Qilei Zhang,Tiantian Zhuang,Hongfei Tong,Hongyin Wang,Dongqiang Lin,Shanjing Yao

Key Laboratory of Biomass Chemical Engineering of Ministry of Education,Department of Chemical and Biological Engineering,Zhejiang University,Hangzhou 310027,China

1.Introduction

Immunoglobulins known as antibodies are glycoprotein molecules produced by plasma cells in response to the presence of foreign antigens[1].These proteins have important biological functions and have been found useful in diagnostic and therapeutic applications[2,3].Immunoglobulin(Ig)can be found and purified from various natural resources,such as animal blood[4-7],milk[8]and poultry eggs[9-10].For example,porcine blood contains up to 2%Ig which show s potential applications as antibacterial and antiviral agents,and it would be valuable if porcine Ig can be purified rather than wasted in the pork industry.Therefore,cost-effective and efficient purification processes are needed for large-scale production of these antibodies,which can benefit human health in addition to economic pro fits.

Currently,affinity chromatography is still the most widely used technique for antibody purification[11],and Protein A affinity chromatography is the most successful method for monoclonal antibody(m Ab)purification due to its excellent selectivity.High degree of purity and recovery can be achieved in a single operation with Protein A resins[12].How ever,high cost of Protein A-based resins de finitely restricts their application in large-scale antibody production.Moreover,Protein A has other limitations such as low re usability,ligand leaching and low tolerance in clean-in-place(CIP)procedures[13].Consequently,a variety of ligands and chromatographic techniques have been developed to purify antibodies over the years[14].Mixed-mode chromatography(MMC)is one type of new techniques which has received great attention in recent years[15].There are multiple interactions between functional ligands of MMC and target biomolecules during purification processes,such as hydrophobic,electrostatic and hydrogen bonding interactions[16].Compared with single-mode chromatography,MMC usually has higher selectivity and loading capacity[17].For example,Wang and co-workers[18]evaluated four mixed-mode resins for the purification of IgG from serum albumin containing feedstock,and high purity(92.3%)and high recovery(95.6%)were achieved after an optimized purification process.

Burton and Harding[19]introduced a form of MMC know n as hydrophobic charge-induction chromatography(HCIC)in 1998,which is based on the p H dependent behavior of ligands[20].HCIC is saltindependent and the adsorption and elution processes can be performed under physiological conditions[21,22].For example,the MEP Hyper Cel resin with 4-mercaptoethyl-pyridine(MEP)as the ligand is the first commercial HCIC adsorbent developed by Pall Corporation.This ligand contains a pyridine ring and a sulfur atom in the hydrophobic chain.Target proteins can be adsorbed on uncharged ligands at neutral p H via hydrophobic forces and eluted due to electrostatic repulsion under acidic conditions.The dynamic binding capacity of MEP Hyper Cel for murine IgG can reach up to 25-35 mg·ml-1[20].HCIC has been considered to be superficially similar to Protein A chromatography which also involves hydrophobic interactions for adsorption and elution because of ionization of histidine residues[23].In addition to the hydrophobic and electrostatic interactions,the thiophilic sites on ligands also play an important role in antibody binding.Research has show n that replacing the sulfur atom with either nitrogen or oxygen leads to much lower IgG binding capacity[20].The mechanism behind the thiophilic effects is not clear and it was postulated that the presence of an electron donor-acceptor pair on both the ligand and the antibody is responsible for this interaction[23-25].Molecular simulation indicates that the molecular structure of ligands is the main factor affecting the binding process,and a sulfonic group on the spacer arm might form additional hydrogen bonds to enhance the binding selectivity[26-28].

A series of HCIC cellulose adsorbents with thiophilic sites and mercaptoheterocyclic ligands were developed in our previous work[29],and the applications of these new resins for the purification of immunoglobulin from egg yolk were studied in both packed and expanded beds[10,30].In this study,new HCIC adsorbents will be evaluated for the purification of porcine Ig from porcine blood.Due to multi-interactions involved in HCIC,the operational conditions will be optimized for better separation/purification performance.The adsorption behaviors of the three adsorbents will be compared under different p H conditions and an optimized multi-p H elution process will be proposed.Moreover,the binding mechanism will be explored with the help of molecular simulation.

