefore, the Granocyte formulation pH (after reconstitution) is higher (pH 6.5). It is somewhat counter-intuitive that even though the intrinsic stability of lenograstim is higher than that of filgrastim, and it is formulated as a dry product, the shelf-life of Granocyte is only 2 years, the shortest of all GCSF products. However, it can be stored at room temperature, whereas the others need to be refrigerated. The formulation contains several excipients: arginine, phenylalanine, methionine, mannitol, polysorbate 20, and hydrochloric acid. The complexity probably arises from reformulation challenges resulting from the removal of albumin from the formulation.117 In 1998, a simpler Granocyte formulation was marketed consisting of HSA (1 mg), mannitol (50 mg), and polysorbate 20 (0.1 mg) and sodium chloride in phosphate buffer.118 Removal of albumin apparently necessitated the introduction of an antioxidant (methionine). Mannitol, phenylalanine, and arginine contribute to the solid-state properties of Granocyte formulation. Mannitol is a commonly used bulking agent in freeze-dried products. Like glycine, it generally crystallizes in the process and results in an DOI 10.1002/jps.24243

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excellent dried cake structure but does not stabilize the protein in the dry state. Additional amorphous stabilizers are needed. Phenylalanine is a hydrophobic amino acid not commonly used as excipient in protein formulations. It has been mentioned in a patent application on inhalable spray-dried four-helix bundle protein powders, along with other hydrophobic amino acids.119 It is believed to increase the glass transition temperature, decrease the surface tension of water, and inhibit aggregation by accumulating on particle surfaces where it protects the protein from the harsh process conditions. The inclusion of phenylalanine in Granocyte may have been influenced by the observations of Mattern et al.120 They found that arginine forms amorphous solids when freeze dried and that phenylalanine inhibited arginine crystallization in vacuum-dried powders by reducing the residual moisture content and increasing the dry product glass transition temperature.120 In the same study, GCSF was vacuum dried in combination with arginine, phenylalanine (1:1 Arg/Phe molar ratio), and a mineral acid (HCl or H3 PO4 ) resulting in effective stabilization of the protein during storage. Therefore, Granocyte seems to be a partially amorphous excipient system where crystalline mannitol provides cake structure, arginine functions as the amorphous stabilizer, and phenylalanine is included to inhibit arginine crystallization during storage. Granulocyte-Macrophage Colony-Stimulating Factor Granulocyte-macrophage colony-stimulating factor is an inflammatory mediator, stimulating proliferation and differentiation of hematopoietic stem cells, and activating granulocytes and macrophages.99 Leukine is an FDA-approved product containing recombinant GM-CSF (sargramostim), for chemotherapy-induced neutropenia or bone marrow transplantation. The indications are the same as for G-CSF products; however, adverse effects are more commonly associated with GM-CSF, making it a much less used product than G-CSF.99 Granulocyte-macrophage colony-stimulating factor is a glycoprotein that consists of 127 amino acid residues and forms a compact globular fold stabilized by "-helices, $-sheets, and two disulfide bridges.121 The $-sheet content is uncommon for bundle cytokines, but overall it has a very similar fold compared with G-CSF, IL-2, IFN-$, and hGH.102 Among the related structures, GM-CSF is the smallest.102 The molecular weight varies depending on the degree of glycosylation, and has been reported to be 14.7 kDa99,121 and around 23 kDa.81 Sargramostim in Leukine is produced in S. cerevisiae. It differs from the natural protein by a single substitution of arginine with leucine at position 23. Yeast cell expression produces glycosylated protein with both O- and N-linked oligosaccharides that are not identical to the ones present in the endogenous human protein. Variation in saccharide chains results in three molecular species (molecular weights of 19.5, 16.8, and 15.5 kDa). Leukine is available both as a liquid and a lyophilized powder product. Excipients in the liquid formulation are mannitol (stabilizer and isotonicity), sucrose (stabilizer and isotonicity), benzyl alcohol (preservative) buffered in tromethamine (Tris) at pH 6.7–7.7. The powder contains mannitol (bulking agent), sucrose (amorphous stabilizer), and tromethamine (reconstituted product pH 7.1–7.7). Tris buffer system remains amorphous during freezing.21 It is noteworthy that there is no surfactant in this formulation, even though the protein concentration is not very high (250–500 :g/mL). The Lipi¨ainen et al., JOURNAL OF PHARMACEUTICAL SCIENCES

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carbohydrate chains probably afford sufficient solubility to this cytokine.

