Journal of Chromatography B, 965 (2014) 72–78

Contents lists available at ScienceDirect

Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb

Protein A affinity precipitation of human immunoglobulin G Lars Janoschek, Matthias Freiherr von Roman, Sonja Berensmeier ∗ Bioseparation Engineering Group, Technische Universität München, 85748 Garching, Germany

a r t i c l e

i n f o

Article history: Received 19 March 2014 Received in revised form 5 June 2014 Accepted 12 June 2014 Available online 19 June 2014 Keywords: Antibody purification Eudragit Affinity precipitation protein A Immunoglobulin G

a b s t r a c t The potential of protein A affinity precipitation as an alternative method for traditional antibody purification techniques was investigated. Recombinant produced protein A from Staphylococcus aureus (SpA) was covalently linked to the pH-responsive copolymer Eudragit® S-100 and used for purification of human immunoglobulin G (hIgG). The Eudragit-SpA conjugate had a static binding capacity of 93.9 ± 2.8 mg hIgG per g conjugate and a dissociation constant of 787 ± 67 nM at 7 ± 1 ◦ C. The antibody was adsorbed rapidly onto Eudragit-SpA and reached equilibrium within 5 min. An excess of hIgG binding sites, provided by the conjugate, as well as adjusted elution conditions resulted in an appropriate hIgG purification performance. In summary, Eudragit-SpA was successfully applied to capture hIgG from a protein mixture with 65% antibody yield in the elution step. Nearly 96% purity and a purification factor of 12.4 were achieved. The Eudragit-SpA conjugate showed a stable ligand density over several cycles, which enabled reusability for repeated precipitation of hIgG. According to this, pH induced affinity precipitation can be seen as a potential alternative for protein A chromatography in antibody purification processes. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Antibodies have become a topic of increasing interest in recent years. Their usage as therapeutics in medical applications as well as for diagnostic purposes places high demands on product purity [1]. The complete removal of impurities and potential contaminants requires extensive downstream processing; including multiple chromatographic operations. A generic process for antibody purification from clarified cell culture supernatant contains a capture step with protein A affinity chromatography; followed by a combination of anion and cation exchange chromatography [2,3]. Despite their wide utilization in research and industry; low dynamic binding capacities and product throughput remain a major drawback of chromatographic purification steps. Following an increasing demand for antibodies; cell culture productivity has substantially improved over the years and antibody titers of about 10 g L−1 can be reached. It requires an enhanced purification performance with respect to product yield and throughput in order to achieve reasonable process economics [2,4]. These demands may be met either by scaling the column dimensions for higher throughput and the use

Abbreviations: hIgG, human immunoglobulin G; SpA, protein A from S. aureus; IPTG, isopropyl-␤-d-thiogalactoside; FBS, fetal bovine serum; EDC, 1-ethyl-3-(3dimethylaminopropyyl)carbodiimide; PBS, phosphate buffered saline. ∗ Corresponding author. Tel.: +49 89 289 15750; fax: +49 89 289 15766. E-mail address: [email protected] (S. Berensmeier). http://dx.doi.org/10.1016/j.jchromb.2014.06.011 1570-0232/© 2014 Elsevier B.V. All rights reserved.

of higher capacity chromatography media and therefore increased raw material costs; or by the search for alternative purification techniques. Low et al. [5] and Roque et al. [6] discuss potential approaches for selective antibody capture and purification; which may give similar yields and purities compared to the currently used chromatography–based techniques. Affinity precipitation is seen as a viable concept for antibody purification; providing product selectivity; easy scalability and a maximum degree of volume reduction during the process [5,7]. Affinity precipitation uses stimuli-responsive polymers or affinity macroligands, which allow antibody purification by simply changing the environmental conditions. Affinity macroligands consist of ligands specific to the target molecule which are conjugated onto polymer supports with a solubility that is controllable by an environmental trigger, such as temperature, salt concentration or pH [8]. Several polymers have been reported to be used for affinity precipitation, for instance polyelectrolyte complexes [9], elastinlike proteins that precipitate with a change in temperature [10] and anionic copolymers that precipitate due to pH changes [11,12]. Affinity ligands for the selective capture of the target molecule vary from chelating agents [13] to antigens with a specificity for the target antibody [9,11,12] or antibody binding proteins [10,14]. Although protein A is extensively used in chromatographic purification of antibodies, it has rarely been applied as an affinity ligand for selective immunoglobulin precipitation. Chen and coworkers [15] as well as Hilbrig et al. [16] presented an approach where protein A was covalently attached to the thermo-sensitive

