Evaluation of protein disulfide conversion in vitro using a continuous flow dialysis system

Analytical Biochemistry 432 (2013) 142–154

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Evaluation of protein disulfide conversion in vitro using a continuous flow dialysis system Xinzhao Grace Jiang a,⇑, Tian Wang a, Oliver Kaltenbrunner a, Kenneth Chen a, Gregory C. Flynn b, Gang Huang a a b

Drug Substance Development, Amgen, Thousand Oaks, CA 91320, USA Product Attribute Sciences, Amgen, Thousand Oaks, CA 91320, USA

a r t i c l e

i n f o

Article history: Received 10 July 2012 Received in revised form 17 September 2012 Accepted 18 September 2012 Available online 27 September 2012 Keywords: Disulfide conversion Quality attributes Recombinant proteins Dialysis

a b s t r a c t Recombinant therapeutic proteins are heterogeneous due to chemical and physical modifications. Understanding the impact of these modifications on drug safety and efficacy is critical for optimal process development and for setting reasonable specification limits. In this study, we describe the development of an in vitro continuous flow dialysis system to evaluate potential in vivo behavior of thiol adducted species and incorrectly disulfide bonded species of therapeutic proteins. The system is capable of maintaining the low-level cysteine concentrations found in human blood. Liabilities of cysteamine adducted species, incorrectly disulfide bonded species, and the correctly disulfide bonded form of an Fc-fusion protein were studied using this system. Results showed that 90% of the cysteamine adduct converted into the correctly disulfide bonded form and incorrectly disulfide bonded species in approximately 4 h under physiological conditions. Approximately 50% of incorrectly disulfide bonded species converted into the correctly bonded form in 2 days. These results provide valuable information on potential in vivo stability of the cysteamine adduct, incorrectly disulfide bonded species, and the correctly bonded form of the Fcfusion protein. These are important considerations when evaluating the criticality of product quality attributes. Ó 2012 Elsevier Inc. All rights reserved.

Quality by design (QbD)1 has been defined in the ICH guideline Q8 [1] and is an ongoing effort in the biopharmaceutical industry. The intent is to build quality into products by a thorough understanding of the product and the process along with knowledge of the risks involved in manufacturing and how to mitigate those risks. Understanding of product quality attributes is foundational for QbD [2–5]. There are many attributes associated with a protein therapeutic that could affect its quality such as posttranslational modifications (oxidation, glycosylation, deamidation, disulfide variants, etc.), aggregation, and biophysical structural variants [6–9]. An understanding of the impact of these attributes on biological activities, such as clearance, toxicology, and immunogenicity in humans, provides important information to assess the criticality of a particular quality attribute [10]. It is a regulatory expectation that potential critical quality attributes associated

⇑ Corresponding author. E-mail address: [email protected] (X.G. Jiang). Abbreviations used: QbD, quality by design; IgG, immunoglobulin G; NEM, Nethylmaleimide; TCEP, tris(2-carboxyethyl)phosphine; TFA, trifluoroacetic acid; DTNB, 50 -dithio-bis(2-nitrobenzoic acid); PBS, phosphate-buffered saline; MWCO, molecular weight cutoff; HPLC, high-performance liquid chromatography; AEX, anion exchange; RT, room temperature; DMSO, dimethyl sulfoxide; RP, reversed-phase; MS, mass spectrometry; ESI, electrospray ionization; MS/MS, tandem MS; LC, liquid chromatography. 1

0003-2697/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ab.2012.09.027

with the drug are identified to allow study and control of process parameters that could affect them [11]. An evaluation of product quality attribute criticality can be performed through a series of in vivo and in vitro experiments [10]. In in vivo studies, an animal model or patient blood/serum samples obtained from clinical trials can be used to evaluate the impact on conversion, clearance, toxicology, and immunogenicity of the product. In vivo product quality attribute studies are normally considered as more relevant than in vitro studies. However, in vivo studies are expensive and complex, and they may require material quality not available at a particular stage of development. Thus, despite other pitfalls, in vitro models of in vivo behavior are attractive alternatives for such attribute conversion studies. One advantage of an in vitro study system is its accessibility. Once established, such a system can be routinely used to predict physiological behavior of a variety of attributes [10,12]. In addition, an in vitro system may be much simpler to use and, therefore, more applicable for modeling studies. In vitro activity assays, using cell-based or receptor binding assays, are also commonly used to evaluate bioactivity of product quality attributes [9]. Several immunoglobulin G (IgG) molecular attributes have been studied using in vitro and/or in vivo approaches. For example, IgG2 is secreted into human blood in the IgG2-A form. As it circulates,

Evaluation of protein disulfide conversion / X.G. Jiang et al. / Anal. Biochem. 432 (2013) 142–154

