Process Biochemistry 46 (2011) 101–107

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Process Biochemistry

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Purification of rabbit polyclonal immunoglobulin G using anion exchangers

Rattana Wongchuphana,e, Beng Ti Teyb,d, Wen Siang Tanc,d, Senthil Kumar Subramanianc,Farah Saleena Taipa, Tau Chuan Linga,∗

a Department of Process and Food Engineering, Faculty of Engineering, Universiti Putra Malaysia, 43400, Serdang, Selangor Darul Ehsan, Malaysiab Department of Chemical and Environmental Engineering, Faculty of Engineering, Universiti Putra Malaysia, 43400, Serdang, Selangor Darul Ehsan, Malaysiac Department of Microbiology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400, Serdang, Selangor Darul Ehsan, Malaysiad Institute of Bioscience, Universiti Putra Malaysia, 43400, Serdang, Selangor Darul Ehsan, Malaysiae Department of Chemistry, Faculty of Science and Technology, Suratthani Rajabhat University, 84100, Surat Thani, Thailand

a r t i c l e i n f o

Article history:Received 25 February 2010Received in revised form 21 July 2010Accepted 22 July 2010

Keywords:Polyclonal IgGSTREAMLINETM DEAERabbit serumAlbumin removalAnion exchange adsorbentsNegative chromatography antibodypurification

a b s t r a c t

Negative chromatography antibody purification (N-CAP) using the weak anion exchanger STREAMLINETM

DEAE to extract impurities while retaining the target antibody is proposed as an effective method for therecovery of antibody from rabbit serum. The effects of pH and initial protein concentration on the removalof albumin were investigated. The optimal pH and initial protein concentration for the efficient removalof albumin from rabbit serum were pH 8.0 and 0.5 mg/ml, respectively. Under optimal binding conditions,DEAE successfully removed more than 90% of the albumin from rabbit serum with less than 20% IgG loss.This process offered good polyclonal IgG yield of 80% with a purity of 83% and a purification factor of 5.5.The use of a strong anion exchanger like STREAMLINETM Q XL for albumin removal was also explored.Under similar optimized conditions, albumin removal by Q XL was as high as 90%. However, IgG recoveryand purity were reduced to about 70% and 62%, respectively. Thus, N-CAP using the anion exchangerDEAE removes albumin from rabbit serum and thereby offers an efficient means of purifying polyclonalantibodies.

© 2010 Elsevier Ltd. All rights reserved.

1. Introduction

Protein A affinity chromatography, which is used widely in anti-body purification, offers specificity and highly efficient productcapture. Protein A has been the standard antibody manufactur-ing method during the past few decades due to its selectiveness,optimal throughput, and its stability to be cleaned and re-used[1,2]. In addition, it has been used for product capture in multistepplatforms [3]. However, since advances in cell culture technol-ogy have been developed, this approach now has a number ofdisadvantages. As the antibody titer in cell culture increases, thedownstream processes become a bottleneck and the product recov-ery cost increases in line with the production scale. In such a case, itmay not be cost-effective to purify antibody using Protein A affinitychromatography. Moreover, the low stability of Protein A is a limi-tation in production scale antibody purification [2]. Therefore, dueto its high cost and concerns over low ligand stability after manycycles of production, there is a genuine need for a replacementfor Protein A affinity chromatography [1]. Hence, the development

∗ Corresponding author. Tel.: +60 603 89466447; fax: +60 603 89464440.E-mail address: ltc555@eng.upm.edu.my (T.C. Ling).

of a cost-effective alternative method for antibody purification isneeded to overcome these concerns.

As recently recommended by some adsorbent manufacturers,negative chromatography antibody purification (N-CAP) can beused for antibody purification from animal serum. Typically, thisrequires passing the clarified antibody feedstock through a seriesof negative chromatography columns to remove all the impuri-ties. This approach could be an efficient way of overcoming thehigh cost and the physical capacity limits of Protein A affinity chro-matography. Ion exchange chromatography (IEC) is a simple, rapidtechnique used in protein separation that depends on the differen-tial ability of various proteins to be adsorbed onto the adsorbent[4].