L. Janoschek et al. / J. Chromatogr. B 965 (2014) 72–78

polymer poly-(N-isoproylacrylamid) (polyNIPAAm) and successfully applied to capture IgG from solution. The use of pH-responsive polymers in protein A affinity precipitation has not been reported so far. Here we describe the application of recombinant staphylococcal protein A (SpA) covalently linked to the polyelectrolyte Eudragit® S-100 for the purification of human immunoglobulin G (hIgG). Eudragit® S-100 is a copolymer of methacrylic acid and methyl methacrylate, showing a sharp pH-dependent phase transition from soluble to insoluble at pH values below 4.5 [17]. Target molecules, bound to the immobilized affinity ligands co-precipitate with the polymer and can be easily separated from other proteins and impurities in the solution by an additional elution step. Contrary to antigen ligands which target only a specific antibody, protein A provides selectivity toward a wide range of antibodies and is therefore more applicable within an industrial scope. We investigated the binding capacity and antibody purification performance of the described Eudragit-protein A macroligand and evaluated whether this precipitation technique can be used as an alternative to protein A affinity chromatography. 2. Materials and methods 2.1. Chemicals Eudragit® S-100 was obtained from Evonik Industries AG (Essen, Germany), fetal bovine serum (FBS) from Sigma-Aldrich (St. Louis, USA). All other chemicals were purchased from AppliChem (Darmstadt, Germany) or Carl Roth GmbH (Karlsruhe, Germany). Gammanorm® polyclonal human immunoglobulin G was obtained from Octapharma (Langenfeld, Switzerland) with an IgG subtype composition of 59% IgG1 , 36% IgG2 , 4.9% IgG3 and 0.5% IgG4 . For additional size fractionation of the hIgG solution, size exclusion chromatography was performed using an ÄKTAexplorer 100 system and a HiLoad Superdex200 16/60 column (both GE Healthcare, Freiburg, Germany). 2.2. Recombinant protein A production Production and purification of five domain protein A from Staphylococcus aureus was carried out according to von Roman et al. [18]. SpA was expressed in Escherichia coli BL21(DE3) with a pET20b-SpA vector (both Novagen, Madison, USA). Seed cultures were grown for 8 h at 37 ◦ C and 220 rpm in LB medium (per liter: 10 g trypton, 5 g yeast extract, 10 g NaCl) supplemented with 100 ␮g L−1 ampicillin. SpA expression was performed in 1 L shake flask with 4 mL of seed culture in 200 mL M9 minimal medium (per liter: 12.8 g Na2 HPO4 , 3.0 g KH2 PO4 , 0.5 g NaCl, 2 g NH4 Cl, 20 g glucose, 0.1 mM CaCl2 , 1.0 mM MgSO4 , 10 ␮M FeCl3 , 1% w/v thiamine) and 100 ␮g L−1 ampicillin [19]. Cells were grown for 16 h at 37 ◦ C and 220 rpm to approximately OD600 nm 3.5. SpA expression and secretion was induced with 1 mM IPTG and 180 mM Tris–HCl pH 8.5 and was allowed to proceed for further 16 h at 30 ◦ C. For SpA purification, cells were removed by centrifugation (3200 × g, 60 min). The protein containing supernatant was concentrated up to 10-fold and dialyzed against 20 mM l-histidine pH 6.5 binding buffer with a Slice200 ultrafiltration device and two 10 kDa MWCO Hydrosart® membranes (0.04 m2 total filtration area, Satorius AG, Göttingen, Germany). Subsequent purification was performed by ion exchange chromatography using an ÄKTAexplorer 100 System and Unosphere Q anion exchange support (Bio-Rad Laboratories GmbH, München, Germany). The column was equilibrated with binding buffer, loaded with the protein solution and followed by a washing step with 80 mM NaCl. Target protein was eluted by an increased salt concentration of 280 mM NaCl. Fractions containing SpA were pooled and dialyzed against phosphate