IgG2-A is first converted into the IgG2-A/B form and then to the IgG2-B form at different rates [13]. Recent in vivo and in vitro studies have demonstrated the same interconversion of disulfide isoforms of recombinant IgG2 antibodies [12,13]. Bioactivity assay results suggested that different IgG2 isoforms may have different binding activities to interleukin-1 cell surface receptor type 1 [12]. An anti-IgE antibody, omalizumab, was found to have unpaired cysteine on the VH domain and Fab region, which was reported to have reduced potency. The serum study suggested the rapid conversion of unpaired cysteine to correct pairing. The study also indicated that thiol-containing reagents in serum provided an environment for disulfide rearrangement [14]. Trisulfide bond in IgG antibody was identified as a common posttranslational modification. The in vivo stability study of trisulfide bond in rat showed conversion to disulfide bond after 24 h of circulation in rat [15]. Other than the disulfide isoforms, the impact of glycosylation on recombinant IgG2 was also evaluated, by analyzing glycan profiles of IgG2 antibodies in patient serum samples and IgG2 samples incubated in serum in vitro. Similar serum incubation studies in vitro have been used to evaluate the biological relevance of deamidation, C-terminal lysine, and N-terminal glutamate to pyroglutamate conversion [16–18]. Although a significant number of in vitro studies have been performed using human serum/blood, maintenance of labile redox levels in human blood remains challenging. Human blood normally contains low concentration of free thiols in the forms of cysteine and glutathione. Free thiols can mediate the disulfide conversion of recombinant protein after administration. Approximately 12.5 ± 3 lM reduced thiol and 250 lM disulfide bonded thiol are found in normal human blood [19,20]. To mimic the redox state in a simplified model, 15 lM cysteine and 250 lM cystine are used in in vitro studies to represent the combined redox potential of cysteine and glutathione in blood. During IgG2 disulfide conversion studies in vitro, conversion was limited due to the rapid oxidation of cysteine in normal environmental conditions [13]. To improve redox control, we developed a continuous flow dialysis system in which physiological cysteine concentration can be maintained. To demonstrate the capability of our system, in vitro conversion of disulfide variants of an Fc-fusion protein was studied. The Fc-fusion protein used was composed of a human Fc portion and a copy of an active peptide moiety linked to each C terminus of the Fc chain. There were two disulfide bonds linking four cysteines on each peptide moiety. This Fc-fusion protein was expressed in Escherichia coli. Recombinant proteins expressed in microbial cells do not form their proper disulfide bonds due to the reducing state of the cytosol [21]. Protein refolding steps, therefore, are necessary to produce active therapeutics. During protein refolding steps, where low-molecular-weight thiol reagents are used to promote oxidation and exchange of disulfides, disulfide variants can be produced along with correctly bonded products. These disulfide variants include incorrectly disulfide bonded species, thiol adducts, and correctly disulfide bonded species [22,23]. It is important to understand their conversion and clearance in the human body because disulfide variants could affect the bioactivity of the molecule [12,13]. In this study, the in vitro conversions of adducted species, the incorrectly disulfide bonded species, and the correctly disulfide bonded form of the Fc-fusion protein were demonstrated using the continuous flow dialysis system.

Materials and methods Materials The Fc-fusion protein drug substance used in this study was produced in-house at Amgen. Lysyl endopeptidase (Wako USA),

143

endoproteinase Glu-C (Roche), N-ethylmaleimide (NEM, Pierce), urea (MP Biomedicals), hydroxylamine (Sigma–Aldrich), sodium phosphate (monobasic, monohydrate) (J.T. Baker), acetonitrile (Burdick & Jackson), tris(2-carboxyethyl)phosphine (TCEP, Pierce), trifluoroacetic acid (TFA, Pierce), 50 -dithio-bis(2-nitrobenzoic acid) (DTNB, Sigma–Aldrich), and L-cysteine free base and L-cystine (MP Biomedicals) were purchased. 10 Phosphate-buffered saline (PBS), Slide-A-Lyzer dialysis cassettes (7000 MWCO [molecular weight cutoff], 0.1–0.5 ml capacity), and Slide-A-Lyzer 1.0-ml syringes and 18-gauge needles were obtained from Thermo Scientific. The adducted species of the Fc-fusion protein investigated in this study was purified from a purification intermediate using anion exchange high-performance liquid chromatography (HPLC) (see Fig. 2 in Results). Incorrectly disulfide bonded species of the Fc-fusion protein was an enriched purification intermediate. Refolding of Fc-fusion protein The Fc-fusion protein was expressed by E. coli. After high-pressure cell homogenization, the inclusion bodies were harvested by centrifugation and solubilized by a solution containing guanidine, urea, and dithiothreitol (DTT). The solubilized inclusion bodies were refolded in a system containing guanidine, urea, and the redox components L-cysteine and cystamine. AEX–HPLC Anion exchange HPLC (AEX–HPLC) was used to analyze the Fcfusion protein forms and to collect specific disulfide isoforms for the in vitro study. AEX–HPLC was performed on a TSK DEAE-5PW column (7.5  75 mm, 10 lm) at a flow rate of 0.5 ml/min and a column temperature of 43 °C. A total of 10 lg of protein was injected into the HPLC column, and the eluting protein was monitored at 230 nm. The autosampler was controlled at 4 °C. AEX– HPLC running buffer A was 25 mM sodium phosphate solution at pH 6.5, and buffer B was 25 mM sodium phosphate with 1.0 M NaCl solution at pH 6.5. For routine analysis, a longer version of the gradient (20% B for 5 min, 20–28.5% B in 5 min, 28.5–66.5% B in 48 min, followed by 99% B for 4 min) was used with a total run time of 86 min. The separation was performed on an Agilent 1100 HPLC system. During fraction collection, a shorter version of the method was used for higher throughput operation. In the short version of the assay, a gradient (32–48% B in 20 min, 48– 60% B in 14 min, followed by 99% B for 2 min) was used with a total run time of 46 min. Static in vitro dialysis system A total of 180 mg of cystine was added to 3 L of PBS buffer for a final concentration of 250 lM. The solution was added to a 5-L container and degassed by purging nitrogen into the solution for 3 h. The container was placed in an incubator at 37 °C supplied with continuous nitrogen flow. The container was covered with Parafilm, and the headspace was continuously purged with nitrogen over the incubation period. The solution was mixed continuously on a stirring plate with a stirring bar inside the container. A total of 0.5 ml of cysteine stock solution of 11 mg/ml (90 mM) was added to the system to make a final concentration of 15 lM. The cysteine level in the container at various time points was measured by using Ellman’s assay. The Fc-fusion protein variants collected from AEX were diluted in PBS buffer to a concentration of 0.2 mg/ml, which mimics the in vivo concentration of the Fc-fusion protein under clinical doses, and injected into dialysis cassettes. The cassettes were placed into the container at the beginning of the experiment.