2.Materials and Methods

2.1.Materials

Fresh porcine blood was kindly provided by a local slaughterhouse.Porcine Ig was supplied by Sonac Co.,Ltd.(Wuhan,China).Buffer solutions with p H 3.0-3.6 were prepared with citric acid/phosphate buffer solutions,and buffer solutions with p H=3.8-5.0 were prepared with acetate buffer solutions.Phosphate buffer solutions were used for preparing buffer solutions with a p H value of 6-8.All of the buffer solutions were pre- filtered with 0.22 μm polymer membranes.The chemicals used were of analytical grade and were purchased from Sinopharm Chemical Reagent Co.,Ltd.(Shanghai,China).

2.2.Feedstock preparation

Fresh porcine blood was first mixed with 0.1 mol·L-1sodium citrate buffer solution(p H 8.7)w ith a ratio of 1:9,and then porcine plasma was obtained via centrifugation(3000 r·min-1,10 min)(Thermo Scientific,Waltham,MA,USA).The plasma was treated with ammonium sulfate to remove fibrinogen using an optimized process reported earlier[31].The supernatant was used as the feedstock for further purification of porcine Ig.

2.3.HCIC adsorbent preparation

Details of the preparation method for the HCIC adsorbents have been reported previously[29]and the structures are show n in Fig.1.Brie fly,macro porous cellulose particles were first prepared and used as matrices.Awater-in-oil suspension thermal regeneration method was used for the preparation,which was similar to the method published earlier[32].Some 5wt% gelatinized cassava starch solution was mixed with cellulose viscose under agitation,and then the mixture was dispersed in 300 g oil phase(vacuum oil:chlorobenzene,5:1)for 20 min.The suspension was heated to 95°C and kept at this temperature for 1.5 h before cooled dow n to 20°C and filtered.The particles formed were washed with boiling water and then ethanol,and treated with 1%amylase solution for 30 min.The final particles were washed with water and screened to obtain particles with a size range of 50-250 μm.These particles were then activated by divinylsulfone(DVS).The activated particles(Cell-DVS)were coupled with 4-mercapto-ethylpyridine(MEP),2-mercapto-methyl-imidazole(MMI)and 2-mercaptobenzimidazole(MBI),respectively.The corresponding adsorbents were denoted as Cell-DVS-MEP,Cell-DVS-MMI and Cell-DVS-MBI.These adsorbents have a size range of 50-250 μm and porosity of 85%.The w et density and the ligand density are approximately 1.8 g·ml-1and 60 μmol·ml-1,respectively.

Fig.1.Schematic diagrams of the chemical structures of the three HCIC adsorbents.

2.4.Adsorption isotherms

The adsorbents were equilibrated with related buffer solutions for 15 min,and then drained and added into five flasks(0.1 g adsorbent in each flask).Each of the flask contained 7 ml related buffer solutions with different initial porcine Ig concentrations of 0.1-0.5 mg·ml-1.The mixture was kept at 25°C for 10 h in a shaking incubator(200 r·min-1).The solutions were then filtered and the supernatant was analyzed via UV spectroscopy at 280 nm(Ultrospec 3300 Pro,Amersham Biosciences,Uppsala)to measure the final protein concentration in the liquids.The weights of protein absorbed onto the adsorbents were obtained by mass balance calculation,and the adsorption equilibrium was fitted by the Langmuir equation,

w here Q*(mg·g-1)and C*(mg·ml-1)are the equilibrium adsorption capacity and protein concentration in liquid phase,respectively,Qm(mg·g-1)is the saturated adsorption capacity and Kd(mg·g-1)is the dissociation constant.

2.5.Chromatographic purification operation

All chromatographic experiments were studied using an ?KTA explorer 100 system(GE Healthcare,Uppsala,Sw eden).A column with 10 cm length and 1 cm I.D.(GE Healthcare,Uppsala,Sw eden)was used with 4 ml adsorbents packed.The adsorbents were first washed with deionized water and then equilibrated with buffer solutions.Thereafter,feedstock was loaded with buffer solutions followed by elution processes.Finally,CIP with 0.5 mol·L-1NaOH and a reequilibrium step with buffer solutions were performed.The elution fractions were collected and analyzed using SDS-PAGE.