73

buffered saline pH 7.4 (PBS, per liter: 8.0 g NaCl, 0.2 g KCl, 1.42 g Na2 HPO4 , 0.27 g KH2 PO4 ) for further use. 2.3. Protein A conjugation to Eudragit® S-100 A Eudragit stock solution (% w/v) dissolved in PBS was prepared as described earlier by Guoqiang et al. [20]. Carbodiimide coupling of protein A to Eudragit® S-100 was carried out essentially following a protocol based on Arasaratnam et al. [21]. 1-ethyl-3(3-dimethylaminopropyyl)carbodiimide (EDC, % w/v) was added to the Eudragit stock solution and mixed for 10 min. 1% v/v Triton X-100 was supplemented to suppress non-covalent binding of SpA on the polymer’s backbone. Subsequently, SpA was added and stirred for 3 h at room temperature. Activated carboxyl groups were blocked with ethanolamine (45% v/v, pH 8.5) for 1 h. The polymer–protein complex was precipitated by lowering the pH to 4.5 with 2 M acetic acid and separated by centrifugation (10,000 × g, 10 min). The precipitate was washed alternately with 0.02 M acetate buffer pH 4.5 containing 0.25 M NaCl and PBS and thereafter was resuspended in PBS pH 7.4 for storage and further use. For the final coupling reaction, a 0.8% w/v Eudragit solution, 0.7% w/v EDC and 80 mg protein A per g polymer were used. At these conditions previously performed experiments pointed out the highest SpA load on Eudragit (data not shown). This was achieved by varying Eudragit and EDC concentrations based on a statistical design approach, as basically described by Silva et al. [22]. A 0.8% w/v Eudragit solution treated in the same way without adding SpA for conjugation was used as a control. 2.4. hIgG binding capacities Static binding capacities of Eudragit-SpA conjugate were determined in batch experiments at different temperatures. 250 ␮L of conjugate were added to 1 mL hIgG solution with concentrations ranging from 0.1 to 3.1 mg mL−1 hIgG. The presence of 1 M NaCl was necessary to avoid unspecific adsorption of hIgG to the polymer. Incubation took place at 1400 rpm at 7 ± 1 ◦ C and 22 ± 1 ◦ C in a Thermomixer (Eppendorf AG, Hamburg, Germany) for 20 min. The conjugate was precipitated by reducing the pH to 5.0, the highest pH at which complete precipitation occurred (not shown), with 2 M acetic acid. The suspension was centrifuged at 17,000 × g for 2 min and the supernatant was neutralized with 1 M NaOH. The equilibrium hIgG concentration in solution (c*) was then measured with SEC–HPLC and the amount of bound antibody (q*) was calculated by mass balance, using the batch volume and the conjugate’s dry mass. Data was fitted with Sigma Plot 8 (Systat Software Inc., San Jose, CA, USA) using a Langmuir isotherm model (Eq. (1)) to obtain the maximum saturation binding capacity (qmax ) and the dissociation constant (KD ). All experiments were performed in triplicate. A negative control was done without hIgG. q∗ =

qmax × C ∗ KD + C ∗

(1)

2.5. hIgG purification procedure The general procedure for hIgG purification using Eudragit-SpA conjugate is shown in Fig. 1. The ratio of accessible binding sites provided by the protein A ligands (mconjugate and qmax ) and the concentration of hIgG in solution (V0 and chIgG ) can be expressed as the capacity ratio CR (Eq. (2)). By varying the hIgG concentration chIgG and the added amount of conjugate mconjugate different capacity ratios of Eudragit-SpA conjugate and hIgG in solution were achieved and incubated for 20 min at 1400 rpm in the presence of 1 M NaCl. The volume of 1 mL was kept constant in 2 mL Eppendorf

74

L. Janoschek et al. / J. Chromatogr. B 965 (2014) 72–78

quantified with a protein A affinity column (SpA covalently linked on ProfinityTM Epoxide resin, Bio-Rad) on an ÄKTAexplorer 100 system. PBS was used as equilibration buffer and 1% w/v phosphoric acid for antibody elution. Purchased polyclonal hIgG was used as standard. In addition, fractions were analyzed by SDS-PAGE [23].

3. Results and discussion 3.1. hIgG binding capacities of Eudragit-SpA

Fig. 1. Scheme of an affinity precipitation process for antibody purification with Eudragit® S-100 and protein A as affinity ligand.

tubes. Supernatant, wash and eluate fractions were collected and analyzed by SEC–HPLC (see Section 2.7). In an additional experimental setup, 4% FBS were added to the final mixture in order to prove the effectiveness of the purification procedure in the presence of impurities. The polymer was precipitated by reducing the pH to 5.0 with 2 M acetic acid and separated by centrifugation. The precipitate was washed for 15 min with 0.02 M acetate buffer pH 5.0, centrifuged again and resuspended in elution buffer. Elution was carried out under constant mixing for 10 min in 0.2 M glycine pH 2.5 containing 0.2 M NaCl or for 1.5 h in 0.5 M arginine pH 3.8, respectively. Centrifugation steps were performed at 17,000 × g for 2 min. Samples for further analysis were neutralized with 1 M NaOH. All experiments were done in triplicate. Eudragit® S-100, treated in the same way but without adding SpA for ligand conjugation was used as a negative control. CR =

mconjugate × qmax V0 × chlgG

(2)