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Continuous flow in vitro dialysis system A diagram of the system is shown in Scheme 1. A high-flow-rate AKTA Explorer 100 system (GE Healthcare) was used to deliver the continuous flow buffer system. Buffer A was 150 lM cysteine in PBS at pH 7.2. It was prepared by adding a thawed stock solution of cysteine (150 mM in PBS and stored at 70 °C) into PBS. Buffer B was 278 lM cystine in PBS at pH 7.2. It was prepared by adding a stock solution of cystine (278 mM in 1.0 N HCl and stored at room temperature [RT]) into PBS. A mixing ratio of approximately 10% A and 90% B was delivered to reach a final concentration of approximately 15 lM cysteine and 250 lM cystine in PBS at pH 7.2. The flow rate was kept at 10 ml/min. An XK50 column (Pharmacia) was used as the dialysis chamber where the samples were incubated. The temperature of the sample chamber was kept at 37 °C by circulating heated water from an external water bath through the column jacket. The buffer inlet was also preheated by a heat exchanger to bring the temperature to 37 °C before entering the chamber. The samples were diluted in PBS buffer to a concentration of 0.2 mg/ml and injected into dialysis cassettes. The cassettes filled with samples were placed into the sample chamber. The sample chamber was covered by aluminum foil to protect the samples from light. The effluent of the sample chamber was going to waste or a fraction collector for cysteine concentration measurement. Analysis of free cysteine by Ellman’s reagent A total of 39.6 mg of DTNB (Ellman’s reagent) was dissolved in 10 ml of dimethyl sulfoxide (DMSO) to make a 10-mM Ellman’s reagent (DTNB) solution. A total of 1.0 ml of fraction collected from AKTA was mixed with 20 ll of the Ellman’s reagent solution. It was mixed gently and incubated at RT for 5 to 20 min. Using a Beckman DU 800 spectrophotometer, the sample was measured at 412 nm. The absorbance reading was divided by the extinction coefficient of 13.6 mM1 cm1 and multiplied by a factor of 1000 to obtain free cysteine concentration in micromolar units. In vitro incubation using the continuous flow dialysis system A pre-peak fraction collected from AEX chromatography was diluted to 0.2 mg/ml using PBS buffer. A total of 400 ll of diluted

10% flow Heat Exchanger

Mixer Dialysis chamber XK50 column

Fractions

Jacket @ 37°C

µM cystine in PBS 278µ 90% flow

RP–HPLC analysis of the Fc-fusion protein The intact Fc-fusion proteins were analyzed on an Agilent 1100 HPLC system equipped with a binary pump. A total of 5 to 10 lg of protein was loaded onto a Zorbax C18 column (2.1  150 mm, 5 lm, Agilent Technologies) and analyzed at a flow rate of 0.2 ml/min. Mobile phases A and B were 0.1% (v/v) TFA in Millipore water and 0.1% (v/v) TFA in acetonitrile, respectively. The gradient was as shown in Table 1. RP–HPLC–MS analysis of the Fc-fusion protein RP–HPLC–MS analysis was performed on an Agilent TOF (timeof-flight) 6224 mass spectrometer with an Agilent 1100 coupled to the electrospray ionization (ESI) source. The injection amount for the RP–HPLC–MS analysis was 5 to 10 lg. The RP–HPLC conditions were the same as those described in the section above. A splitter was added after the UV (ultraviolet) detection and before the ESI source to divert 20% of the flow into the mass spectrometer. The mass spectra were collected and deconvoluted using MassHunter software from Agilent Technologies. The relative quantity of each disulfide isoform was calculated using the ratio of the integrated peak areas over the total peak area in the deconvoluted mass spectra. Lys-C digestion of the Fc-fusion protein

