International Journal of Pharmaceutics 472 (2014) 356–361

Antimicrobial preservatives induce aggregation of interferonalpha-2a: The order in which preservatives induce proteinaggregation is independent of the protein

Regina L. Bis, Krishna M.G. Mallela *Department of Pharmaceutical Sciences & Center for Pharmaceutical Biotechnology, Skaggs School of Pharmacy and Pharmaceutical Sciences,University of Colorado Anschutz Medical Campus, 12850 E Montview Blvd, C238, Aurora, CO 80045, United States

A R T I C L E I N F O

Article history:Received 29 March 2014Received in revised form 11 June 2014Accepted 25 June 2014Available online 27 June 2014

PubChem classifications:Interferon alpha-2aBenzyl alcoholm-CresolPhenolPhenoxyethanolAntimicrobial preservatives

Keywords:Interferon alpha-2aPreservativesAggregationFormulationBenzyl alcoholm-Cresol

A B S T R A C T

Antimicrobial preservatives (APs) are included in liquid multi-dose protein formulations to combat thegrowth of microbes and bacteria. These compounds have been shown to cause protein aggregation,which leads to serious immunogenic and toxic side-effects in patients. Our earlier work on a modelprotein cytochrome c (Cyt c) demonstrated that APs cause protein aggregation in a specific manner. Theaim of this study is to validate the conclusions obtained from our model protein studies on apharmaceutical protein. Interferon a-2a (IFNA2) is available as a therapeutic treatment for numerousimmune-compromised disorders including leukemia and hepatitis C, and APs have been used in itsmulti-dose formulation. Similar to Cyt c, APs induced IFNA2 aggregation, demonstrated by the loss ofsoluble monomer and increase in solution turbidity. The extent of IFNA2 aggregation increased with theincrease in AP concentration. IFNA2 aggregation also depended on the nature of AP, and followed theorder m-cresol > phenol > benzyl alcohol > phenoxyethanol. This specific order exactly matched with thatobserved for the model protein Cyt c. These and previously published results on antibodies and otherrecombinant proteins suggest that the general mechanism by which APs induce protein aggregation maybe independent of the protein.

ã 2014 Elsevier B.V. All rights reserved.

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International Journal of Pharmaceutics

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1. Introduction

Multi-dose protein formulations comprise approximately onethird of protein-based pharmaceuticals available on the globalmarket (Meyer et al., 2007). These formulations are beneficial interms of economics and patient compliance, and require theinclusion of at least one antimicrobial preservative (AP) in order toinhibit the growth of microbes and bacteria during administration(Akers et al., 2002; Meyer et al., 2007).

It has become increasingly important to study APs in proteinpharmaceuticals because these necessary compounds have beenlinked to protein aggregation in the liquid state. One of the earliest

Abbreviations: BA, benzyl alcohol; Cyt c, cytochrome c; CR, m-cresol; IFNA2,interferon alpha-2a; PE, phenoxyethanol; PH, phenol; SE-HPLC, size exclusion highpressure liquid chromatography.* Corresponding author. Tel.: +1 303 724 3576; fax: +1 303 724 7266.E-mail address: Krishna.mallela@ucdenver.edu (K.M.G. Mallela).

http://dx.doi.org/10.1016/j.ijpharm.2014.06.0440378-5173/ã 2014 Elsevier B.V. All rights reserved.

reports demonstrated that the addition of various aromaticcompounds induced the aggregation of recombinant humangrowth hormone (Maa and Hsu, 1996). Numerous studies haveshown the ability of APs to cause destabilization and aggregation ofmany proteins (Gupta and Kaisheva, 2003; Tobler et al., 2004;Zhang et al., 2004; Roy et al., 2005). Aggregates in theseformulations cause a decrease in the effective concentration of thedelivered drug as well as result in immunogenic and toxicresponses in patients (Ratner et al., 1990; Bucciantini et al.,2002; Hermeling et al., 2006; Rosenberg 2006; Fradkin et al., 2009;Sauerborn et al., 2010; Vazquez-Rey and Lang, 2011). In order tominimize AP-induced protein aggregation, an understanding of theinteractions between APs and proteins is critical.