2.6.SDS-PAGE analysis

The loading feedstock and the elution fractions were analyzed by 8%SDS-PAGE gel.Some 5 μl of protein marker and 10 μl of samples were used.The protein migration was run at a constant voltage of 220 V.The gel was stained with Coomassie Blue R-250 and later de stained.The resulted protein gel was scanned using the Gel Doc 2000 imaging system(Bio-Rad,Hercules,USA).

2.7.Molecular simulation

The molecular interactions between ligands(MEP,MMI,and MBI)and IgG were studied with molecular simulation using a software platform Discovery Studio(DS,Accelrys Inc.,San Diego,USA)[27].The ligand molecule models were constructed by DS,and the protein structure of the Fc domain of IgG was obtained from the Protein Data Bank(PDB,www.rcsb.org/pdb/)with the ID code of 1FC1.The binding sites were identified using a similar protocol discussed in our earlier w ork[26].The ligands were first docked onto the surface of the protein with the LibDock module,and the potential binding sites were identified.The ligand-protein binding modes were further evaluated with molecular dynamics(MD)at neutral p H using the CHARMm module.Moreover,the interaction energies were analyzed and the mechanism of adsorption and desorption was discussed.

3.Results and Discussion

3.1.Adsorption isotherms of porcine Ig

Adsorption behaviors with HCIC adsorbents are strongly related to solvent p H.In this study,the adsorption capacities of these three HCIC adsorbents were measured by adsorption isotherms under different p H conditions and the experimental results are show n in Fig.2.Data were fitted with the Langmuir equation[Eq.(1)],which further provides the saturated adsorption capacity(Qm)and the dissociation constant(Kd)values(Table 1).

Fig.2(a)show s the adsorption isotherms with Cell-DVS-MMI under different p H conditions.There sults indicate that Cell-DVS-MMI had the highest adsorption capacity at p H 5.0.When the p H value increased,the adsorption capacity decreased to almost zero and the Langmuir equation was not applicable under such conditions.How ever,higher p H value solutions(p H 7.0 and p H 8.0)may be used for the elution of porcine Ig from Cell-DVS-MMI.Furthermore,Fig.2(b)and(c)show s the adsorption isotherm pro files for Cell-DVS-MEP and Cell-DVS-MBI,respectively.Similarly,the fitting pro files in both figures indicate that the highest adsorption capacity happened at p H 5.0.Table 1 show s that the Qmvalue at p H 5.0 increased in an order of Cell-DVSMMI＜Cell-DVS-MEP＜Cell-DVS-MBI,which indicates the adsorption capacity difference among these adsorbents,although the Kdvalues are in the same magnitude.

Fig.2.In fluence of p Hon the adsorption of porcine Ig with(a)Cell-DVS-MMI,(b)Cell-DVSMEP and(c)Cell-DVS-MBI.(■)p H 4.0;(○)p H 5.0;(Δ)p H 6.0;(▼)p H 7.0;(×)p H 8.0.

HCIC ligands can have multi-interactions with target biomolecules,such as hydrophobic,thiophilic and electrostatic interactions.Adsorption and desorption in HCIC usually result from these interactions working together.Since p H can affect the charging properties of both ligands and target molecules,it is a key parameter to control the separation with HCIC.A p H controlled adsorption mechanism of HCIC adsorbents has been discussed earlier in detail[30].In this study,the p Kavalues of MMI,MEP and MBI are around 5.3,4.3 and 4.1,respectively,while porcine Ig has an isoelectric point(p I)value of approximately 7.When a buffer solution with p H 4.0 was used,both the ligands and the porcine Ig were positively charged.The electrostatic repulsion force between the ligands and the target molecules leads to low adsorption capacity.Meanwhile,the hydrophobic and thiophilic effects may dominate the binding process w hen p H is higher than 5.How ever,as porcine Ig is a polyclonal antibody,the interaction between ligands and porcine Ig is complicated.The experimental results shown in Fig.2 and Table 1 indicate that p H 5.0 is the optimal adsorption p Hcondition for these three HCIC adsorbents tested,consistent with results reported when similar HCIC adsorbents were employed to separate immunoglobulin of egg yolk(IgY)[29,30].