Water bath 37°C

150µM cysteine in PBS

pre-peak fraction was injected into each of five 0.5-ml dialysis cassettes. Fc-fusion protein drug substance was prepared in the same way in five dialysis cassettes as a control. The cassettes containing the pre-peak fractions or drug substance were placed into the dialysis chamber. The cassettes were arranged in three layers with the adjacent layers perpendicular to each other (Fig. 1). There were three cassettes in each layer, and one extra cassette lay on top of the three layers. This arrangement of the cassettes allowed efficient buffer refreshment around each cassette. Each cassette was 4 cm high, and the total sample chamber height was 14 cm. The total volume of the sample chamber was 275 ml. The sample chamber was continuously filled with PBS buffer with cysteine (15 lM) and cystine (250 lM) at 37 °C. One cassette containing pre-peak fraction or drug substance was taken at each time point of the incubation. The samples were collected from the cassettes and analyzed by AEX–HPLC, intact reversed-phase–HPLC–mass spectrometry (RP–HPLC–MS), and peptide mapping. This experiment was repeated with incorrectly disulfide bonded species.

Scheme 1. Schematic diagram of the continuous flow dialysis system.

The Lys-C digestion coupled with RP–HPLC–tandem MS (MS/ MS) analysis was used to characterize the disulfide structure of the Fc-fusion protein in the AEX pre-peak and main peak fraction. The Fc-fusion protein was denatured and alkylated by mixing 0.2 mg of protein with denaturation and alkylation buffer that was composed of 8 M guanidineHCl and 0.1 mM NEM in 0.1 M Tris buffer at pH 7.5. The final concentration of the solution contained 10 mg/ml protein, 4 M guanidineHCl, and 0.1 mM NEM in a 20ll volume. The solution was incubated at RT for 30 min. After the denaturation and alkylation, this solution was mixed with 90 ll of digestion buffer (8 M urea, 40 mM NH2OHHCl, and 0.2 M sodium phosphate at pH 7.0), 80 ll of water, and 10 ll of 1 lg/ll Lys-C. The final protein concentration was 1 mg/ml with an enzyme-to-substrate ratio of 1:20. The solution was incubated at 37 °C for 5 h and quenched with 50 ll of 5% TFA. The samples generated using the in vitro incubation system described earlier in Materials and Methods had a relatively low protein concentration. Therefore, these samples were concentrated and buffer exchanged into 0.1 M phosphate buffer using a 10,000MWCO filtration device (VivaScience) to achieve a final protein

Evaluation of protein disulfide conversion / X.G. Jiang et al. / Anal. Biochem. 432 (2013) 142–154

145

Fig.1. Dialysis cassettes packing in the dialysis chamber. Samples are stored in dialysis cassettes. The buffers containing cysteine and cystine continuously flow through the dialysis chamber to maintain a constant concentration.

min. The data were analyzed both manually and by MassAnalyzer software [24].

Table 1 RP–HPLC gradient for intact molecular analysis. Time (min)

%A

%B

Flow rate (ml/min)

0 25 26 29 30 44 45

67 63 5 5 67 67 67

33 37 95 95 33 33 33

0.2 0.2 0.4 0.4 0.4 0.4 0.2

concentration of approximately 5 mg/ml. The denaturation and alkylation were carried out by mixing appropriate amounts of concentrated protein and digestion buffer and 1 mM NEM to make a final solution composed of 2 mg/ml protein, 4 M guanidine, and 0.1 mM NEM. The solution was incubated at RT for 30 min and then mixed with an equal volume of digestion buffer and an appropriate amount of 1 lg/ll Lys-C to make a final mixture of 1:20 enzymeto-substrate ratio. The mixture was then incubated for 5 h at 37 °C and finally quenched with 25 ll of 5% TFA for every 100 ll of digestion.

LC–MS/MS analysis of protein digest Liquid chromatography (LC)–MS/MS analysis of the protein digest was executed on a Thermo LTQ ion trap mass spectrometer or an LTQ-Velos Orbitrap mass spectrometer coupled with an Agilent 1100 HPLC system. Mobile phases A and B were 0.1% TFA in water and 0.1% TFA in acetonitrile, respectively. The gradient was from 2 to 22% B in 40 min, followed by 22 to 42% B for an additional 80 min. Approximately 30 lg of protein digest was injected onto a C18 column (250  2 mm, 5 lm, Phenomenex) for separation. The column temperature was set at 50 °C, and the flow rate was 0.2 ml/

Results Characterization of disulfide variants in the Fc-fusion protein A pre-peak in the AEX analysis of a purified Fc-fusion protein was observed (Fig. 2A). To characterize the species, the pre-peak was collected using a short version of the AEX–HPLC method (described in Materials and Methods) from a purification intermediate (Fig. 2B). The main peak was also collected as a control. Collected proteins were characterized using RP–HPLC–MS intact mass analyses. The main component of the pre-peak had a mass increase of 152 Da over the mass of the protein in the main peak (Fig. 3). Theoretically, each cysteamine adduct increases the mass of the molecule by 75 Da. Therefore, this result suggested a twocysteamine adduction of the Fc-fusion protein, which likely forms during refolding/redox step. To test this hypothesis, the collected pre-peak was proteolyzed by endoproteinase Lys-C and characterized using RP–HPLC coupled with MS. The collected main peak was used as a control. Several cysteamine adduct-containing peptides were identified in the pre-peak fraction digest (Fig. 4A and B) but were absent in the main peak fraction digest. Unexpected cleavage sites at cysteine residues 275 and 282 were observed in both the reduced and nonreduced peptide maps. The cysteamine adduction sites were identified to be on cysteine residues 275 and 282 on the peptide moiety based on LC–MS/MS data. Multiple miscleaved peptides were also observed in the nonreduced and reduced peptide maps. These miscleaved peptides were cleaved on the C-terminal sides of cysteamine modified cysteine residues 275, 282, and 285. Other pre-peaks between the cysteamine adduct pre-peak and main peak were also collected using AEX chromatography and characterized using intact LC–MS and Lys-C digest followed