Previous work from our laboratory has demonstrated that aspecific order exists in which individual APs induce the aggrega-tion of a model protein cytochrome c (Cyt c) (Hutchings et al.,2013). The aim of the present study is to examine the effects ofvarious APs on a pharmaceutically relevant protein and validatethe results obtained with the model protein. We chose interferon

R.L. Bis, K.M.G. Mallela / International Journal of Pharmaceutics 472 (2014) 356–361 357

a-2a (IFNA2) (Fig. 1) for this purpose, which is available as atherapeutic treatment for numerous immune-compromised dis-orders including leukemia and hepatitis C. IFNA2 has been shownto aggregate in its formulation state (Braun and Alsenz, 1997;Braun et al., 1997; Hochuli 1997; Ryff, 1997). APs have been used inIFNA2 multi-dose as well as single-dose liquid formulations.However, it is unknown whether APs cause IFNA2 aggregation.Here, we demonstrate that IFNA2 aggregation is enhanced in thepresence of APs and that the extent and order of these effectsmatch exactly to what we observed earlier in the case of the modelprotein Cyt c (Hutchings et al., 2013).

2. Materials and methods

2.1. Materials

Synthetic cDNA corresponding to IFNA2 was obtained fromOperon (Huntsville, Alabama), and was cloned into the pET-SUMOexpression vector (a generous gift from Christopher Lima, Sloan-Kettering Institute). Protein was expressed in Escherichia coli BL21(DE3) cells, and the soluble protein was purified using a NickelSepharose 6 Fast Flow column (GE Healthcare Life Sciences,Pittsburgh, Pennsylvania). The SUMO tag was cleaved using theUlp1 protease, leaving no additional amino acids. The final proteinsequence is identical to that of the pharmaceutical protein.Detailed expression and purification protocols, and biophysicalcharacterization of the protein were described in our earlierpublication (Bis et al., 2014).

Preservatives were obtained in their highest available purity(Table 1). All experiments were performed in the buffer conditionsused for IFNA2 formulations (0.01 M ammonium acetate, 0.12 Msodium chloride, pH 5.0), unless otherwise noted.

Fig. 1. Molecular structure of interferon a-2a (IFNA2; 1ITF.pdb). The protein isa-helical in nature. In the structure, helices are colored according to theirorganization in the three-dimensional structure: helix A, residues 11–21 (orange);helix B, residues 52–68 (green); helix B’, residues 70–75 (yellow); helix C, residues78–100 (purple); helix D, residues 110–132 (cyan); helix E, residues 137–157 (blue).Residues 22–51 comprise the AB-loop, with residues 40–43 usually found in a 310helix (red). (For interpretation of the references to color in this figure legend, thereader is referred to the web version of this article.)

2.2. Preservative efficacy test

To confirm the antimicrobial activity of APs, a simplifiedpreservative efficacy test was performed (Sutton and Porter, 2002;Hutchings et al., 2013). A primary culture of E. coli BL21(DE3) cellswas incubated overnight at 37 �C in a shaker. Aliquots of 0.5 mlwere transferred into five 50 ml culture flasks containing either nopreservative (control), or one of the four APs (Table 1). Cultureswere incubated at 37 �C with shaking for 6 h, and optical density at600 nm was used to measure the cell count.

2.3. Size exclusion chromatography

To monitor the effects of various APs on protein aggregation,IFNA2 (10 mM in 0.01 M ammonium acetate, 0.12 M sodiumchloride, pH 5) was incubated at 50 �C in borosilicate vials (KimbleChase Life Science, #60910 L-12, Vineland, New Jersey) andsamples were taken at desired intervals. Samples were centrifugedto remove insoluble aggregates prior to HPLC injection. Concen-tration of monomer was estimated by injecting 70 ml onto a TSKgel5 mM G3000SWxl column (Tosoh Bioscience LLC, San Francisco,California) on an Agilent 1100 HPLC (Santa Clara, California) atroom temperature. The mobile phase used was 0.01 M ammoniumacetate, 0.12 M sodium chloride, pH 5, at a flow rate of 1 ml min�1.Absorbance at 280 nm was used to determine the monomercontent.