2.6. Conjugate reusability In order to investigate the reusability of Eudragit-SpA conjugate, seven consecutive precipitation cycles were performed. Each cycle comprised the precipitation procedure described above as well as an additional washing step in PBS and subsequent resolubilization of the conjugate in PBS. The amount of protein A which was covalent linked to Eudragit was determined after 1, 3, 5 and 7 cycles. Precipitation of hIgG from a hIgG/FBS mixture with recycled conjugate was carried out as previously described at a capacity ratio of 6.4. Untreated Eudragit-SpA conjugate was used as a reference. All experiments were performed in triplicate. 2.7. Analytical procedures Quantification of protein A, Eudragit-SpA conjugate and total protein in FBS containing samples was carried out in 96-well plates with Pierce® BCA-assay (Thermo Fisher Scientific, Waltham, USA). All samples were diluted in PBS and measured in triplicate. Bovine serum albumin (BSA) was used as standard.hIgG concentrations of binding capacity experiments were determined with SEC–HPLC, using an Agilent 1100 system (Agilent Technologies, Santa Clara, USA) and an ENrich SEC 650 column (Pt. 780-1650, Bio-Rad, Hercules, USA). PBS was used as mobile phase at a flow rate of 0.75 mL min−1 . hIgG in FBS containing samples was

In order to investigate maximum loading capacity of the compounded Eudragit with protein A as affinity ligand, adsorption equilibrium data was determined at two different temperatures. Eudragit-SpA conjugate with a ligand density of 35.5 mgSpA gconjugate −1 (0.4 mgSpA mL−1 ) and a dry weight of 11.2 mgconjugate mL−1 was used in all experiments. Obtained adsorption isotherms of hIgG to Eudragit-SpA are shown in Fig. 2A. Langmuir fitting resulted in a static binding capacity qmax of 85.9 ± 1.8 mghIgG gconjugate −1 at 22 ± 1 ◦ C, which is equivalent to 3.8 ± 0.08 mghIgG mL−1 Eudragit-SpA conjugate solution. The covalently linked protein A showed a high binding affinity toward hIgG with a dissociation constant KD of 0.25 mghIgG mL−1 (equ. 1.7 ␮M). In addition, incubation of antibody in conjugate solution was performed at 7 ± 1 ◦ C in order to stabilize hIgG and the conjugate solution and to prevent antibody aggregation. At this temperature an increased static binding capacity of 93.9 ± 2.78 mghIgG gconjugate −1 (4.2 ± 0.12 mghIgG mLconjugate −1 ) was achieved and the dissociation constant was reduced to 0.12 mghIgG mL−1 (equ. 0.8 ␮M). The measured differences in binding capacity and dissociation constant at a reduced temperature are well in accordance with observations made by Tu et al. [24], who reported stronger binding of immunoglobulin toward protein A at low temperatures. As a possible explanation of this observation, a stabilization of the Eudragit-SpA conjugate structure at reduced temperature was assumed, enabling a better accessibility of the protein A ligands for hIgG molecules and therefore higher amounts of bound antibody. Comparison of binding capacities had to be done based on the carrier material dry weight. For commercially available chromatographic protein A affinity resins, Bak et al. [25] reported binding capacities of 57.5 mgIgG (MabSelect) to 72.5 mgIgG (rProtein A Sepharose) per g carrier. With an 18% increased amount of bound hIgG, the Eudragit-SpA used in this study, showed a significantly higher binding capacity, even with a reduced ligand density of 0.4 mg mL−1 conjugate solution compared to 5–6 mg mLresin −1 for both chromatographic materials. At a reduced temperature, the amount of bound hIgG could be increased even up to 30%. The determined dissociation constant at 22 ± 1 ◦ C is in the same order of magnitude compared to those reported for chromatographic resins (3.0 ␮M) [25,26], indicating that covalent binding of protein A did not significantly affect the ligand affinity. For 7 ± 1 ◦ C a better KD -value was achieved, which is in the same order of magnitude like reported by Hilbrig et al. [16] for the polyNIPAAm-protein A macroligand (0.3 ␮M). Also, the heterogeneous and amorphous structure of the ligand-carrying polymer seemed to not be a drawback in regards to ligand accessibility and steric hindrance when compared to porous and spherical shaped chromatographic particles. Furthermore, the Eudragit-SpA conjugate facilitated rapid adsorption of hIgG at 22 ± 1 ◦ C (Fig. 2B). Equilibrium concentration of 86% of the initial 0.78 mg mL−1 hIgG in batch experiments was reached within 5 min. Incubation over a longer time period did not improve the amount of bound hIgG. This allows short incubation times during the precipitation procedure, which is crucial for an efficient and economic process for antibody purification.

L. Janoschek et al. / J. Chromatogr. B 965 (2014) 72–78

75

Fig. 2. Binding capacities and kinetics of Eudragit-SpA conjugate. (A) Adsorption equilibrium data of Eudragit-SpA conjugate at 22 ± 1 ◦ C and 7 ± 1 ◦ C. A 0.8% Eudragit conjugate with a SpA load of 35.5 mgSpA gconjugate −1 was used. Data was fitted with Langmuir isotherms with R2 = 0.998 for high temperature and R2 = 0.994 for low temperature, respectively. (B) Adsorption kinetics for hIgG binding to Eudragit-SpA conjugate at 22 ± 1 ◦ C with varied incubation time. Initial hIgG concentrations were 0.78 mg mL−1 .