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A Norm. 25

20

pre-peak of question

15

10

Lot 2 5

Lot 1 0 25

30

35

40

45

50

55

Main Peak

mAU 1750

Start collection

1500

End collection

min

B

1250 1000

pre-peak

750 500 250 0 0

5

10

15

20

25

30

35

40

min

Fig.2. (A) A long version of the AEX–HPLC chromatogram of purified Fc-fusion protein from two different lots. (B) A short version of the AEX–HPLC chromatogram of a prepeak enriched fraction from a purification intermediate.

by LC–MS/MS analysis. These peaks mostly contain incorrectly disulfide bonded species, where misfolds of the disulfide bonds in the Fc-fusion peptide region formed during refolding processes. Methionine oxidation species were also identified in these prepeaks as a minor form in the samples studied (data not shown).

ately after 10 dialyzer units were added into the container, and the cysteine concentration was found to be 16 lM. After 2 h, the cysteine level dropped to 5.5 lM and then to 0 lM at 4 h (Table 2). These results indicated complete oxidation of 16 lM cysteine in 4 h in a static system.

Performance of a static in vitro dialysis system

Performance of the continuous flow in vitro dialysis system

The capability of maintaining cysteine concentrations in a static system was evaluated with a DTNB assay. The cysteine level was found to be 16 lM at the 0-h time point when the solution was freshly prepared. The cysteine level was measured again immedi-

Similar experiments were performed in a continuous flow dialysis system to evaluate the capability of maintaining cysteine concentrations. The sample chamber was flushed with 90% cystine buffer and 10% cysteine buffer at 10 ml/min to reach equilibrium

Evaluation of protein disulfide conversion / X.G. Jiang et al. / Anal. Biochem. 432 (2013) 142–154

147

Total ion count

63665 (+152 Da, + 2 cysteamine)

Pre Peak

Elution time (min)

63513 (main)

Total ion count

Main Peak

Elution time (min)

Fig.3. Total ion chromatogram of RP–HPLC–MS analysis of the pre-peak fraction and main peak fraction collected from AEX–HPLC separation of an enriched fraction from a purification intermediate.

as described in Materials and Methods. The effluent buffer was collected into 15-ml tubes before and after adding the sample cassettes using the fraction collector and cysteine concentrations measured. The cysteine concentration was further monitored at 1.4, 3.9, 6.8, 24, and 48 h after the addition of the sample cassettes. Cysteine concentrations were measured in triplicate at each time point. The standard deviation and average measurement at each time point are shown in Table 3. The continuous flow dialysis system maintained the cysteine level between 13.3 and 14.8 lM during incubation over 48 h. The standard deviation of the cysteine concentration was less than 0.3 lM. These results indicated that the in vitro dialysis system is capable of maintaining the cysteine concentration near the target level of 15 lM. To maintain constant cysteine concentrations, the buffer volume needs to be optimized based on the liquid volume in the sample chamber. Because a 10-ml/min buffer flow rate was sufficient to maintain a constant cysteine concentration in a sample chamber with 167 ml of liquid, an experiment was performed to explore the total volume required to completely refresh the whole sample chamber at 10 ml/min. The system was filled with six dialysis cassettes and 167 ml of water. The chamber was continuously refreshed with the cysteine/cystine PBS buffer at a flow rate of 10 ml/min, and the conductivity was measured. Fig. 5 shows the measured conductivity versus the volume being pumped through the sample chamber. The equation illustrates the relationship between conductivity (y) and the volume being pumped through the sample chamber (x). It was assumed that the conductivity reached a plateau when the cysteine and cystine concentrations reached a steady state in the sample chamber. Therefore, the volume needed to fully replenish the sample chamber can be determined as the volume when the curve began to plateau. As shown in Fig. 5, approximately 400 ml of buffer was required to pump through the system for the conductivity value to reach a plateau. Because the flow rate was 10 ml/min, the duration of volume exchange was calculated to be 40 min (400 ml  10 ml/min) for a chamber with a 167-ml liquid volume. The buffer exchange rate inside the sample chamber could be calculated as total time that is needed to replenish the full chamber (40 min) divided by total liquid volume inside the chamber (167 ml). The buffer exchange rate was calculated to be 0.24 min/ml at a flow rate of 10 ml/ min. Assuming that the same volume exchange rate is needed to maintain the constant cysteine concentration in the sample