2.4. Isothermal incubation experiments

IFNA2 (10 mM) was incubated at the desired temperature withvarious APs, and the changes in optical density at 350 nm weremeasured as a function of the incubation time (Eberlein et al.,1994;Eckhardt et al., 1994). Buffer and protein do not absorb at thiswavelength. The aggregation kinetics were monitored until thesignal reached a plateau. At longer incubation times, theaggregates started to settle down to the bottom of the cuvette,resulting in decreased optical density. At that point, the experi-ment was stopped.

Table 1Antimicrobial preservatives (AP) used in this study.

AP and typical useconcentration

Molecular structure Molecularweight (Da)

Source Purity(%)

Benzyl alcohol(BA) 1% v/v

108.1 Merck 97

m-Cresol(CR) 0.3% v/v

108.1 Sigma 99

Phenol(PH) 0.5% v/v

94.1 Sigma 99.5

2-Phenoxyethanol(PE) 1% v/v

138.2 Fluka 99.5

358 R.L. Bis, K.M.G. Mallela / International Journal of Pharmaceutics 472 (2014) 356–361

2.5. Thermal scanning method

The aggregation temperature (TmAgg) of the protein wasmeasured on an UV–visible spectrophotometer (Agilent Technol-ogies, Santa Clara, California). The temperature was increased at arate of 1 �C/min followed by 90 s equilibration, and changes in theoptical density at 450 nm were recorded (Charman et al., 1993;Kurganov, 2002). Tm

Agg was determined as the temperature at halfthe maximum optical density. For these experiments, 10 mm IFNA2in formulation buffer was used with varying AP concentration.

3. Results

3.1. APs cause IFNA2 aggregation

Four commonly employed APs in liquid protein formulationswere used in this study. Table 1 lists the concentrations of theseAPs used in formulations. Fig. 2A shows the antimicrobial efficacy

Fig. 2. Preservative efficacy test of APs on BL21(DE3) E. coli cells (Sutton and Porter,2002; Hutchings et al., 2013). The cell count was monitored by measuring the relativeoptical density at 600 nm as a function of the growth time. Addition of APs inhibitedbacterial growth. (A) Preservative efficacy at the AP concentrations used in proteinformulations. Closed circles represent the data untreated with APs, while the othersymbols represent the data treated with various APs: 1% v/v benzyl alcohol (BA, opencircles), 0.3% v/v m-cresol (CR, closed triangles), 0.5% v/v phenol (PH, open triangles),and 0.5% v/v phenoxyethanol (PE, closed squares), respectively. (B) Preservativeefficacy at 0.3% v/v AP concentrations. Closed circles, untreated control; open circles,BA; closed triangles, CR; open triangles, PH; closed squares, PE.

of these APs, measured using a simplified test (Sutton and Porter,2002; Hutchings et al., 2013). We tested their effect on the growthof E. coli bacteria. For this purpose, we used BL21(DE3) cells in LBmedia and monitored the cell count by measuring changes in theoptical density at 600 nm as a function of the growth time. WithoutAPs, the growth curve showed an exponential increase (Fig. 2A).However, the optical density increase was minimal with theaddition of any of the four APs when compared with the growthcurve with no AP, indicating that these molecules inhibitedbacterial growth. Table 2 lists the minimum inhibitory concentra-tion (MIC) values for these APs against various organisms (Lucchiniet al., 1990; Simpson and Wuthiekanun, 2000; Meyer et al., 2007;Rowe et al., 2009; Abd-Elsalam et al., 2011).

In previous studies, APs have been shown to induce proteinaggregation over a period of days or months (Maa and Hsu, 1996;Katakam and Banga, 1997; Remmele et al., 1998; Gupta andKaisheva, 2003; Tobler et al., 2004; Roy et al., 2006; Thirumanga-lathu et al., 2006). In order to test the effects of different APs onprotein aggregation on a convenient laboratory timescale, weaccelerated aggregation kinetics by conducting isothermal incu-bation studies at an elevated temperature. Using elevated temper-atures to accelerate protein aggregation is becoming a commonlyused method for scanning the effect of a large number of solutionconditions on protein stability and aggregation (Brummitt et al.,2011; Nashine et al., 2013; Chaudhuri et al., 2014). Initially, wedemonstrated that APs induce IFNA2 aggregation. For comparingthe effect of the four APs on IFNA2 aggregation, we chose aconcentration of 0.3% v/v AP due to the low solubility of m-cresolabove this concentration. At this concentration of 0.3% v/v, all theAPs retained their antimicrobial efficacy (Fig. 2B). Isothermal

Table 2Minimum inhibitory concentration (MIC) values of APs measured using bacterialand microbial organisms (Lucchini et al., 1990; Simpson and Wuthiekanun, 2000;Meyer et al., 2007; Rowe et al., 2009; Abd-Elsalam et al., 2011).