3.2. Optimization of hIgG binding and elution conditions Next to the conditions during binding and elution, the amount of added conjugate was expected to have the highest impact on the purification efficiency of hIgG with Eudragit-SpA. The ratio of possibly accessible binding sites provided by the protein A ligands and the concentration of hIgG in solution can be expressed as the capacity ratio (CR, Eq. (2)). The implication of the static binding capacity qmax allows the evaluation of the required amount of conjugate, independently from parameters like the ratio of actual bound antibody molecules per ligand molecule which is given by the hIgG binding domains of SpA. A capacity ratio of 1.0 implies that there are as many hIgG molecules in solution as accessible binding sites on the conjugate, at the same conditions the static binding capacity was determined. Different capacity ratios were investigated in order to find out at which ratio an efficient antibody capture and thus a high purification performance can be achieved. Elution of bound hIgG was carried out with a 0.5 M arginine buffer at pH 3.8. It was reported that arginine buffer achieved a similarly efficient elution compared to the commonly used glycine buffer at pH 2.5 [14,27]. In addition, it was pointed out earlier that arginine might suppress protein aggregation [28]. Capacity ratios were varied in a range between 0.1 and 6.4. hIgG was precipitated and protein concentrations were determined for all collected fractions (Fig. 3). In the control experiment, where no SpA was conjugated to the polymer, all hIgG was recovered in the supernatant and wash fraction, as expected. No hIgG was detected in the eluate fraction. With a 10-fold excess of hIgG (CR 0.1), 75% of the applied hIgG was found in the supernatant and hardly any antibody was eluted. By increasing the capacity ratio to 1.0, which means as much binding sites as hIgG molecules in the solution, unbound hIgG decreased to 45% of the initial concentration and up to 38% were recovered in the eluate fraction. At a 6.4-fold excess of binding sites provided by the Eudragit-SpA conjugate, 34% of the hIgG remained unbound in the supernatant and 54% was found in the eluate. For all capacity ratios, no antibody was detected in the washing fraction. The total antibody recovery in all fractions increased with rising capacity ratios from 75 to 88%. An equal amount of hIgG and available binding sites was not sufficient enough for an efficient capture of the present antibody. The need for a multiple excess of binding sites and precipitation agent, respectively, to facilitate high antibody capture was also reported earlier for SpA domains attached to elastin-like polypeptides [29] and polyNIPAAm [15,16]. The authors argue with alterations of the binding regions of protein A due to a possible interference with the

carrier polymer, which is in our opinion the most probable explanation for the herein used Eudragit-SpA conjugate. Even a 6.4-fold excess of hIgG binding sites was insufficient to bind most of the initial hIgG (without hIgG3 ). The difference of approximately 11% of unbound hIgG in the supernatant between capacity ratios of 1.0 and 6.4 is not proportional to the increased amount of possible binding sites. It was assumed, that the amount of residual hIgG in the supernatant is partly caused by the precipitation process itself. Although KD -values and therefore binding affinity is in the same range like for chromatographic resins, the binding might not be sufficiently strong for the soluble-insoluble transition during precipitation and therefore leading to a dissociation of bound antibody. Despite the temperature as shown above, interaction and binding affinity of hIgG and protein A is highly dependent on the pH of the solution. Lowering the pH to 5.0 in order to precipitate the antibody-conjugate complex might cause a partial premature elution of the bound hIgG, which was then detectable in the supernatant and discarded. Regarding the obtained total antibody recovery, the achieved maximum of 88% indicated that the bound hIgG was not completely eluted from the Eudragit-SpA conjugate. Hence, further investigations were focused on varying the elution conditions during the

Fig. 3. Precipitation of hIgG with different capacity ratios for hIgG concentration and Eudragit-SpA conjugate. Shown is the hIgG recovery in the supernatant fraction, after washing with acetate buffer and after elution with arginine buffer at pH 3.8. EDC-activated Eudragit without protein A for conjugation, was used as control.

76

L. Janoschek et al. / J. Chromatogr. B 965 (2014) 72–78

Fig. 5. Coomassie blue stained 12% SDS PAGE of hIgG purification from a FBS/hIgG protein mixture. Lane M: NEB Broad Range Protein Marker (2–212 kDa); Lane 1: FBS/hIgG mixture; Lane 2: unbound protein in supernatant after precipitation; Lane 3: recovered protein after washing with 0.02 M acetate buffer pH 5.2; Lane 4: eluate fraction after elution with 0.5 M arginine buffer pH 3.8 with hIgG light (25 kDa) and heavy chain (50 kDa) bands (arrows).