IEC is used commonly in protein purification due to its high bind-ing capacity and cost-effectiveness [5–8]. Commercially availableionic matrices for protein purification (including DEAE sepharoseand Q XL) are highly activated with a high density of ionic lig-ands such as diethylaminoethyl and quaternary amine groups [9].These matrices have high adsorption capacity towards proteinseven in the presence of Escherichia coli (E. coli) biomass and/orcell debris [4,10,11]. Recently, Pessela and colleagues successfullypurified immunoglobulin G (IgG) from whey proteins concentrate(WPC) by eliminating BSA with DEAE-agarose adsorbent [9]. BSAwas adsorbed strongly on highly activated DEAE-agarose, while

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102 R. Wongchuphan et al. / Process Biochemistry 46 (2011) 101–107

IgG was adsorbed on and desorbed from anion exchange adsor-bents weakly activated with mild monoaminoethyl-N-aminoethyl(MAMAE) ionic groups. Such weakly activated adsorbents havebeen developed and used for the selective adsorption of only largeproteins such as !-galactosidase (Mr, 70 kDa for each monomer;with a predominant tetrameric form) [4] and IgG (Mr = 150 kDa)[9]. BSA was also found to be adsorbed onto weakly activatedadsorbents. This phenomenon is related to the tendency of BSAto form aggregates either with itself or other proteins [4,12–14].The mechanism of adsorption of these matrices is based on mul-tipoint adsorption [9,15]. Aggregates of albumin complexes andalbumin–IgG complexes exhibit pseudo-affinity for different lig-ands on either weakly or strongly activated adsorbents [4]. Thepresence of albumin in animal serum can hinder the purificationof an antibody. Thus, albumin must be eliminated before purifyingantibodies with anion exchange adsorbents. Removal of albuminfrom any antibody-containing source can be achieved via positivelycharged adsorbents.

In the present study, anion exchange matrices were used topurify polyclonal antibodies from rabbit serum by removing albu-min in a batch binding system. Polyclonal antibodies againsthepatitis B core antigen (anti-HBcAg IgG) were generated in rab-bits. The albumin removal capacity of two anion exchange matrices(STREAMLINETM DEAE and STREAMLINETM Q XL) was evaluated.The albumin-adsorbing capacity of DEAE sepharose was assessedas functions of the pH of the medium and the initial protein con-centration. The recovery of polyclonal IgG as well as its puritywas determined after the removal of albumin. The strong anionexchanger Q XL, which is known to offer a higher binding capacityfor albumin, was also studied. The efficiency of both the weak andstrong anion exchangers were compared and discussed in terms ofyield and purity of the polyclonal antibodies recovered.

2. Materials and methods

2.1. Materials

The STREAMLINETM DEAE and STREAMLINETM Q XL adsorbents were purchasedfrom GE Healthcare (Uppsala, Sweden). DEAE, which is a modified sepharose matrix,is a weak anion exchanger that provides about 0.13–0.21 mmol/ml of total ionicadsorbent capacity. It has high binding capacity towards BSA, at least 40 mg/mladsorbent. On the other hand, the matrix structure of Q XL is based on highly cross-linked 6% agarose beads modified by including a crystalline quartz core and bounddextran. The strong Q ion exchange groups are then coupled to these dextran chainsthrough chemically stable ether bonds. This causes an increase in the effective inter-acting volume as well as in the steric availability of the ligands for the substance tobe adsorbed. The Q XL adsorbent offers a total ionic capacity of 0.23–0.33 mmol/mladsorbent. The binding capacity of Q XL for BSA is up to 110 mg/ml adsorbent. Bovineserum albumin (BSA) was purchased from Sigma (St. Louis, MO, USA). The isoelectricpoint (pI) of BSA given in the product information is 4.7. Serum samples collectedfrom immunized rabbits were used as the source of polyclonal anti-HBcAg IgG.

2.2. Production of HBcAg in E. coli

E. coli W31101Q cells carrying the plasmid pTacpcore encoding full-lengthHBcAg [16] were cultured in Luria Bertani (LB) broth containing ampicillin(100 "g/ml). The bacteria were grown at 37 ◦C until the OD600nm reached about0.6, followed by adding isopropyl-!-d-thiogalactopyranoside (IPTG; final concen-tration of 0.5 mM) to induce HBcAg expression. Then, the cells were allowed to growfor another 16–20 h. The cells were harvested by centrifugation at 3752 × g (AvantiJLA-16.250 rotor, Beckman, USA) at 4 ◦C for 15 min.