Preservative and typical useconcentration

Typical useconcentration(mg/ml)

Organism tested MIC(mg/ml)

Benzyl alcohol, 1% v/v 10400 Aspergillus nigerCandida albicansEscherichia coliPseudomonasaeruginosaStaphylococcusaureus

500025002000200025

m-Cresol, 0.3% v/v 3090 Aspergillus nigerBacillus subtilisBurkholderiapseudomalleiCandida albicansPseudomonasaeruginosaStaphylococcusaureus

20001000125250010001000

Phenol, 0.5% v/v 5350 Aspergillus nigerBacillusaerogenesEnterococcusfaeciumEscherichia coliPseudomonasaeruginosaStaphylococcusaureus

3116973600250018001800

Phenoxyethanol, 1% v/v 11000 Aspergillus nigerCandida albicansEscherichia coliPseudomonasaeruginosaStaphylococcusaureus

33005400360032008500

Fig. 3. Effects of APs on IFNA2 monomer concentration under isothermalconditions. Soluble monomer remaining in solution during 24 h of incubation at50 �C in the presence and absence of 0.3% (v/v) AP as measured by size exclusionchromatography. Black – native (no AP); red – m-cresol (CR); green – phenol (PH);yellow – benzyl alcohol (BA); blue – phenoxyethanol (PE). Error bars indicatetriplicate data. Individual time points are normalized with respect to the samplewith no AP at hour zero. (For interpretation of the references to color in this figurelegend, the reader is referred to the web version of this article.)

Fig. 5. Variation in the optical density at 450 nm as a function of increasing solutiontemperature at different concentrations of phenol (PH). (For interpretation of thereferences to color in this figure legend, the reader is referred to the web version ofthis article.)

R.L. Bis, K.M.G. Mallela / International Journal of Pharmaceutics 472 (2014) 356–361 359

aggregation kinetics were performed at 50 �C and the concentra-tion of IFNA2 monomer was monitored as a function of theincubation time (Fig. 3). With all the APs, only soluble monomerwas detected using SE-HPLC. After a period of one day in theabsence of AP, approximately 90% IFNA2 monomer remained insolution. No monomer was detected after 8 h in the presence of0.3% v/v m-cresol (CR) and after 12 h in the presence of 0.3% v/vphenol (PH). Both benzyl alcohol (BA) and phenoxyethanol (PE)exerted a marginal effect on monomer loss, losing approximately20% monomer content over the course of 24 h. To differentiate theimpact of BA and PE on IFNA2 monomer loss, an isothermalincubation study was performed using 0.9% v/v AP concentration(Fig. 3 inset). In 0.9% BA, all IFNA2 monomer disappeared within 8 hat 50 �C, while monomer was detected up to 8 h in PE samples.These results followed the order CR > PH > BA > PE in terms of theireffects on IFNA2 aggregation. We also monitored the change inoptical density as a function of the incubation time, in order tocorrelate monomer loss with protein aggregation (Fig. 4). In thesecases, the same pattern of AP-induced destabilization wasobserved: CR > PH > BA > PE. No IFNA2 aggregation was observedin the absence of APs. This order in which APs induce IFNA2aggregation is identical to what we observed earlier in the case ofthe model protein Cyt c: CR > PH > BA > PE (Hutchings et al., 2013).

Fig. 4. Isothermal kinetics of IFNA2 aggregation at 50 �C as measured by thechanges in optical density at 350 nm in the presence of 0.3% v/v APs.

Fig. 6. Dependence of aggregation temperature TmAgg on AP concentration.

Individual panels indicate the slope of this variation.