Fig. 4. hIgG recovery in supernatant and eluate under varied binding and elution conditions. Reference was handled as described in Section 2.5 in the absence of FBS. Other experiments were performed by altering binding, or elution parameters for precipitation.

Table 1 Purification performance of hIgG affinity precipitation from a protein mixture. Initial concentrations were 0.1 mg mL−1 hIgG and 1.2 mg mL−1 FBS (4% v/v). Total protein concentration was measured by BCA-assay. hIgG concentrations were determined with SEC–HPLC and protein A affinity chromatography (PF: purification factor). Supernatant

precipitation procedure. As already pointed out, it was suggested that partitions of the bound hIgG elute during the precipitation step. A pH dependent elution of hIgG from protein A ligands was reported earlier by Duhamel et al. [30], where different hIgG subtypes elute at different pH in a chromatographic run with a pH gradient. In own chromatographic experiments with herein used hIgG-SpA system a similar observation was made as parts of the loaded hIgG already eluted at approx. pH 5.0 (not shown). Therefore a fraction of hIgG might be bound at neutral pH conditions but subsequently elute while lowering the pH for precipitation. To minimize this premature elution of hIgG from the Eudragit-SpA conjugate, the pH for precipitation of the antibody-conjugate complex was shifted to 5.2. Higher pH values would have caused a conjugate loss during the purification procedure due to an incomplete precipitation (data not shown). As an alternative elution buffer, 0.2 M glycine HCl with 0.2 M NaCl and a lower pH of 2.5 as commonly used in protein A affinity chromatography was applied. In addition, the pH for incubation of antibody and conjugate was slightly increased from 7.4 to 8.0, based on a report that optimal binding of hIgG to protein A can thus be achieved [31]. All experiments were carried out with a capacity ratio of 6.4. As can be seen in Fig. 4, the glycine elution buffer at pH 2.5 revealed a 13% reduced elution performance compared to the primarily used arginine buffer at pH 3.8. This observation fits well with other reports, where arginine was successfully applied for the elution of antibodies from protein A columns [27,32]. A repeated elution step with arginine pH 3.8 and glycine pH 2.5, respectively, did not elute any further hIgG and a shift to pH 8.0 of the incubation solution did not significantly reduce the amount hIgG found in the supernatant. This observation is well in agreement with previously made assumptions, that the precipitation step itself prevents a more efficient hIgG capture. This could be confirmed when precipitation was carried out at a pH of 5.2, which led to a 9% decrease in the supernatant concentration of hIgG and therefore a 8% increased antibody recovery in the eluate fraction. Considering this observation, it can be assumed that complete binding of hIgG was achieved with the use of Eudragit-SpA conjugate, but bound hIgG elute as a consequence of the subsequent precipitation step. This problem may be circumvented by shifting the pH range in which precipitation of Eudragit-SpA occurs

mg mL Total proteina 1.19 hIgG 0.024 a b

−1

Wash

%

mg mL

91.5 24.0

0.03 0.00

Eluate −1

%

mg mL

2.3 0.0

0.065 0.065

−1

% 5.0 65.0

Recovery PF (%) (–) 98.8 89.0

– 12.4b

Initial total protein: 1.30 mg mL−1 . Initial purity of hIgG: 7.7%.

to higher pH values. Dainiak et al. [9] suggested an approach where anionic and cationic polymers form a nonstoichiometric polyelectrolyte complex. In this context, it was reported that the addition of the positively charged polyelectrolyte polyethylenimine (PEI) to polymethacrylate (PMA), which is also part of Eudragit® -S-100, can shift the pH range for precipitation toward higher pH values [33]. 3.3. Purification of hIgG from a protein mixture In order to investigate if the Eudragit-SpA conjugate is suitable for hIgG purification in the presence of impurities precipitation was performed with 1.2 mg mL−1 FBS (4% v/v). The FBS was used to create a protein mixture and to examine the influence of foreign protein on binding and elution performance of hIgG toward the Eudragit-SpA conjugate. Results are shown in Fig. 4. Only a small decrease in detectable hIgG in the supernatant and a 4% increase of hIgG in the eluate fraction were achieved. hIgG purification procedure was carried out with a capacity ratio of 6.4 at 7 ± 1 ◦ C and precipitation was performed at pH 5.2. Fractions were at first analyzed by SDS-PAGE (Fig. 5). Results indicated that the Eudragit-SpA conjugate was highly selective for hIgG, leaving most of the FBS unbound in the supernatant. Furthermore, no contaminating protein seemed to be co-eluted with hIgG. Purification performance was evaluated in detail, in terms of protein recovery, purity of hIgG and the purification factor (Table 1). It can be seen that a majority of the total initial foreign protein (97%) remained in the supernatant and only 2.3% hIgG, which corresponds to 24% of initial hIgG, remained unbound. Residual 3% FBS was removed during the washing step with 0.02 M acetate buffer pH 5.2 whereas no antibody could be detected in this fraction. Overall recovery of total protein and hIgG was 98.8% and 89.0%, respectively. SEC–HPLC analysis of the eluted antibody revealed a purity of more than 95.4% and