2.3. Purification of HBcAg by sucrose density gradient ultracentrifugation

Sucrose gradient density ultracentrifugation was employed for the purificationof HBcAg [17,18]. The cell pellets were resuspended in Tris–HCl buffer (50 mM, pH8.0 containing 0.1% Triton X-100) and then lysed with lysozyme (0.2 mg/ml) in thepresence of MgCl2 (4 mM) and DNase (20 "g/ml) at room temperature for 2 h. Thecell suspension was clarified by centrifugation at 12,096 × g (Avanti JA-25.50 rotor,Beckman, USA) for 20 min at 4 ◦C, the HBcAg was precipitated with ammoniumsulphate (35% saturation) and it was centrifuged at 12,096 × g. The pellet was sus-pended in dialysis buffer (50 mM Tris–HCl, pH 8.0 containing 150 mM NaCl) anddialyzed in the same buffer (2 L) for 16 h at 4 ◦C. The dialyzed sample was loaded on

a 8–40% continuous sucrose gradient and centrifuged at 210,053 × g (SW 41 rotor,Beckman, USA) for 5 h at 4 ◦C. The gradient was fractionated (0.5 ml per fraction)and fractions containing HBcAg were concentrated with a Vivaspin concentrator(300,000 MWCO, Vivascience, UK) by centrifugation at 3024 × g for several hours at4 ◦C.

2.4. Preparation of sera

New Zealand white rabbits were injected subcutaneously with HBcAg (250 "g;0.5 ml) containing 0.5 ml of complete Freund’s adjuvant (Millipore, USA). Four weeksafter first injection, the rabbits were injected with a mixture of HBcAg and incom-plete Freund’s adjuvant (Millipore, USA) in subsequent boosters. Two boosters wereadministered every 2 weeks and the rabbits were bled 10 days after each booster.Rabbit serum was collected by centrifugation at 2700 × g for 10 min. Sera containingpolyclonal anti-HBcAg IgG from rabbits were pooled as the antibody feedstock forN-CAP.

2.5. Protein assays

2.5.1. The Bradford assayThe total protein content of serum protein solutions before and after albumin

removal was determined by the Bradford method [19]. BSA was used as a proteinstandard.

2.5.2. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)Serum protein solutions before and after albumin removal were separated on

10% polyacrylamide gel under non-reducing condition [20]. The gel was stained withCoomassie Brilliant Blue (CBB) R-250 and destained with destaining solution (30%methanol and 10% glacial acetic acid). The intensity of the protein band on SDS-PAGEwas determined using a Gel-Doc system [21,22] with some minor modifications.

2.5.3. Enzyme-linked immunosorbent assay (ELISA)The amount of polyclonal anti-HBcAg IgG present in serum before and after the

albumin depletion process was quantified with ELISA [23]. A microtiter plate wasfirst coated with HBcAg (100 ng/well, 100 "l) overnight at 4 ◦C. The coated platewas first washed with TBST (50 mM Tris–HCl and 150 mM NaCl, pH 7.5 containing0.05% Tween 20). The plate was then blocked with 10% milk diluent/blocking solu-tion (KPL, USA) for 2 h. After washing, 100-"l samples containing anti-HBcAg IgGdiluted in TBS buffer were added to the plate and then incubated at room tempera-ture for 1 h. The plate was washed once more and anti-rabbit antibody conjugatedto alkaline phosphatase (KPL, USA; 1:5000) was loaded into the wells. The plate wasfurther incubated at room temperature for 1 h. After giving six washes with TBST, p-nitrophenyl phosphate (pNPP 1 mg/ml, Sigma) in 1 M diethanolamine buffer (DEA,pH 9.8 containing 0.5 mM MgCl2·6H2O) was added to the plate. The absorbance at405 nm was measured using a universal microplate reader (Bio-Rad, USA). All mea-surements were performed in quadruplicate. Mouse anti-HBcAg IgG monoclonalantibody (Millipore, USA) was used as a standard.