360 R.L. Bis, K.M.G. Mallela / International Journal of Pharmaceutics 472 (2014) 356–361

3.2. IFNA2 aggregation increases with AP concentration

Another method used in the literature to monitor the effect ofsolution conditions on protein aggregation is thermal scanning(Brummitt et al., 2011; Nashine et al., 2013; Chaudhuri et al., 2014).This is very similar to using denaturant melts to determine theprotein stability, although denaturant is not present in proteinformulations. Here, we used temperature as the perturbant, ratherthan the denaturant. We utilized the thermal scanning techniqueto determine the effects of preservative concentration on IFNA2aggregation. We measured the changes in the optical density at450 nm as a function of increasing solution temperature. Thiswavelength was selected because neither the protein nor anysolution components absorb in this range, and the changes inoptical density can be attributed solely to protein aggregation. Inbuffer alone without APs, the optical density initially increasedwith temperature in a sigmoidal variation (Fig. 5). At highertemperatures, aggregated protein particles began to settle to thebottom of the cuvette, causing an observable decrease in theoptical density. We performed these thermal scanning experi-ments in the presence of increasing concentrations of PH (Fig. 5).With the inclusion of PH, the midpoint aggregation temperature(TmAgg) decreased. In the absence of PH, the Tm

Agg of IFNA2 was63.9 � 0.9 �C. The addition of 0.5% PH decreased the Tm

Agg to56.7 � 0.7 �C, indicating that the presence of PH accelerated theaggregation of IFNA2. A similar phenomenon was observed withCyt c whose aggregation increased with an increase in APconcentration (Hutchings et al., 2013).

3.3. Aggregation temperature decreases linearly with APconcentration

As seen with PH, the other APs also caused a decrease in TmAgg of

IFNA2 with the increase in AP concentration (Fig. 6). Interestingly, alinear correlation was observed between the Tm

Agg and theconcentration of AP. This relationship is significant in that it is aqualitative measurement of how efficiently each AP causes IFNaggregation, which is represented in their individual slopes. Thisslope is analogous to the m-value commonly used in analyzingprotein denaturant melts (Santoro and Bolen, 1988), whichrepresents the efficiency of a denaturant to denature a protein.Comparison of the slopes of Tm

Agg variation with the APconcentration demonstrates an effective order of CR > PH > BA > PE,with CR being the most effective AP in causing protein aggregation.These experiments indicate that for every percent concentrationchange of CR, the Tm

Agg of IFNA2 decreases by 22 �C. In contrast, PEhas a slope of 8.2 �C/%v/v, nearly three-fold less in its effectivenessto aggregate IFNA2 than CR. This trend is identical to that observedin the isothermal incubation studies (Figs. 3 and 4), and matchesour earlier observations on the model protein Cyt c (Hutchingset al., 2013).

4. Discussion

Protein therapeutics are marketed in a variety of dosage forms,including multi-dose formulations, which require the inclusion ofat least one antimicrobial preservative in order to combat thegrowth of microbes and bacteria during repeated contact betweenthe solution and a syringe needle (Cleland et al., 1993; Akers et al.,2002; Meyer et al., 2007). However, it has been shown that thesepreservatives cause protein aggregation (Maa and Hsu, 1996;Katakam and Banga, 1997; Remmele et al., 1998; Gupta andKaisheva, 2003; Tobler et al., 2004; Roy et al., 2006; Thirumanga-lathu et al., 2006). We have demonstrated previously that APs usedin liquid protein formulations lead to protein destabilization andaggregation using the model protein Cyt c (Singh et al., 2010, 2011;

Hutchings et al., 2013). The extent of this effect was dependentupon the nature of the AP, and the pattern of aggregation observedwas CR > PH > BA > PE.

In this study, we tested the conclusions drawn from our modelprotein studies on a pharmaceutically relevant protein interferona-2a. IFNA2 belongs to a family of cytokines that play crucial rolesin the innate immune response, and is one of the numerousinterferon-a subtypes found in humans. A number of interferon-aproducts exist in the pharmaceutical market and are used to treatvarious debilitating diseases including hairy cell leukemia andhepatitis C (Hiscott et al., 1984; Diaz et al., 1993; Kirkwood, 2002).