Possible Upcoming Cytokine Pharmaceuticals Most of the cytokine products have been approved to market a number of years ago and new products are duly expected. Long-term treatment and frequent administrations, required with MS disease therapy, for example, is believed to result in an antibody response over time.122 This causes reduction in therapeutic efficacy and may lead to fatal immunogenic reactions. Several strategies to reduce administration frequency and immunogenicity are being explored. Simultaneously, patient compliance could be improved. Pegylation is one promising strategy; pegylated IFN-$-1a and IFN-$-1b formulations have been in clinical trials123,124 and a pegylated IFN-$ product (Plegridy) is in the filing stage.125 Albumin-interferon aggregates have also been proposed to be responsible for immunogenicity.126 Therefore, albumin-free formulations could provide reduced immunogenicity. On the contrary, albumin-IFN-" fusion protein (albinterferon), intended for a prolonged-action treatment has been developed. Albinterferon shows decreased aggregation tendency during agitation and storage.127 More cytokine biosimilars will also emerge in the near future. European Medicines Agency (EMA) has published a guideline on IFN-$ biosimilars in 2013 and Neulasta patent expires in 2015. Interleukin-2 has been at the forefront for cytokine delivery system development. It is commonly used to treat latestage cancers, in high-dose i.v. injections that cause severe, life-threatening side effects.81,128 Polymeric microsphere formulations, liposomes, pegylation, and pulmonary administration are some strategies evaluated to improve IL-2 therapy.81 Toxicity could be reduced by local delivery and/or by achieving lower plasma concentrations, whereas therapeutic efficacy may be improved by prolonging half-life and better reaching the target site. Sustained and local delivery of IL-2 might be achieved by injecting IL-2-loaded microspheres to the target site.81 New stabilizing excipients are studied for these purposes, such as choline dihydrogen phosphate intended for IL-2 liposomes or nanoparticles.128 In addition, future may show promise for combination products, such as a controlled-release microparticle formulation with a mixture of IL-2, transforming growth factor $ and rapamycin, implicated for treatment of autoimmune diseases.129 For rhIL-11, attempts to maximize the clinical potency and reduce the frequency of administration include developing a hyaluronic acid matrix for sustained release,130 pegylation,131 and site-directed mutagenesis.132 New expression systems are being developed, such as modified yeasts.133 For some cytokines, glycosylation is critical for stability and activity, but mammalian cell expression systems are costly and generally have low expression levels. Overexpression is difficult to achieve because high cytokine levels are toxic to the host cell. Further still, enhanced protein forms are being developed by hyperglycosylation, to provide higher solubility and half-life.134 Alternative manufacturing processes may be introduced. A dry formulation containing IFN-"-2a, trehalose, and mannitol was prepared by in situ precipitation by PEG followed by vacuum drying.135 It was said to have comparable stability against aggregation and oxidation than a similar lyophilized formulation. Lipi¨ainen et al., JOURNAL OF PHARMACEUTICAL SCIENCES

Many previously unknown cytokines have been discovered during the last decades. With greater understanding of the complex cytokine biology and improved genomics-related methods, cytokines provide a pool of new potential therapeutics. IFN-8 is the most promising candidate and it is in Phase 3 clinical trials (PEG-IFN-8).136 Interleukins IL-7 and IL-21 are already approved by EMA for rare disease indications. In addition, rhIL-21 (denenicokin) is in Phase 1 clinical trials.136 A few cytokines that previously failed in clinical trials (e.g., IL-12 and IL-10) are now being reinvestigated in light of new information and better delivery strategies.2 Cytokine-antibody fusion proteins are in company pipelines; IL-2,137 IL-12,137,138 and IL-10 (dekavil)139 are in Phase 1 clinical trials. There is also a long list of potential new indications, such as G-CSF for the treatment of noise-induced hearing loss140 and chronic obstructive pulmonary disease,141 rhIL-11 for HIV therapy,142 and IFN-"-2b for the treatment of viral conjunctivitis by extemporaneously prepared eye drop formulation.143 Because of their immunomodulatory activities, cytokines, especially IFN-( and GM-CSF have potential applications as vaccine adjuvants.73,99,144