2.6. BSA adsorption on DEAE sepharose

At pHs beyond its pI (4.7), BSA is negatively charged and is able to bind DEAEsepharose carrying a positive charge. On the other hand, at pHs lower than its pI, verylittle or no binding of BSA is observed. Thus, the binding capacity of DEAE sepharosefor BSA was tested in binding buffers at different pH ranging from 4.0 to 8.0 (25 mMsodium acetate buffers pH 4.0–5.0; 25 mM sodium phosphate buffers pH 6.0–8.0).The binding capacity of DEAE sepharose for BSA was estimated and compared tothat recommended by the manufacturer. BSA (10 mg) was allowed to bind to DEAEsepharose adsorbent (0.25 ml) on a roller mixer until no more protein was adsorbed.Absorbance readings at 280 nm were taken before and after binding. The amount ofBSA was determined using a U-2900 spectrophotometer (Hitachi, Japan). At 280 nm,the extinction coefficient of BSA is 6.67 for a 1% solution (E1%

279 = 6.67). Therefore, theextinction coefficient used to calculate the concentration of BSA solutions (1 mg/ml)was fixed at 0.667 ml/(mg cm). The measurements were carried out in duplicate.

2.7. Albumin removal at various pHs

The effect of pH on albumin removal by DEAE sepharose was investigated at pHsranging from 5.0 to 8.0. Briefly, DEAE sepharose adsorbent (0.25 ml) was incubatedwith a serum sample (0.75 mg/ml initial concentration, 10 ml) at various pHs. Abatch binding system was used with a universal bottle that was mixed well on aroller mixer. A fraction was collected every 15 min for 1 h. Samples were analyzedfor total protein content with the Bradford assay. The intensity of the albumin andIgG bands on SDS-PAGE was determined using a Gel-Doc system (Quantity One,Bio-Rad, USA). The protein profile was used to assess the optimal pH for efficientalbumin extraction with minimal antibody binding.

2.8. Albumin removal at various initial protein concentrations

A serum sample (10 ml) was prepared in 25 mM sodium phosphate buffer, pH8.0. Solutions with two initial protein concentrations (0.5 and 0.75 mg/ml) were

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mixed separately with 0.25 ml DEAE sepharose or 0.1 ml Q XL. The binding continuedfor 1 h on a roller mixer. A fraction was collected every 15 min and then analyzedquantitatively by three protein assays. Total protein concentration was estimatedby the Bradford assay. The intensity of the albumin band was estimated using a Gel-Doc system. ELISA was used to determine the quantity of polyclonal anti-HBcAg IgGin the serum protein solution before and after albumin removal.

2.9. Calculations

Yield is stated as the percentage of polyclonal IgG in the albumin-depleted serumdivided by the initial amount of polyclonal IgG in the serum sample:

Yield = Amount of IgG in albumin-depleted serumAmount of IgG in serum sample

× 100% (1)

The purity (or relative quantity) of polyclonal anti-HBcAg IgG is expressed asthe ratio of the amount of polyclonal anti-HBcAg IgG to the total amount of proteinin the serum sample either before or after albumin removal:

Purity = Amount of polyclonal anti-HBcAg IgGAmount of total protein

(2)

The purification factor (PF) is calculated from the purity of polyclonal IgG in thealbumin-depleted serum divided by the purity of polyclonal anti-HBcAg IgG in theserum sample:

Purification factor = Purity of polyclonal IgG in albumin-depleted serumPurity of polyclonal IgG in serum sample

(3)

3. Results and discussion

3.1. Adsorption of BSA on DEAE sepharose

DEAE sepharose 6BCL is a commercially available anionexchanger that has high binding capacity for albumin (up to40 mg/ml adsorbent). In this study, DEAE sepharose was incubatedwith BSA solution under varying pHs ranging from acidic to basic.Fig. 1 shows the percentage of BSA adsorbed onto DEAE sepharoseat various pHs. There was no adsorption of BSA onto the adsorbentat pH 4.0. This can be explained by the fact that at pH below itspI (around 4.7), BSA assumes a net positive charge, and there is anelectropositive repulsion between positively charged BSA and thecationic surface of DEAE sepharose, an observation similar to onepresented earlier in the literature [5]. On the other hand, greaterthan 80% BSA adsorption was observed at pHs higher than its pI.At pH 8.0, the highest amount of BSA was bound to the DEAEsepharose, 10.1 ± 0.1 mg (Fig. 1). At pHs beyond its pI (4.7), BSA isnegatively charged and is able to bind DEAE Separose, activatedwith ionized cationic groups. It also has been reported that thetertiary structure of BSA bound to DEAE sepharose is relativelyunaltered in the pH range between 5.0 and 8.0 at room temperature[5].

