Xianxiu Li,Yan Sun,2,Xiaoyan Dong,2,

1 Department of Biochemical Engineering,School of Chemical Engineering and Technology,Tianjin University,Tianjin 300354,China

2 Key Laboratory of Systems Bioengineering and Frontiers Science Center for Synthetic Biology(Ministry of Education),Tianjin University,Tianjin 300072,China

Keywords:Alginate Sulfonation γ-Globulin Adsorption capacity Uptake rate

ABSTRACT Our previous work proved that high adsorption capacity and uptake rate of lysozyme were achieved on alginate(Alg)-grafted resin with an ionic capacity(IC)of 240 mmol·L?1(Alg-FF-240).Moreover,the salt-tolerant feature of Alg-FF-230 was improved by using sequential alginate grafting and sulfonation strategy.Inspired by the enhanced adsorption performance of lysozyme,we have herein proposed to investigate the static and dynamic adsorption behaviors of γ-globulin on a series of Alg-grafted resins with different grafting densities and sulfonation degrees.The adsorption capacity of γ-globulin decreased with increasing alginate-grafting density(IC)from 160 to 230 mmol·L?1 at 0 mmol·L?1 NaCl because of the steric hindrance caused by the alginate-grafting layer.Effects of ionic strength(IS)indicated that the adsorption capacities of the resins with the IC value of 230–370 mmol·L?1 were much higher than CM Sepharose FF at 50–100 mmol·L?1 NaCl,and the uptake rate of Alg-FF-230 was about twice as much as that of CM Sepharose FF.This work demonstrated the important effects of alginate-grafting layer and IS in γ-globulin adsorption behavior,which would be helpful in the design of Alggrafted resins and the selection of proper IS condition for protein purification.

1.Introduction

Ion exchange chromatography (IEC) has been widely applied in biopharmaceutical protein purification due to its advantages of high capacity,ease of cleaning,and long lifetime[1–3].In order to pursue optimized IEC resins,polymer grafting procedure has been introduced as an efficient way for developing ion exchangers of high binding capacities and uptake rates[4–6].In our previous work[7],protein adsorption behavior on alginate(Alg)-grafted resins of different ionic capacities(ICs)was investigated using lysozyme as a model protein.It was found that the adsorption capacity and uptake rate of lysozyme increased with increasing IC value.The accelerated uptake rate was similar to the phenomenon occurred in many polymer-grafted resins where there existed“chain delivery”effect of bound proteins[8,9].“Chain delivery”belongs to a kind of surface diffusion of adsorbed proteins on the polymer chains by the swings of the flexible chain and is driven by the chemical potential toward the bead center as well as the interactions between neighboring flexible chains mediated by the adsorbed proteins[10,11].Therefore,Alg-grafted resin with the highest IC value(IC=240 mmol·L?1),Alg-FF-240,was chosen to be compared with CM Sepharose FF at various ionic strengths(ISs).It revealed that both adsorption capacity and uptake rate of lysozyme on Alg-FF-240 were significantly higher than those on CM Sepharose FF at salt concentrations lower than 100 mmol·L?1,which resulted in higher dynamic binding capacity (DBC) of Alg-FF-240 at the low salt concentration range and high flow rate.However,Alg-FF-240 only exhibited favorable adsorption performance at low salt concentrations because of its low charge density.For the purpose of increasing salt tolerance of the Alg-FF resins,a sequential alginate grafting and sulfonation strategy was adopted to synthesize two resins of higher ICs (270 and 370 mmol·L?1,named as S-Alg-FF-IC) [12].High-charge-density resin showed high salt-tolerance in protein adsorption,as reflected by the high DBC presented by S-Alg-FF-370 at a wide IS range.

As compared to the commercial resin CM Sepharose FF,both Alg-FF and S-Alg-FF resins showed more preferable adsorption performance for lysozyme.However,different proteins are likely to behave different adsorption behaviors on ion exchangers,depending on their molecular characteristics.For example,high binding capacity of bovine serum albumin(BSA)on poly(ethylenimine)(PEI)-4 FF resin was observed at pH 5.0–8.9,while very little adsorption of immunoglobulin G(IgG)was obtained over a pH range from 5.0 to 8.0 [13].Additionally,the influence of protein properties on bound protein transport behavior on PEI-grafted anion exchangers was studied by using BSA and γ-globulin[14].It was notable that the IC range where the hopping of protein uptake rate(effective pore diffusion coefficient,De)occurred for γ-globulin was earlier than that for BSA,owing to its larger size and less net charge.