chamber, the flow rate for a sample chamber with a different liquid volume can be calculated. Prior to protein incubation, the system was flushed with the mixture of 10% cysteine and 90% cystine for at least one full volume exchange cycle to reach equilibrium. In vitro exchange of disulfide variants in an Fc-fusion protein Samples containing purified adduct species or drug substance were individually filled into five dialysis cassettes and placed into the sample chamber of the continuous flow dialysis system. After incubation, samples were collected at five different time points and analyzed by AEX–HPLC. The chromatogram overlay showed that, when exposed to the simulated physiological temperature, pH, and redox conditions, cysteamine adducted species quickly converted to the main peak species and incorrectly disulfide bonded species within the first 4 h (Fig. 6A). The AEX chromatogram was integrated, and the percentage abundances of three major species—cysteamine adduct, incorrectly disulfide bonded, and correctly bonded product—were quantified. By plotting the percentage peak areas versus the incubation time, conversion time courses for these three species (Fig. 6B) can be illustrated. The conversion appeared to reach a plateau after 4 h. Based on the integration results, cysteamine adducted species levels were 93.4, 31.3, and 9.0% at 0-, 1.7-, and 4-h incubation times, respectively. Accompanying the decrease of the adducted peak, there were increases of the correctly disulfide bonded product and incorrectly disulfide bonded species. The correctly bonded product levels were 0.8, 41.0, and 50.7% at 0, 1.7, and 4 h, respectively. Incorrectly disulfide bonded species levels were 0, 20.5, and 30.9% at 0, 1.7, and 4 h, respectively. These results indicated that cysteamine adduct species converted quickly into other species in a redox environment similar to human blood. The majority of adduct species converted into the correctly bonded product, whereas approximately 30% of adduct species converted into incorrectly disulfide bonded species. AEX analysis of drug substance incubated in vitro showed that the adduct species disappeared in 61.7 h and incorrectly disulfide bonded species levels increased with the increase of incubation time (Fig. 7A). The chromatogram was integrated to quantify the relative abundance of incorrectly disulfide bonded species and the correctly bonded product. The relative amounts of incorrectly disulfide bonded species and correctly bonded product were plotted against incubation time (Fig. 7B). The correctly bonded product

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A mAU 2000 1750

Pre-peak

1500 1250 1000 750 500

Main peak

250 0 20

25

30

35

40

45

50

55

60

Norm.

min

B

3000

2000

1000

0

-1000

-2000

-3000

20

30

40

50

60

min

Fig.4. (A) RP–HPLC separation of nonreduced Lys-C digest of collected pre-peak fraction and main peak fraction from AEX–HPLC separation. (B) Comparison of RP–HPLC separation of nonreduced and reduced Lys-C digest of collected pre-peak fraction.

Table 2 Measured cysteine levels in a 3-L static system protected from oxygen and maintained at 37 °C.

Table 3 Measured cysteine levels in the continuous flow dialysis system during incubation of adduct species.

Incubation time (h)

Cysteine concentration (lM)

Time (h)

Cysteine concentration (lM)

Standard deviation (n = 3)

T = 0 before adding dialyzer T = 0 after adding dialyzer T=2 T=4

16.0 16.1 5.5 0

0 1.4 3.9 6.8 24 48

14.7 14.0 14.8 13.3 14.5 14.1

0.3 0.1 0.2 0.2 0.2 0.1

Note: The measurements were obtained using Ellman’s reagent.

levels decreased from 92.6 to 82.8% after 48 h; incorrectly disulfide bonded species levels increased approximately 8.0% after 48 h. The trends of changes started to reach a plateau after a 48-h incubation. Adduct samples incubated in vitro were analyzed by intact RP– HPLC–MS. The assay measures the molecular weight of the species

in the samples. Fig. 8A shows the overlay of deconvoluted mass spectra of adduct samples. The peaks at molecular weights of 63,512 and 63,665 Da matched the unmodified product and cysteamine adduct species, respectively (Fig. 3). The peaks at molecular weights of 63,705 and 63,750 Da match the molecular weights of a

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isoform to the sum of all isoforms. Fig. 8B shows the plotted changes of the three adduct species during the 2-day incubation time span. The cysteamine adduct decreased from approximately 100% to undetectable levels. The one cysteine/one cysteamine adduct species was not detected in the starting material but increased to approximately 20% at the 1.7-h time point before decreasing to an undetected level at the 7-h time point. No cysteine/cysteine adduct was observed during the first 2 h, but approximately 17% of this adduct was observed at the 7-h time point before decreasing to 7% at the 48-h time point. To further characterize the adduct sites during the incubation, the adduct samples collected at 2 and 48 h were analyzed by peptide mapping coupled with MS. In both samples, amino acid residues cysteine 275 and 282 were observed to be the cysteamine adduct sites, whereas the cysteine adduct was observed only on the cysteine 282 residue. Because cysteine/cysteine adducts were observed in the intermediate sample collected at 48 h using intact molecule LC/MS, the fact that only one cysteine adduct site was detected could be attributed to the symmetrical double chain structure of the Fc-fusion protein.