The high adsorption of BSA onto DEAE sepharose is due to thefact that this highly activated adsorbent has high-density ionic lig-ands so that it can adsorb large quantities of protein in solutions

Fig. 1. Amount of BSA adsorbed onto DEAE sepharose (0.25 ml) as a function of pHof the medium. The pH was varied as follows: 25 mM sodium acetate buffer at pH4.0 and 5.0; 25 mM sodium phosphate buffer at pH 6.0–8.0. The results are from twoindependent measurements.

and even in crude extracts of E. coli [24,25]. Serum albumins aresmall and water-soluble proteins that are able to interact withmany different compounds or even other serum albumins, exhibit-ing a tendency to form homo or hetero protein aggregates even atlow concentrations [15]. Hence, this multipoint protein–adsorbentinteraction forms the basis of the adsorption of these proteins tomost chromatographic adsorbents [4,9,11,26]. It has been notedthat protein aggregation in solutions is pH-dependent [27,28].The result obtained here might reinforce the perception that theBSA aggregation was induced strongly by attractive ionic forcesat the pHs beyond its pI (4.7) as it was adsorbed onto the DEAEsepharose.

3.2. Effect of pH on serum albumin depletion

Initially, 10 mg of total protein in a serum sample at pH 8.0 wassubjected to 0.25 ml DEAE sepharose. It was found that about 80%of the serum albumin was removed. The ELISA result, however,showed that there was no improvement in the relative quantity ofanti-HBcAg IgG (data not shown) in the albumin-depleted serum.This suggests that IgG molecules were removed along with thealbumin. Band intensity analysis showed that the albumin deple-tion was associated with 25% IgG loss. About 75% of total serumprotein was adsorbed onto the adsorbent. Moreover, when theamount of total serum protein was increased to 12.5 mg, reducedalbumin binding (from 80% to 50%) was observed (data not shown).This smaller amount of bound albumin (30% less) corresponds toa lower degree of albumin aggregation. Indeed, a previous studyshowed that aggregate size can be controlled by the albuminconcentration [15]. This result suggests that initial protein concen-tration is another important parameter that impacts the retentionof albumin by adsorbents. This parameter was further investigatedto improve albumin removal from the serum sample while retain-ing more antibodies.

Therefore, rabbit serum albumin binding to DEAE sepharose wasassessed as a function of both pH and initial protein concentration,and the latter was fixed at 0.75 mg/ml. Due to the fact that albuminbinding did not increase even when the binding time was increasedbeyond 1 h, binding was performed within 1 h in this study.

After adsorption of serum proteins onto DEAE sepharose for 1 hat the indicated pHs, unbound proteins were separated on 10% SDS-PAGE and the levels of albumin and IgG were estimated by bandintensity analysis (see Fig. 2). Most of the albumin was removedefficiently at pH 7.0 and pH 8.0. SDS-PAGE gels presented in Fig. 2aand b show a qualitative analysis of rabbit serum proteins beforeand after albumin removal under both pH conditions. With 1 h ofbinding time, less serum albumin was expected to be removed atpH 7.0 than at pH 8.0. However, although the majority of the albu-min was removed under both conditions, the kinetics of albuminbinding were different. By comparing lane 5 of Fig. 2a with lane3 of Fig. 2b, the band intensity of albumin at pH 7.0 after 1 h ofadsorption was similar to that at pH 8.0 after 30 min of adsorption.Thus, albumin adsorption onto DEAE sepharose occurs with fasterkinetics at pH 8.0.

This observation was further supported by quantitative analy-sis Fig. 2c shows the amount of albumin removed from the serumsample (as determined by band intensity analysis) with 1 h of bind-ing and at pH 7.0 and 8.0. At pH 8.0, rapid albumin depletion wasobserved in which about 80% of the albumin was depleted fromthe initial protein solution within the first 15 min. Approximately89 ± 1% of the albumin was removed observed at pH 8.0 after 1 h ofadsorption, whereas at pH 7.0, 85 ± 1% of the albumin was removedafter 1 h. Fig. 2d shows the percentage of IgG loss at each time pointunder different conditions. Band intensity analysis revealed thatIgG was adsorbed onto the DEAE sepharose at the two pHs withdifferent efficiencies. About 20% of the IgG was lost at pH 8.0, while