L. Janoschek et al. / J. Chromatogr. B 965 (2014) 72–78

77

7 repetitive cycles. The decreased affinity of protein A to the antibody is supposed to be based on a proceeding denaturation of the ligand. Repeated precipitation, centrifugation and continuous mixing of the conjugate solution might have caused conformational rearrangements of the polymer and the bound protein A, which led to a loss of affinity to hIgG. Milder process conditions, such as filtration instead of centrifugation, might avoid this problem. Also additional washing and regeneration steps after a complete cycle might contribute to a more stable hIgG purification performance over repeated cycles. 4. Conclusions

Fig. 6. Reusability of Eudragit-SpA conjugate for hIgG purification. SpA leakage from Eudragit and hIgG eluted from the conjugate were determined after 1, 3, 5 and 7 precipitation cycles. SpA was measured with BCA-assay and hIgG by SEC–HPLC, respectively. Results are average values gained in triplicate experiments and adjusted by conjugate mass loss. Conjugate mass loss during precipitation was determined after 7 cycles and linearized for each cycle. Error bars were obtained from triplicate measurement and conjugate loss determination.

therefore a purification factor of 12.4 could be achieved within the process. In previously published works, where affinity precipitation with polyelectrolytes was used for antibody purification, factors of 1.5 and 6.2 were reported [9,12]. In comparison, the herein presented Eudragit-SpA conjugate significantly improved the purification efficiency by the use of protein A as an affinity ligand and therefore enables the application of affinity precipitation for a wide range of IgGs and antibodies. 3.4. Reusability of Eudragit-SpA conjugate As cost effectiveness plays a major role in downstream process development, recycling and reusability of raw material for product purification is an important aspect. The reusability of EudragitSpA for repeated precipitation of hIgG was rated by means of two parameters. First, protein A leakage from Eudragit was measured to investigate the conjugate’s stability and second, the hIgG concentration in the eluate fraction was determined in order to evaluate purification efficiency of the recycled conjugate. 7 sequenced precipitation cycles were performed and the mentioned parameters were determined after 1, 3, 5 and 7 cycles (Fig. 6). SpA leakage and hIgG purification efficiency were adjusted by the conjugate mass loss, which occurred due to the precipitation procedure. A significant decrease in protein A linked to Eudragit can be observed only after the first precipitation cycle. It is assumed that still non-covalent bound protein A was attached to the polymer and therefore removed within the first precipitation cycle. An additional washing step after the conjugate preparation might prevent this one-time leakage. During further cycles, the amount of SpA remained stable within variance at around 80% of the primarily bound SpA. A nearly stable amount of bound SpA after 3 and more cycles is also in accordance to earlier reported 1.0 to 1.5% leakage of Eudragit-coupled protein [17]. Nevertheless, protein A leakage is an important issue in the development of affinity material and further investigations have to be made to ensure that no ligand bleeding occurs during the process. Efficiency of hIgG purification and elution decreased steadily over repeated precipitation. While after 3 cycles 75% of hIgG elution efficiency compared to freshly prepared Eudragit-SpA was still achieved, additional purification passages reduced the elution performance down to approximately 21% after