Fig.5. Measured conductivity of buffer outlet from the continuous flow dialysis system as a function of total buffer volume.

variant with one cysteine adduct/one cysteamine adduct and a variant with two cysteine adducts, respectively. The relative amount of each isoform can be calculated from the peak area ratio of each

Cysteamine Adduct

mAU

Correctly Bonded Product

A

120

100

Incorrectly Disulfide Bonded Species

80 48 h 60

24 h 7h

40 4h 1.7 h

20

0h 0 10

15

20

25

30

35

min

B

Fig.6. (A) Overlay of AEX–HPLC chromatogram of an enriched adduct species at six different incubation time points: 0, 1.7, 4, 7, 24, and 48 h. (B) Plot of AEX–HPLC purity changes of each integrated peak as a function of incubation time.

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Correctly Bonded Product

A

mAU 120 Incorrectly Disulfide Bonded Species 100

48 h 80

24 h

60

7h 4h

40

1.7 h 20 0h 0 10

15

20

25

30

35

min

% Abundance

B

Fig.7. (A) Overlay of AEX–HPLC chromatogram of drug substance at six different incubation time points: 0, 1.7, 4, 7, 24, and 48 h. (B) Plot of AEX–HPLC purity changes of each integrated peak as a function of incubation time.

Incorrectly disulfide bonded species were also studied in the same fashion as the cysteamine adduct species. The incorrectly disulfide bonded species is a mixture of two major disulfide isoforms that appear as two peaks in AEX–HPLC (Fig. 9A). Samples were incubated in vitro for 4, 8, 24, 48, and 96 h and then analyzed by AEX–HPLC. The main peak and pre-peaks containing incorrectly disulfide bonded species were integrated to quantify the changes during the time course. Results showed a continuous decrease in the level of incorrectly disulfide bonded species as they converted into the correctly bonded product during the 96-h incubation (Fig. 9B). The two peaks containing incorrectly disulfide bonded species showed similar trends. Incorrectly disulfide bonded species

1 levels decreased from 57.9 to 34.6% during the 96-h incubation. Incorrectly disulfide bonded species 2 levels decreased from 40.2 to 23.5% during the same time period, whereas the correctly bonded product levels increased from 2.0 to 41.9%. The plot shows that the species conversion nearly reached equilibrium in 96 h.

Discussion In this study, we have described the development of a continuous flow incubation system for the evaluation of criticality of cysteine related modifications of an Fc-fusion protein. We demon-

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Fc-fusion Protein/Incorrectly Disulfide Bonded Species 63512 5

A

x10

+2 Csm (63665, + 154*)

1.2

+2 Cys (63750, + 239*)

+1 Csm/1Cys (63705, + 194*)

1.0 0.8

48 h 24 h

0.6

7h 0.4

4h 1.7 h

0.2 0.0

0h 63500

63540

63580

63620

63660

63700

63740

63780

63820

Counts vs. deconvoluted mass (Da) *Delta mass was calculated as the differences between measured mass and the theoretical mass of the correctly disulfide bonded Fc-fusion protein

B

Fig.8. (A) Deconvoluted LC–MS analysis of cysteamine adduct species at different incubation times. ⁄Delta mass was calculated as the differences between measured mass and the theoretical mass of the correctly disulfide bonded Fc-fusion protein. (B) Plot of percentage abundance of each species versus incubation time.

strated that this system is able to effectively maintain low cysteine concentrations similar to those of human blood. Cysteine is a labile reagent when dissolved in water, readily oxidizing to form cystine. Maintaining a 15-lM cysteine concentration in an in vitro system is a very challenging task. Complete oxygen removal is difficult, but without removal free cysteine levels are rapidly depleted. Although extensive efforts were made to remove oxygen in a static dialysis system, which included nitrogen purging of the system and keeping the entire static system in a nitrogen-filled incubator, cysteine levels still quickly decreased over time. Therefore, manually changing the dialysis buffer every few hours was required to maintain cysteine concentrations within acceptable ranges in a static system. Compared with the static system, the continuous flow dialysis system is automated and could maintain the cysteine levels with less variability. This system provides a platform for potential high-throughput evaluation of disulfide related quality

attributes. The rates of conversion can be compared with expected lot-to-lot variability and serum clearance to influence criticality decision making. There are several major advantages associated with the continuous flow dialysis system described in this article. First, the dialysis chamber is continuously refreshed with cysteine/cystine buffer stock to maintain the cysteine level. Second, the cysteine buffer is prepared as a 10 concentrated stock, which minimizes the percentage loss of cysteine during its preparation and handling. Third, the cysteine and cystine in PBS buffer were delivered from two separate buffer systems that offer the flexibility to adjust the mixing ratio to achieve the target concentration. Finally, the chamber is insulated from air, significantly slowing the oxidation rate in the buffer as compared with the static system. In this continuous flow system, the dialysis sample chamber is continuously replenished with fresh buffer at 10 ml/min. This flow rate has proven to be sufficient to maintain stable cysteine

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mAU

Incorrectly Disulfide Bonded Species 1

A

Correctly Bonded Product

50 Incorrectly Disulfide Bonded Species 2 40 96 h 30 48 h 24 h

20

8h 4h

10

0h 0 16

18

20

22

24

26

28

30

32

min

B

Fig.9. (A) Chromatogram overlays of AEX analysis of incorrectly disulfide bonded species at various incubation times. (B) Plot of disulfide variant purity as a function of incubation time.