104 R. Wongchuphan et al. / Process Biochemistry 46 (2011) 101–107

Fig. 2. Non-reducing SDS-PAGE (10%) analysis of protein adsorption from rabbit serum onto DEAE sepharose as a function of pH of the medium. The adsorption conditionswere: 0.25 ml settled bed volume of DEAE sepharose; 0.75 mg/ml initial protein concentration; room temperature. Albumin removal was carried out in 25 mM sodiumphosphate buffers at (a) pH 7.0 and (b) pH 8.0 for 1 h. Lanes: (M) protein markers in kDa; (1) initial serum proteins; (2) serum proteins adsorbed in 15 min; (3) serum proteinsadsorbed in 30 min; (4) serum proteins adsorbed in 45 min; (5) serum proteins adsorbed in 60 min. Quantitative analysis of albumin removal (c) and total unbound IgG (d)was performed based on the intensity of the bands. The results are from duplicate measurements.

up to 30% was lost at pH 7.0. Hence, pH 8.0 was better than pH 7.0for removing albumin from rabbit serum with minimal IgG loss.

Based on these results, it can be concluded that rabbit serumalbumin is adsorbed rapidly onto highly activated DEAE sepharoseat pH 7.0. However, the binding is more efficient at pH 8.0. Asexplained earlier, the extent of protein–protein interactions waspossibly highest at pH 8.0, and hence this was the optimal pHfor removing albumin. In addition, the IgG loss at this pH couldbe explained by the formation of albumin–IgG complexes [9].The greater loss of IgG at pH 7.0 could be explained by the factthat the overall protein charge might be such that it allowed thealbumin–IgG interactions ensuing in the formation of large proteincomplexes. As a result, the better IgG adsorption by DEAE sepharoseoccurred at this pH [9]. As pH 8.0 is higher than the pI of rabbit IgG(around 7.8) [29], it is likely that the formation of albumin–IgGcomplexes at this pH is low. It should be noted that the aggregationstate of the protein may be altered as the effect of pH. Apparently,at pH 8.0, there was minimal IgG loss and albumin elimination wasadequate.

3.3. Effect of initial protein concentration on albumin depletion

The initial protein concentration also significantly impacted thequantity of albumin removed from rabbit serum (our earlier obser-vation in Section 3.2). At high protein concentrations, albuminremoval by DEAE sepharose diminished. Hence, low initial pro-tein concentrations (0.5 and 0.75 mg/ml) were finalized upon foralbumin removal. Table 1 shows that, at pH 8.0, the amount oftotal protein bound to the DEAE sepharose decreased as the ini-tial concentration of the serum sample increased. At the initialprotein concentration of 0.5 mg/ml, about 80% of the total pro-tein was bound, while only 66% bound at 0.75 mg/ml. In otherwords, when the initial protein concentration was low (0.5 mg/ml),the total unbound protein was found to be only 20%. If all the

serum IgG remained in this 20% unbound total protein fraction,then this adsorption condition at 0.5 mg/ml initial protein wouldhave been adequate for purifying antibodies. Quantitative analysisin this regard was required and is discussed below.

To estimate the efficiency of albumin removal by DEAEsepharose at pH 8.0, the SDS-PAGE gels shown in Fig. 2b(0.75 mg/ml) and Fig. 3 (0.5 mg/ml) were analyzed and compared.As illustrated in Fig. 2b, with the former condition, about 90% of thealbumin was adsorbed onto the adsorbent. The adsorption of IgGwas found to be about 20 ± 2% at that pH. According to the bandintensity analysis (Fig. 4a), at the initial protein concentration of0.5 mg/ml, a dramatic reduction of albumin (82 ± 1%) was observedwithin 15 min of binding. Further removal of albumin (95 ± 1%)was obtained after 1 h. Similarly, rapid albumin adsorption ontoDEAE adsorbent was observed by Pessela et al. [9]. As stated previ-ously, albumin tends to form self-aggregates. The aggregation rateof BSA in solution decreases with increasing protein concentra-tion [30]. The rise of the aggregation rate is associated with theincrease in water mobility. It was noted that greater than 90% ofthe albumin was efficiently removed at initial protein concentra-tions of both 0.5 and 0.75 mg/ml. At the former concentration, theremoval of albumin was accompanied by low IgG adsorption ontothe DEAE sepharose (about 16 ± 3%). In other words, 80% of therabbit polyclonal IgG was recovered and about 90% of the albuminwas removed at the low initial protein concentration. Therefore,

Table 1Total protein adsorbed onto DEAE sepharose as a function of initial protein concen-tration at pH 8.0.