However,the prior research on Alg-FF and S-Alg-FF resins was done using only one model protein(lysozyme)[7,12].Two key problems presented herein needs to be solved in a scientific and rigorous way.Namely,(1) what about the adsorption behavior of other protein on the resins mentioned above,and(2) is there an optimal IC(alginate grafting density or charge density)at each IS exhibits the best performance for protein adsorption?In order to solve these problems,this paper was designed to investigate γ-globulin adsorption performance on Alg-grafted resins and their sulfonate derivatives.The experimental results are expected to provide more in-depth understanding on the effects of IC and IS on protein adsorption for guiding the selection of resins suitable for the purification of various proteins from different feedstocks.

2.Materials and Methods

2.1.Chemicals and protein preparations

Sepharose FF and CM Sepharose FF were purchased from GE Healthcare(Uppsala,Sweden).Bovine γ-globulin(≥99%,MW～150,000,pI～6.9)was from RuitaiBio(Beijing,China).Sodium alginate of low viscosity(4–12 cP),BSA(≥96%,Mw～66,400,pI～4.9)and lysozyme(from chicken egg white,～96%,MW～ 14,400,pI～11) were received from Sigma-Aldrich(St.Louis,MO,USA).All other chemical reagents were of analytical grade and were obtained commercially.

Alg-FF cation exchangers and S-Alg-FF resins used in this work were prepared following the procedure described in our previous work[7,12]and Fig.S1.Protein solutions were prepared by dissolving protein powder into 20 mmol·L?1acetate buffer(pH 5.0)with different sodium chloride(NaCl)concentrations.

2.2.Resin characterization

Ionic capacities of all the cation exchangers were measured by acid-based titration as described previously [15].The effective porosities(εp)of the resins for γ-globulin,BSA and lysozyme were determined in 20 mmol·L?1Tris–HCl(pH 8.0)containing 1.0 mol·L?1NaCl and determined by batch diffusion measurements as described in literature[12].

2.3.Protein adsorption properties

Adsorption experiment for γ-globulin on the cation exchangers was carried out in 20 mmol·L?1acetate buffer(pH 5.0)with different NaCl concentrations.The effect of pH was carried out in 20 mmol·L?1Na2HPO4-KH2PO4(pH 5.0–7.0).A mass balance was used to calculate the protein adsorption density (q) in the solid phase[16].Langmuir equation was applied to describe the adsorption isotherms and determine the adsorption capacity(qm)and dissociation constant(Kd)[17].

Adsorption kinetics of γ-globulin on the resins were determined by the dynamic batch adsorption method described by Wang et al.[18].The uptake rate was expressed by the effective diffusion coefficient(De)in the effective pore diffusion model(PDM)[19],and the Devalues under different conditions were determined by fitting the model to the dynamic curves determined by the dynamic batch adsorption method.The diffusivity of γ-globulin in the free solution(D0)is 4.4×10?11m·s?1at 25°C[20].

3.Results and Discussion

3.1.Properties of the resins

Three Alg-FF resins of different IC values were prepared and named after their IC values,denoted as Alg-FF-IC with IC value in mmol·L?1(Table 1).The four sulfonate resins derived from the three Alg-FF resins were named as Alg-FF-mSn,where m stands for the IC value of the basic Alg-FF resin and n represents the total IC value of sulfonate derivative(Table 1).The εpvalues of the resins for γ-globulin(εp,γ),BSA(εp,BSA)and lysozyme(εp,Lys)are also listed in Table 1.

Table 1 shows that the εpof the Alg-FF resins decreased with increasing protein size from 1.95 nm(lysozyme)[14]to 5.9 nm(γ-globulin)[21],which should be caused by the unreachable narrow pores for the larger protein molecule.The εp,γof the Alg-FF resins decreased with increasing alginate-grafting density(IC value),similar with the trend reported in PEI-grafted resins[14].Moreover,the εp,γof the two sulfonate derivatives(Alg-FF-110S230 and Alg-FF-160S340)was obviously lower than their corresponding counterparts(Alg-FF-110 and Alg-FF-160)due to the narrowed inner pore volume caused by the enhancement of electrostatic repulsion between the alginate chains,while the εp,Lysof the resins kept almost unchanged after sulfonate modifications.Both of the phenomena indicated that the procedures of alginategrafting and sulfonation affected the accessible pore space only for the large protein(γ-globulin),and would further influence the adsorption capacity and diffusion of the protein into the pores.The latter will be discussed in the next section.