IFNA2 multi-dose formulations have been shown to aggregate inthe liquid state (Braun and Alsenz, 1997; Braun et al., 1997; Hochuli,1997; Ryff,1997) and contain APs. In ordertovalidate the conclusionsdrawn from our studies on a model protein Cyt c, we studied theeffectof multiple APsonIFNA2aggregation using various biophysicaltechniques. Isothermal incubationstudiesshowedthatAPscause theloss of IFNA2monomer (Fig. 3) and the increasein opticaldensity dueto protein aggregation (Fig. 4). The order in which APs induce IFNA2aggregation was CR > PH > BA > PE, which is the same order weobserved earlier in the case of the model protein Cyt c (Hutchingset al., 2013). Further, the addition of AP caused a decrease in Tm

Agg.The slopes indicate that CR was the most efficient in aggregatingIFNA2, whereas PE was the least (Fig. 6). Again, this order exactlymatched the patternwe observed in our earlier studies on the modelprotein Cyt c (Hutchings et al., 2013). Based on these results, PEappears to be the best preservative choice for pharmaceuticalformulations in causing less protein aggregation when comparedwith the commonly used BA. However, the perfect AP and itsconcentration in a multi-dose formulation should be such that itcauses less protein aggregation and has sufficient antimicrobialactivity (Hutchings et al., 2013).

Similar comparisons, although not as extensive as demonstrat-ed in the case of IFNA2 or Cyt c, were made earlier on otherproteins. Recombinant human growth hormone (rhGH) aggrega-tion was monitored during freezing, high-temperature incubation,and agitation using changes in optical density and the percentagemonomer loss by SEC. In this case, the three APs followed a similarorder in causing rhGH aggregation: CR > PH > BA (Maa and Hsu,1996). In the case of a monoclonal antibody (IgG) (Gupta andKaisheva, 2003), aggregation was monitored during isothermalincubation using visual inspection of the samples, percentmonomer loss using SEC, and light scattering. The three APsexamined induced protein aggregation in the order: CR > PH > BA.Changes in interleukin-1 receptor (IL-1R) melting temperatureassessed by DSC, and monomer content measured using SEC in thepresence of preservatives also showed a similar relationship(Remmele et al., 1998). Taken together, the data on these fiveproteins (Cyt c, IFNA2, rhGH, IgG, IL-1R), suggest that the order inwhich APs induce protein aggregation may be independent of thenature of protein. Cyt c, IFNA2, and rhGH are a-helical proteins,whereas the IgG is primarily b-sheet and IL-1R contains botha-helix and b-sheet. The proteins also differ in their function: Cyt cplays a role in electron transport; IFNA2, IgG, and IL-1R have criticalfunctions in the immune system; and rhGH is a multi-functionalprotein with emphasis on growth and regeneration.

The observation that the extent of AP effects on proteinaggregation is independent of the nature of protein implies thatAPs may interact with common structural groups present inproteins. APs do not have strong binding sites on proteins (Maa andHsu, 1996; Roy et al., 2006; Singh et al., 2010, 2011). APs mayhydrogen bond with the peptide backbone, suggested by earlierstudies on BA and polyproline monitoring changes in amide I,amide II, and hydroxyl bands using infrared spectroscopy(Strassmair et al., 1969). The extent of aggregation seems toqualitatively correlate with the hydrophobicity of the AP (Singh

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et al., 2011; Hutchings et al., 2013). APs also have been suggested tointeract with hydrophilic regions of proteins (Alford et al., 2011).Perhaps the mechanism by which APs affect protein aggregation isa combination of hydrogen bonding, hydrophobic interactions, andelectrostatics, and further examination of the exact mechanism isnecessary in order to develop stable and effective pharmaceuticals.

Acknowledgments

The authors would like to acknowledge John Carpenter,Theodore Randolph, David Bain, and LaToya Jones Braun for manyhelpful discussions and critical comments. This work was fundedby the University of Colorado Skaggs School of Pharmacy andPharmaceutical Sciences. Regina Bis was partially supported by aNIH Leadership Training Grant in Pharmaceutical Biotechnology(T32GM008732) and a predoctoral fellowship from the PhRMAFoundation (AWD-120487).

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