Initial proteinconcentration(mg/ml)

Amount of totalprotein bound to DEAEsepharose (mg)

Amount oftotal proteinunbound (%)

0.5 4.09 ± 0.04 190.75 5.15 ± 0.21 34

R. Wongchuphan et al. / Process Biochemistry 46 (2011) 101–107 105

Fig. 3. Non-reducing SDS-PAGE (10%) analysis of proteins from rabbit serum onDEAE sepharose. The adsorption conditions were: 0.25 ml settled bed volume ofDEAE sepharose; 0.5 mg/ml initial protein concentration, pH 8.0; 1 h at room tem-perature. Lanes: (M) protein markers in kDa; (1) initial serum proteins; (2) proteinsadsorbed in 15 min; (3) proteins adsorbed in 30 min; (4) proteins adsorbed in45 min; (5) proteins adsorbed in 60 min.

both initial protein concentrations seem to be good for albumindepletion and antibody yield, but these parameters are somewhatbetter at the lower protein concentration (0.5 mg/ml). Contamina-tion with unbound albumin, however, would affect the purity ofthe IgG. It was found that less than 5% of the albumin remainedin albumin-depleted serum at the initial protein concentration of0.5 mg/ml, while more than 5% of it remained in the solution at0.75 mg/ml. Hence, the purity of the recovered IgG was better at0.5 mg/ml than at 0.75 mg/ml initial protein concentration.

The adsorption was performed for 1 h because no furtherimprovement in purity was observed as the binding time was pro-longed. Fig. 4b shows the relative amount of polyclonal anti-HBcAgIgG after adsorption onto DEAE sepharose as a function of initialprotein concentration. It was found that 83 ± 4% pure polyclonalanti-HBcAg IgG was obtained at the initial protein concentration of0.5 mg/ml. On the other hand, at 0.75 mg/ml, there was no improve-ment in the relative quantity of polyclonal IgG, which remainedsteady at about 30%. These results indicate that the rabbit poly-clonal IgG was purified to about 80% purity by using the DEAE anionexchanger to remove albumin at low initial protein concentrations.

By eliminating the major contaminant (albumin) with DEAEsepharose, a high yield of IgG antibodies was obtained from whey,as reported earlier by Pessela and colleagues [9]. The loss ofIgG molecules was explained by the formation of some type ofalbumin–IgG complex [9]. The retention of some albumin afterconsiderable albumin removal affects the purity of an antibody aswell as its yield. Notably, DEAE sepharose showed a good capacityfor purifying polyclonal IgG by eliminating albumin at low serumprotein concentrations, giving 83% purity and 82% recovery. Fora scaled-up process, the ratio of total protein to DEAE sepharoseadsorbent can be fixed to 5 mg/0.25 ml. However, different serumsources and batches might alter the efficiency of albumin removaland hence the purity of the antibody. Based on our experience,IgG should be at 15% or higher in the serum for efficient antibodypurification via the negative chromatography approach.

3.4. Capacity of Q XL for albumin depletion

The capacity of the STREAMLINETM Q XL strong anion exchangerwas also tested under similar optimized conditions for albuminremoval. Initially, according to the maximum binding capacity of QXL, 10 mg of total protein in serum sample was prepared in pH 8.0binding buffer for binding with 0.1 ml Q XL. The adsorbent showedinefficient albumin removal (<50%) at 1.0 mg/ml total protein, incontrast to that of the single protein binding system in which ∼95%of the BSA was adsorbed onto Q XL (data not shown). These results

Fig. 4. (a) Serum albumin adsorption (estimated by band intensity analysis) ontoDEAE sepharose as a function of initial protein concentration. Two initial proteinconcentrations (0.5 and 0.75 mg/ml) of serum samples were prepared individuallyin 25 mM sodium phosphate buffer at pH 8.0. (b) Relative quantity of polyclonalanti-HBcAg IgG (determined by ELISA) adsorbed onto DEAE sepharose as a functionof initial protein concentration. The adsorption conditions were: 0.25 ml settled bedvolume of DEAE sepharose; 1 h at room temperature. The results are from triplicatemeasurements.

suggest that the presence of a protein in different environmentsthat include other components (such as serum) affects its adsorp-tion onto the ionic exchange adsorbent. Since the total proteinconcentration has an influence on protein aggregation [15,32], thebinding capacity of Q XL for the removal of albumin was carried outat the lower initial protein concentration (0.5 mg/ml).