Table 1 Saturated adsorption capacity(Q m)and dissociation constant(K d)of the adsorption of porcine Ig under different p H conditions

3.2.Porcine Ig purification

The three adsorbents were used separately to evaluate the performance in their porcine Ig separation from porcine plasma.The supernatant of porcine plasma after ammonium sulfate precipitation was used as the feedstock.

3.2.1.Cell-DVS-MBI

Fig.3 shows two typical chromatographic pro files of the elution process w hen Cell-DVS-MBI was used as the adsorbent.It was found that the proteins could not be efficiently eluted under acidic p Hand extreme conditions(p H 10.0)were needed.This is probably due to the strong adsorption capability of Cell-DVS-MBI as show n in Fig.2 and Table 1.MBI has a benzene ring on its molecule structure,which can facilitate the hydrophobic interaction between the ligand and porcine Ig.This is likely the reason why it show ed higher adsorption capacity w hen comparing to MMI and MEP resins.Similar results have been reported by Porath and Oscarsson[33,34].How ever,the extreme elution conditions may be undesirable for immunoglobulins to keep their bioactivity and thus are not suitable for efficient separation.Therefore,although Cell-DVS-MBI had the highest adsorption capability for porcine Ig,its selectivity was poor and proteins cannot be easily eluted.

3.2.2.Cell-DVS-MEP

Fig.3.Typical chromatogram of immunoglobulin separation with Cell-DVS-MBI at different elution p Hs.

Similar to the case of Cell-DVS-MBI,experiments were conducted with Cell-DVS-MEP.The results are shown in Fig.4.The elution peaks at different p H conditions indicate that porcine Ig can be eluted using acidic buffer solutions.With the increase of buffer solution acidity,the electrostatic interaction became intensive and the repulsion force between the ligand and the target protein increased.In order to evaluate the separation performance,SDS-PAGE analysis was carried out and the results are also show n in Fig.4.It could be found that porcine Ig was collected in the elution,but there were impurities co-existing in the fraction.Currently,MEP-related ligands are used in commercial adsorbents such as MEP HyperCel(MEP HyperCel has a similar ligand functional group of DVS-MEPas show n in Fig.1 but without the sulfonic group),and it would be useful to compare the performance of MEP-based adsorbents.A cellulose based adsorbent was prepared earlier in our group to separate IgY from egg yolk,which had the same functional ligand as MEP Hyper Cel[30].The home-made adsorbent and MEP HyperCel had different size range,porosity and ligand density.However,the adsorption results show ed that the two adsorbents had almost the same adsorption capacity.When an additional sulfonic group was added in the spacer arm as Cell-DVS-MEP,the adsorption of IgYshow ed an increase from 115 mg·ml-1to 130 mg·ml-1.This may indicate that the sulfonic group can have a positive contribution to the protein adsorption.Molecular simulation results also indicated that the sulfonic group on the spacer arm might improve the hydrogen bonding and thiophilic interaction with immunoglobulin and result in high adsorption capacity[27].

3.2.3.Cell-DVS-MMI

The chromatographic pro files of Cell-DVS-MMI study are show n in Fig.5.It can be found that stronger acidic conditions are preferred for the elution of porcine Ig.How ever,strong acidic conditions such as p H 3.0 may not be suitable for immunoglobulin to keep its bioactivity.Therefore,p H 3.8 was chosen for the separation of porcine Ig using Cell-DVS-MMI.Although the elution peaks show high intensity at p H 3.0 and 3.4,SDS-PAGE show ed that the fractions collected contained substantial impurities.The purity of porcine Ig was 65.7%for the p H 3.8 elution.Interestingly,the SDS-PAGE analysis indicated that with p H 7.0 elution,there was little amount of porcine Ig in the fraction and the majority of the proteins were impurities such as albumin.This result indicates that p H 7.0 can be used as a pre-elution condition to remove impurities.A dynamic binding capacity measurement showed that the IgG dynamic binding capacity on Cell-DVS-MMI was 10.1 mg·ml-1gel at 10%breakthrough when the flow rate was 0.5 ml·min-1,which was equal to 25.3%of the equilibrium binding capacity.The residence time under this flow rate was about 8 min,which indicates that adsorption process was relatively slow.Although increasing the flow rate to 1.0 ml·min-1can shorten the residence time to 4 min the dynamic binding capacity would be decreased to 14%of the equilibrium binding capacity.Therefore,a flow rate of 0.5 ml·min-1was used in the following investigation.In summary,Cell-DVS-MMI was more sensitive to p H,which can benefit the efficient charge-induced elution with p H change.Moreover,Fig.5 show s higher and more symmetric elution peaks than that of Cell-DVS-MEP and Cell-DVS-MBI.Therefore,Cell-DVS-MMI may be more suitable for porcine Ig separation.