In this study we investigated the potential of a protein A affinity macroligand for direct antibody recovery by affinity precipitation. Recombinant protein A was covalently linked to the pH-responsive polyelectrolyte Eudragit® S-100 in order to create a conjugate which is highly selective for antibodies and also easy controllable by an environmental trigger. The conjugated Eudragit-SpA showed a high affinity toward human immunoglobulin G and significantly increased binding capacities in comparison with two commercially available protein A chromatographic resins. Reduced temperature facilitates even higher loading of Eudragit-SpA with an increased hIgG binding affinity and also contributes to antibody and conjugate stability. Using an excess of conjugate as well as adjusted elution conditions, it was demonstrated that hIgG can be successfully captured from a cell culture supernatant mimicking protein mixture. Purification of immunoglobulines was achieved with a high recovery and >95% purity in a fast and simple procedure. It was shown that Eudragit-SpA can be recycled without significant leakage of affinity ligand and that it is reusable for a number of precipitation cycles, enabling more reasonable process economics. With these results, and also considering that precipitation is regarded as an easy scalable and robust purification technique, we see the application of Eudragit-protein A macroligands for affinity precipitation as a promising alternative in antibody purification processes. Acknowledgements The authors gratefully acknowledge the support of the TUM Graduate School, Technische Universität München, Germany. Eudragit® S-100 was a gracious gift from Evonik Industries AG, Essen, Germany. The ENrich SEC 650 column was kindly provided by Bio-Rad Laboratories, Hercules, USA. References [1] A.A. Shukla, B. Hubbard, T. Tressel, S. Guhan, D. Low, J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 848 (2007) 28. [2] B. Kelley, Mabs 1 (2009) 443. [3] R.L. Fahrner, H.L. Knudsen, C.D. Basey, W. Galan, D. Feuerhelm, M. Vanderlaan, G.S. Blank, Biotechnol. Genet. Eng. Rev. 18 (2001) 301. [4] F.M. Wurm, Nat. Biotechnol. 22 (2004) 1393. [5] D. Low, R. O’Leary, N.S. Pujar, J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 848 (2007) 48. [6] A.C.A. Roque, C.S.O. Silva, M.A. Taipa, J. Chromatogr. A 1160 (2007) 44. [7] P. Gagnon, J. Chromatogr. A 1221 (2012) 57. [8] F. Hilbrig, R. Freitag, J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 790 (2003) 79. [9] M.B. Dainiak, V.A. Izumrudov, V.I. Muronetz, I.Y. Galaev, B. Mattiasson, Bioseparation 7 (1998) 231. [10] J.Y. Kim, A. Mulchandani, W. Chen, Biotechnol. Bioeng. 90 (2005) 373. [11] M.A. Taipa, R. Kaul, B. Mattiasson, J.M.S. Cabral, J. Mol. Recognit. 11 (1998) 240. [12] M.A. Taipa, R. Kaul, B. Mattiasson, J.M.S. Cabral, Bioseparation 9 (2000) 291. [13] A. Kumar, P.O. Wahlund, C. Kepka, I.Y. Galaev, B. Mattiasson, Biotechnol. Bioeng. 84 (2003) 494. [14] B. Madan, G. Chaudhary, S.M. Cramer, W. Chen, J. Biotechnol. 163 (2013) 10. [15] J.P. Chen, A.S. Hoffman, Biomaterials 11 (1990) 631.

78

L. Janoschek et al. / J. Chromatogr. B 965 (2014) 72–78

[16] F. Hilbrig, G. Stocker, J.M. Schlappi, H. Kocher, R. Freitag, Food Bioprod. Process. 84 (2006) 28. [17] A. Kumar, M.N. Gupta, J. Mol. Catal. B: Enzym. 5 (1998) 289. [18] M. Freiherr von Roman, A. Koller, D. von Rüden, S. Berensmeier, Protein Expr. Purif. 93 (2014) 87–92, http://dx.doi.org/10.1016/j.pep.2013.10.013, ISSN 1046-5928. [19] J.J. Sambrook, D.D.W. Russell, Molecular Cloning: A Laboratory Manual, Vol. 2, Cold Spring Harbor, New York, 2001. [20] D. Guoqiang, A. Lali, R. Kaul, B. Mattiasson, J. Biotechnol. 37 (1994) 23. [21] V. Arasaratnam, I.Y. Galaev, B. Mattiasson, Enzyme Microb. Technol. 27 (2000) 254. [22] C.J.S.M. Silva, G. Gübitz, A. Cavaco-Paulo, J. Chem. Technol. Biotechnol. 81 (2006) 8. [23] U.K. Laemmli, Nature 227 (1970) 680. [24] Y.Y. Tu, F.J. Primus, D.M. Goldenberg, J. Immunol. Methods 109 (1988) 43.

[25] H. Bak, O.R.T. Thomas, J. Abildskov, J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 848 (2007) 131. [26] R. Hahn, R. Schlegel, A. Jungbauer, J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 790 (2003) 35. [27] T. Arakawa, J.S. Philo, K. Tsumoto, R. Yumioka, D. Ejima, Protein Expression Purif. 36 (2004) 244. [28] T. Arakawa, K. Tsumoto, Biochem. Biophys. Res. Commun. 304 (2003) 148. [29] R.D. Sheth, B. Madan, W. Chen, S.M. Cramer, Biotechnol. Bioeng. 110 (2013) 2664. [30] R.C. Duhamel, P.H. Schur, K. Brendel, E. Meezan, J. Immunol. Methods 31 (1979) 211. [31] B. Akerstrom, L. Bjorck, J. Biol. Chem. 261 (1986) 240. [32] D. Ejima, R. Yumioka, K. Tsumoto, T. Arakawa, Anal. Biochem. 345 (2005) 250. [33] S.K. Chatterjee, D. Yadav, S. Ghosh, A.M. Khan, J. Polym. Sci., A: Polym. Chem. 27 (1989) 3855.