DISCUSSION Cytokine therapeutics on the market include different subtypes of IFN-"s, IFN-$, IFN-(, IL-2, IL-11, G-CSF, and GM-CSF. Their protein characteristics are summarized in Table 2. They are all small proteins with molecular weight ranging between 15 and 20 kDa, except for IFN-(, which is a 30–50-kDa dimer. The cytokines have a helical bundle fold, generally associated with marked hydrophobicity.6 Therefore, challenges because of aggregation and surface adsorption are common with cytokines. However, the degree of hydrophobicity is quite different among the cytokines and production host affecting the degree of glycosylation can have significant impact, as in case for IFN-$. Residual impurities, which can be present in excipients, as well as product contact with packaging materials (e.g., glass surfaces and rubber stoppers) can cause protein degradation.7,113 Oxidation is another major degradation pathway for cytokines. To prevent loss of activity as well as formation of immunogenic species, attention must be paid to preventing aggregation, surface adsorption, and oxidation of cytokine proteins. The formulation excipients used in currently marketed cytokine products are listed in Table 4. The most common buffer systems are phosphate and acetate. Protein-stabilizing excipients include sucrose, mannitol, sorbitol, albumin, and amino acids. Surfactants (polysorbate 20/80, poloxamer, or SDS) or albumin are used in almost all cytokine products. The exceptions are Infergen (optimized sequence), Neumega (high protein concentration), and Leukine (highly glycosylated protein). As more therapeutic cytokine products are expected to be developed in the coming years, this review should serve as a useful guide for the formulation scientist. As a first, it is important to understand the structural properties of the cytokine and the possible degradation mechanisms that might exist. Most likely, the stability concerns relate to hydrophobicity and low solubility, whereas chemical instability arises from oxidation. Liquid formulations provide convenience of manufacture and use, and many of the cytokines in market are stable in solution. Therefore, development of a liquid formulation is a justified starting point. A liquid formulation requires an optimal buffer system and pH to ensure conformational stability. Dependency R

R

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Table 4.

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Summary of Cytokine Formulation Excipients Product Name

Excipient Buffers/pH adjustment Phosphate

Acetate

Glutamate Succinate Tromethamine (TRIS) Hydrochloric acid No buffer Salts Sodium chloride

Solution

IntronA (IFN-") Infergen (IFN-") Alferon (IFN-")

Roferon-A (IFN-") Pegasys (PEG-IFN-") Avonex (IFN-$) Rebif (IFN-$) Neupogen (G-CSF) Biograstim/Ratiograstim/Tevagrastim (G-CSF) Nivestim (G-CSF) Neulasta (PEG-GSF) Lonquex (LiPEG-G-CSF) Filgrastim Hexal/Zarzio (G-CSF) Actimmune/Imukin (IFN-() Leukine (GM-CSF)

Lyophilized Powder

IntronA (IFN-") PegIntron/ViraferonPeg (PEG-IFN-") Sylatron (PEG-IFN-") Avonex (IFN-$) Proleukin (IL-2) Neumega (IL-11)

Leukine (GM-CSF) Granocyte (G-CSF) Betaferon/Betaseron/Extavia (IFN-$) Betaferon/Betaseron/Extavia (IFN-$) Avonex (IFN-$)

Potassium chloride

IntronA (IFN-") Roferon-A (IFN-") Pegasys (PEG-IFN-") Infergen (IFN-") Alferon (IFN-") Alferon (IFN-")

Sugars, Sugar Polyols Sucrose

Leukine (GM-CSF)

PegIntron/ViraferonPeg (PEG-IFN-") Sylatron (PEG-IFN-") Leukine (GM-CSF) Betaferon/Betaseron/Extavia (IFN-$) Proleukin (IL-2) Granocyte (G-CSF) Leukine (GM-CSF)

Mannitol

Rebif (IFN-$) Actimmune/Imukin (IFN-() Leukine (GM-CSF)

Sorbitol

Neupogen (G-CSF) Biograstim/Ratiograstim/Tevagrastim (G-CSF) Filgrastim Hexal/Zarzio (G-CSF) Nivestim (G-CSF) Neulasta (PEG-GSF) Lonquex (LiPEG-G-CSF)