Fig.4.Chromatogram of immunoglobulin separation with Cell-DVS-MEP at different elution p Hs.Inserted(right):SDS-PAGE analysis of elution fractions at different p Hs.

Fig.5.Chromatogram of immunoglobulin separation with Cell-DVS-MMI at different elution p Hs.Inserted(right):SDS-PAGE analysis of elution fractions at different p Hs.

3.3.Process optimization with Cell-DVS-MMI

Amulti-pH elution process was proposed to separate porcine Ig with Cell-DVS-MMI.The procedure was as follow s in brief:(1)feedstock(4.0 ml)loading at p H 5.0 onto a Cell-DVS-MMI column;(2)preelution with p H 7.0 buffer solution;(3)elution with p H 3.8 buffer solution;(4)elution with p H 3.0 buffer solution and(5)regeneration with 0.5 mol·L-1NaOH and buffer solutions.Atypical chromatographic pro file is show n in Fig.6.The results show that although three buffer solutions were used in sequence,the major peak appeared at p H 3.8 w here porcine Ig was collected.The SDS-PAGE analysis of different fractions at each stage is shown in Fig.7.The raw feedstock had about 16.7%porcine Ig.The p H 7.0 elution could remove most albumin and other impurities.For the p H 3.8 elution,the majority of proteins in the fraction was porcine Ig,and the purity was approximately 94.3%with a yield of 9.8 mg·(ml plasma)-1.The p H 3.0 elution could further elute the remaining porcine Ig.Compared with the raw feedstock,the purity of porcine Ig after the multi-p H elution was 5.6 times higher.Adding a pre-elution process(p H 7.0)can have an almost 1.5 times increase of the porcine Ig purity,which makes it a promising process for porcine Ig separation with Cell-DVS-MMI.

Fig.6.Chromatogram of immunoglobulin separation with Cell-DVS-MMI with multi-pH elutions.

Fig.7.SDS-PAGE analysis of different fractions with Cell-DVS-MMI chromatography under multi-p H elution conditions.

3.4.Molecular simulation analysis

Molecular simulation is a useful technique to explore binding mechanisms between ligand molecules and proteins.Based on our previous work,the pocket located on the CH2domain(around TYR319 and LEU309)of the Fc fragment of IgG was identified as the most stable binding site for the MEP ligand[26].At neutral p H,the pyridine ring at the end of the MEP ligand can provide hydrophobic association and hydrogen bonds,while the sulfonic group on the spacer arm might form additional hydrogen bonds,thus enhancing the binding of ligands with IgG.Moreover,it was also found that the MEP ligand departing from IgG under acidic conditions was via electrostatic repulsion.Here similar molecular simulation methods were used for the DVS-MBI and DVS-MMI ligands.The results show that the DVS-MBI and DVS-MMI ligands were docked onto the same pocket(around TYR319 and LEU309)located on CH2domain of the Fc fragment of IgG.At neutral p H,these tw o ligands could bind stably during the 4 ns MD simulation.Typical binding modes of DVS-MMI and DVS-MBI are show n in Fig.8,which is similar to that of DVS-MEP[27].It can be found that the main functional groups of the ligands were totally embedded into the target pocket.The amino acid residues surrounding the ligands within the distance of 0.3 nm were VAL279,VAL284,ASN 286,ALA287,LYS 288,LEU306,VAL308,LEU309,and TRY319,and most of them are hydrophobic residues.In addition,the spacer arm of DVS on the DVSMMI ligand could approach ASN286 and form hydrogen bonds as show n in Fig.8(a).For MBI,the spacer arm of DVS could also move near LYS288 of Fc and form hydrogen bonds(see Fig.8(b)).These additional hydrogen bonds were likely to enhance the binding process.