concentrations based on the adduct incubation study. For studies requiring different chamber volumes or those maintaining different labile chemical concentrations, optimization of the flow rate would be needed to ensure constant reagent concentrations. During characterization of cysteamine adducts using Lys-C peptide mapping, cleavage sites at the C terminus of cysteamine modified cysteine residues were observed. Similar results were observed with other Fc-fusion proteins (data not shown). This phenomenon can be explained by the structural resemblance of cysteamine modified cysteine residue and lysine residue (Fig. 10). Both side chains have a primary amine on the end with a carbon chain on the lysine residue and a carbon/sulfur chain on the cysteamine modified cysteine residue. We hypothesize that the Lys-C enzyme

may recognize the cysteamine modified cysteine residue as lysine and cleave on the C-terminal side of cysteine. This hypothesis can be supported by the previous studies on aminoethylation of cysteines, which selectively convert cysteine into lysine analogues S-aminoethylcysteine (Fig. 10) [25,26]. The method of aminoethylation of cysteines has been used to artificially generate trypsin substrate for protein sequencing as well as other biological purposes [27,28]. A similar phenomenon of structural resemblance in amino acids has been observed with the structural similarity between methionine and norleucine [29]. Although cysteamine modified cysteine residues were recognized by Lys-C, cleavage was not as efficient as lysine site-specific cleavage. As a result, a cysteamine modified cysteine residue may reside in more than one enzymatic

Evaluation of protein disulfide conversion / X.G. Jiang et al. / Anal. Biochem. 432 (2013) 142–154

O

HS

153

O

OH

S

Cysteamine H2N

S

OH

NH2

NH2

Cysteamine modified cysteine

Cysteine

O H2N OH NH2

Lysine

Aminoethylation H2N

H2N

NH2

NH2

Cysteine

S-aminoethylcysteine

Fig.10. Structural similarity among lysine residue, cysteamine modified cysteine residue, and S-aminoethylcysteine.

peptide after digestion. This incomplete nonspecific enzymatic cleavage has posed challenges in product characterization, especially in quantification of posttranslational modifications due to the increased heterogeneity of the enzymatic peptides. Therefore, tracing the site-specific changes of adduct species quantitatively during the incubation study has been difficult. Due to the nature of the cysteamine adduct, it can be detected only under nonreduced environments. The protease chosen must be active under nonreducing conditions in the presence of a denaturant such as urea or guanidine. This fact limited the choices of enzymes for peptide mapping characterization. Alternatively, the intact LC–MS coupled with the information from peptide mapping provides an estimate on the quantitative trending information of the posttranslational modifications. Cysteine adducts and cysteine/cysteamine adducts were also formed during the dialysis process. These cysteine-containing adduct species existed transiently during in vitroincubations, indicating that cysteine adduct was the intermediate product of the conversion reaction. As a control, an incubation study was carried out in PBS buffer only without a redox couple, and no significant changes in the disulfide variant levels were observed at 37 °C up to 2 days (data not shown). In vitro study results using drug substance showed a small amount of conversion of correctly bonded protein into incorrectly disulfide bonded species, whereas in vitro study results of incorrectly disulfide bonded species showed major conversion of the incorrectly disulfide bonded species into the correctly bonded species. These results indicated that equilibrium favors the correctly bonded Fc-fusion protein over the disulfide variant forms under physiological conditions. These results suggest that the Fc-fusion protein, which contains various isoforms, will mainly convert into the correctly bonded disulfide species in 2 to 3 days. Further in vivo biological studies of these disulfide variant forms will be crucial to understand their impact on immunogenicity, efficacy, and stability. Because the half-life for clearance of this Fc-fusion protein in humans is 4 to 6 days and adduct species convert in a couple of hours, the criticality of cysteamine adduct of this particular Fc-fusion protein is diminished. Peptide mapping analysis of the disulfide vari-

ants at the beginning and end of incubation showed that the conversion of the disulfide structure was limited to the peptide moiety of the Fc-fusion protein. There was no obvious change observed in the disulfide structure of the Fc domain. This finding can be explained by the greater solvent accessibility of cysteine residues on the peptide moiety compared with the Fc domain [30–32]. The in vitro incubation study provides valuable information on the behavior and prevalence of the biopharmaceutical product and its impurity species in an environment mimicking human physiological conditions. The study results provide an initial evaluation of the criticality of the quality attributes before any costly endeavor such as clinical studies with humans or animals. This continuous flow dialysis system can be readily adopted to predict the behavior of a variety of thiol related modifications such as disulfide shuffled species, trisulfide species, and free thiols in protein molecules in vivo. Lability of an attribute in vivo can factor into the range setting of minor thiol species between different production lots. In addition, the design of this system enables improved control of labile thiol reagent in the in vitro study, providing more confident and reliable results for in vitro studies. Besides thiol related protein modifications, the system can potentially be used for evaluation of quality attributes in the presence of other types of labile reagents. Acknowledgments The authors thank Tom Dillon for his generous help and knowledge on the in vitro incubation system. We also extend our appreciation to Ronald Keener III for his help with the development of the continuous flow dialysis system. We thank Izydor Apostol for a critical review of the manuscript. References [1] Food and Drug Administration (FDA), Guidance for industry: Q8(R2) pharmaceutical development, US Department of Health and Human Services, November 2009. .

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