Fig. 5a shows the SDS-PAGE analysis of total protein before andafter serum albumin was removed in a time course (1 h). Albu-min was removed efficiently up to 89 ± 1% as estimated by bandintensity analysis. However, the loss of IgG was as high as 27 ± 1%.Fig. 5b shows the relative quantity of polyclonal anti-HBcAg IgGbefore and after albumin removal as determined by ELISA. IgG wasadsorbed as well as albumin at the mentioned time points, resultingin antibodies only 62% pure. These results show that albumin wasremoved efficiently by Q XL (about 90%), but only 73 ± 1% of the IgGwas recovered. Q XL demonstrated binding capacity for albumin ashigh as that of DEAE sepharose (90–95% of the albumin removed).The adsorption of albumin and IgG molecules onto the highly acti-vated adsorbent Q XL occurs due to the formation of aggregates insolution. In fact, albumin is capable of interacting with numerousdifferent species of molecules [12–14]. Albumin aggregates as wellas the albumin-IgG aggregates are adsorbed quickly onto Q XL, asreported earlier [9].

A comparative analysis of albumin removal using DEAEsepharose and Q XL is summarized in Table 2. Under similar con-ditions, greater than 90% of the albumin was removed by DEAEsepharose within 1 h. The loss of IgG in the presence of DEAEsepharose adsorbent was less than 20%. Thus, the purity of the IgGantibody in the serum after albumin removal by DEAE sepharose

106 R. Wongchuphan et al. / Process Biochemistry 46 (2011) 101–107

Fig. 5. (a) Non-reducing SDS-PAGE (10%) analysis of protein adsorption from rabbitserum onto Q XL. Lanes: (M) protein markers in kDa; (1) initial serum proteins; (2)proteins adsorbed in 15 min; (3) proteins adsorbed in 30 min; (4) proteins adsorbedin 45 min; (5) proteins adsorbed in 60 min. (b) The relative quantity of anti-HBcAgIgG adsorbed onto Q XL as determined by ELISA. The adsorption conditions were:0.10 ml settled bed volume of Q XL; 0.5 mg/ml initial protein concentration, pH 8.0;1 h at room temperature. The results are from triplicate measurements.

Table 2A comparison of STREAMLINETM DEAE and STREAMLINETM Q XL adsorbents in neg-ative chromatography antibody purification.

Polyclonal IgG DEAE sepharose Q XL

Yield (%) 82 ± 3 68 ± 3Purity in serum sample 0.15 0.15Purity after albumin removal 0.83 0.62Purification factor 5.5 4.1

was higher than that for Q XL. In summary, DEAE sepharose is bet-ter than the strong anion exchanger Q XL for purifying polyclonalIgG via negative chromatography in terms of both yield and purity;the former brings about a yield of 80% with a purification factor 5.5.

4. Conclusions

Negative chromatography, antibody purification usingSTREAMLINETM DEAE anion exchanger was used to removecontaminants from rabbit serum. The pH of the binding mediumand the initial protein concentration were optimized. Underthe optimized conditions, DEAE sepharose efficiently removedalbumin and other contaminants from rabbit serum. Depletionof albumin increased the purity of the polyclonal IgG (83%) andresulted in recovery as high as 80% at pH 8.0 with 1 h of bindingtime. The protein/adsorbent ratio of 5.0 mg total protein and0.25 ml DEAE sepharose was very useful for scaling-up the process.The method proposed here for removing albumin is simple andinexpensive. Furthermore, it is a single-step purification thatcan be easily scaled up. This method might be applicable forantibody purification from other animal sera as well. The use ofSTREAMLINETM DEAE anion exchanger offers a simple method

for purifying polyclonal antibodies from serum, which achievedby eliminating contaminating proteins with the N-CAP approach.Furthermore, DEAE sepharose is superior to Q XL for the removalof albumin and thus for augmenting the purity and recoveryof IgG.

Acknowledgements

The authors acknowledge Suratthani Rajabhat University, Thai-land and Grants 02-01-04-SF0808 and RUGS 91937 for financialsupport. The authors thank Suet Lin Chia and Ayele Taddese fortechnical support in the immunization work and Khai Wooi Lee forthe purification of HBcAg.

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