In addition,electrostatic interaction energy(EIE)and van der Waals interaction energy(VIE)between the ligands and Fc were calculated during the4 ns simulation and the results are shown in Fig.9.The values of VLE were in the range of 10-15 kcal·mol-1(1 cal=4.1840 J)while ELEs were of 0-5 kcal·mol-1,which indicates that non-polar(hydrophobic)interactions dominated the binding process.The average contributions of VIE were-13.74 and-11.69 kcal·mol-1for MBI and MMI,respectively(it was-13.54 kcal·mol-1for MEP ligand[27]).The absolute value of VIE increased in an order of Cell-DVSMMI＜Cell-DVS-MEP＜Cell-DVS-MBI,which is consistent with the adsorption capacity of the three adsorbents studied.Moreover,lg P is an important parameter to indicate the hydrophobicity of chemical molecules.The lg P values of MBI,MEP,and MMI are 2.247,1.367 and 0.824,respectively,which indicates that MBI is the most hydrophobic molecule,followed by MEP and MMI.In addition,the EIE value of MBI is higher than that of MMI,which might result in a stronger binding between MBI and IgG.

Fig.8.Simulation snapshot of the binding of MMI-DVS(a)and MBI-DVS(b)on the surface of Fc-A.Ligands are shown by the thick stick,w hile key residues are shown by the ball and stick,and the hydrophobic amino acids surround ligands within 0.3 nm are show n by the orange thin stick.The surface of protein is displayed by green transparent surface.The hydrogen bonds are marked with black dotted line.

The influence of p H on ligand binding was also investigated using molecular simulation.Since the acid dissociation constant values(p Ka)of MBI,MEP,MMI are 4.1,4.8 and 5.3,respectively,most MEP and MMI molecules are protonated at p H 4.0.How ever,the sulfonic group on the spacer arm would weaken the electrostatic repulsion and thus stronger acidic condition is needed in elution processes w hen such ligands are used.The results show that DVS-MMI can depart quickly from the surface of Fc during the 1 ns simulation.For DVS-MBI(p Ka=4.1),there were about 50%MBI molecules protonated at p H 4.During the 2 ns MD simulation,MBI in the protonated state departed quickly from Fc due to the electrostatic repulsion,but MBI in the deprotonated state is still stably bound to Fc.The EIE and VIE results between the ligands and Fc during the 2 ns simulation at p H 4 are show n in Fig.10.Due to strong interactions between MBI and Fc,the desorption of IgG from MBI-based adsorbents became difficult and stronger acidic condition was needed for efficient elution.In general,the molecular simulation results are consistent with the experimental studies.

Fig.9.EIE and VIE between the ligand and Fc-A during 4 ns simulation at p H 7.

Fig.10.EIE and VIE between the ligand and Fc-A during 2 ns simulation at p H 4.The protonated ligand:MBI(+1);non-protonated ligand:MBI.

4.Conclusions

Three HCIC adsorbents(Cell-DVS-MMI,Cell-DVS-MEP and Cell-DVS-MBI)were evaluated for their potential application in the purification of immunoglobulin from natural resources.Adsorption isotherms were characterized at different p H conditions,and the results show ed that the adsorption process was largely affected by solution p H.The highest adsorption capacity appeared at p H 5.0 due to the synergetic interaction of thiophilic,hydrophobic and electrostatic effects between the ligands and target proteins.Moreover,chromatographic results demonstrate that Cell-DVS-MBI was not suitable for the porcine Ig purification due to low selectivity,while Cell-DVS-MMI showed reasonable adsorption capacity with great selectivity.A multi-p H step elution process was proposed with Cell-DVS-MMI to purify porcine Ig from porcine blood and the purity of porcine Ig could reach to 94.3%w ith a yield of 9.8 mg·(ml plasma)-1.Binding mechanisms of the three ligands were studied using molecular simulation,and the results show ed that both Cell-DVS-MBI and Cell-DVS-MMI ligands were docked on the same pocket of the Fc fragment of IgG,with Cell-DVS-MBI showing stronger binding interactions.

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