Surfactants Polysorbate 80

Polysorbate 20

Poloxamer 188 SDS Albumin HSA

IntronA (IFN-") Roferon-A (IFN-") Pegasys (PEG-IFN-") Neupogen (G-CSF) Biograstim/Ratiograstim/Tevagrastim (G-CSF) Filgrastim Hexal/Zarzio (G-CSF) Nivestim (G-CSF) Avonex (IFN-$) Actimmune/Imukin (IFN-() Neulasta Neulasta (PEG-GSF) Lonquex (LiPEG-G-CSF) Rebif (IFN-$) (EU)

PegIntron/ViraferonPeg (PEG-IFN-") Sylatron (PEG-IFN-")

Granocyte (G-CSF)

Proleukin (IL-2) Alferon (IFN-") Rebif (IFN-$) (US)

IntronA (IFN-") Betaferon/Betaseron/Extavia (IFN-$) Avonex (IFN-$) Continued

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Table 4.

Continued Product Name

Excipient

Solution

Amino Acids Glycine Arginine Methionine Phenylalanine Chelating Agents Edetate disodium Preservatives Benzyl alcohol

M-cresol Phenol

Avonex (IFN-$) Rebif (IFN-$) (EU)

IntronA (IFN-") Neumega (IL-11) Granocyte (G-CSF) Granocyte (G-CSF) Granocyte (G-CSF)

IntronA (IFN-") Roferon-A (IFN-") Pegasys (PEG-IFN-") Rebif (IFN-$) (EU) Leukine (GM-CSF) IntronA (IFN-") Alferon (IFN-")

of structural stability on pH varies between cytokines. Some have increased stability in acid, whereas others have decreased stability.6 Optimal pH might be in the neutral or slightly acidic range, where phosphate (pKa = 7.21) and acetate (pKa = 4.76) buffers have proven to be good options. It is possible that pH adjustment is enough to alleviate hydrophobicity related concerns. A general rule in protein formulation development is to set the formulation pH at least 0.5 units above or below the protein isoelectric point.21 However, for example, Pegasys (a pegylated formulation) is stable even at the isoelectric point of IFN-". In cases where sodium chloride does not adversely affect stability, the formulation may be as simple as that in Infergen (Table 3). To achieve desired stability, additional stabilizers such as sucrose or mannitol may be required and the presence of a surfactant might prove helpful. For polysorbates, attention should be paid to peroxide impurities. To mitigate oxidationrelated instability, antioxidants such as methionine or a metal chelator such as EDTA could be useful agents in formulation. Albumin has several useful properties such as being a stabilizer, surfactant, and even antioxidant; however, its use in formulations can complicate the analytics (with two proteins in the formulation) and also raise concerns of HSA–cytokine aggregate-induced immunogenicity. In case a multidose product is developed and a preservative is needed, attention to the potential of benzyl alcohol causing cytokine degradation should be paid. M-cresol and phenol are other preservative options previously used in cytokine products. If liquid formulations do not achieve the desired long-term stability, dry formulations with suitable buffer system (e.g., phosphate or Tris) and a bulking agent (e.g., mannitol or glycine) can first be investigated. A surfactant might be needed in order to prevent aggregation during the manufacturing process and rehydration. Phosphate buffer is not generally recommended for lyophilized formulations because of the risk of pH shifts owing to crystallization of the less soluble dibasic salt component during freezing. Several lyophilized cytokine products containing phosphate buffer are in the market, though, and a pH drop could even prove to be advantageous as some cy-

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Lyophilized Powder

tokines have improved stability under acidic conditions.6 Usually, an amorphous stabilizer is required for lyophilized protein formulations. However, stable cytokine products have been formulated without them (e.g., Proleukin and Neumega), and the formulation with minimum amount of excipients necessary should be selected. For proteins that otherwise degrade during lyophilization, excipients that remain in the amorphous state (e.g., sucrose or arginine in combination with phenylalanine) may provide the additional protection required.

CONCLUSION Cytokines have shared properties such as small size and helical bundle tertiary structure. They are often vulnerable to aggregation, surface adsorption, and oxidation. However, there is intrinsic variation between these molecules because of differences in amino acid sequences and in glycosylation patterns produced by different expression hosts. The significance of these variations on solubility and susceptibility to aggregation or chemical degradation is cytokine dependent. Consequently, the optimal formulation must be uniquely developed based on extensive understanding of individual stability characteristics of the cytokine of interest. However, advice for best first choices in cytokine formulation development can be adopted from this review.

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DOI 10.1002/jps.24243