3.2.Protein adsorption equilibria and kinetics

Fig.1 shows the effect of salt concentration on the equilibrium γ-globulin adsorption density(q)on the cation exchangers obtained at an initial protein concentration of 3.0 mg·ml?1.The data are listed in Table S1.

It is seen that the cation exchangers displayed different favorable properties for use in protein adsorption at different salt concentrations.For γ-globulin adsorption at 0 mmol·L?1NaCl,the q value increased firstly and then decreased with the increase of alginate-grafting density in the IC range of 110–230 mmol·L?1.For the three alginate-grafted resins,although the number of protein binding sites increased with increasing grafting density(IC),the εp,γdecreased monotonically in the IC range(Table 1),so the appearance of a maximum q was a result of the interplay between the different changes of number of protein bindingsites and εp,γas a function of IC.For example,the q value of Alg-FF-160 was about four times higher than that of Alg-FF-110 because of its more protein binding sites.The εp,γof Alg-FF-230 was obviously lower than Alg-FF-160(Table 1),so it is considered that the lower q value of Alg-FF-230 was due to the decrease of available pore volume,which was in accordance with those occurred in PEI-grafted resins[8,22].By the same token,the q value of Alg-FF-110S230 was about six times higher than the other resins in the IC range of 230 to 370 mmol·L?1.For the resins with high alginate-grafting densities,the electrostatic repulsion between grafting polymers was enhanced by the increase of charge density,which resulted in the more occupied pore volume caused by the extended grafting layer,so the q value decreased with the increase of sulfonation degree.For example,the q value of Alg-FF-160 was significantly higher than that of Alg-FF-160S340 at 0 mmol·L?1.However,the adsorption capacity of lysozyme increased with increasing sulfonation degree[12].As we know,the molar molecular weights of lysozyme(14.4 kDa)and γ-globulin(150 kDa)are quite different,and the size of lysozyme is much smaller than that of γ-globulin,so the stronger steric hindrance caused by the increased sulfonation degree was not significant for lysozyme.With the increase of salt concentration,the q value decreased rapidly in the IC range of 110 to 160 mmol·L?1,while it appeared the opposite change at IC=370 mmol·L?1.At 230 ≤IC ＜370 mmol·L?1,the q value firstly increased and then decreased with increasing IS,which was similar to the phenomenon observed from PEISepharose FF resins[23].That is to say,the salt tolerance degree of the resin was enhanced with increasing IC.It is considered that the increase of IS led to the increase of pore accessibility and thus the decrease of electrostatic exclusion to free proteins in their entering into the pores[23],leading to the increase of q value.Thereafter,the decrease of q value was mainly caused by the screening effect of salt ions on the electrostatic interaction between protein and ion exchange groups[9].For the resins of low IC values (Alg-FF-110 and Alg-FF-160),it was easy for the electrostatic interaction between their charged groups and γ-globulin to be screened by salt ions,which could offset the positive effect of the increased pore accessibility,leading to the decreased q values.For Alg-FF-230S370,it was difficult for the electrostatic interaction between ion exchange groups and protein to be shielded by salt ions because of its high charge density,giving rise to the increased q value in the salt concentration range of 0–100 mmol·L?1.While for the other resins in the IC range of 230 to 340 mmol·L?1,the charge density was lower than Alg-FF-230S370,leading to the higher q value with increasing NaCl concentration in the salt concentration range lower than 75 mmol·L?1.For lysozyme,however,the adsorption capacity of Alg-FF-230S370(S-Alg-FF-370)decreased continuously with increasing salt concentration [12].The difference in protein size between γ-globulin and lysozyme led to the difference in the extents of the decrease of effective pore volume(Table 1),resulting in a downtrend of adsorption capacity in the salt concentration range of 0–100 mmol·L?1for lysozyme.

Table 1 Physical properties of Sepharose FF and the cation exchange gels

Fig.1.Adsorption density of γ-globulin at different salt concentrations in 20 mmol·L?1 acetate buffer(pH 5.0).

Because Alg-FF-160 presented the highest q value at 0 mmol·L?1NaCl,it was selected as representative for the effect of pH on protein adsorption density.For the given protein,extremely lowering pH could induce conformational changes and protein aggregation [24],so the investigation was conducted at mild conditions,pH 5.0–7.0.The data are listed in Table 2.

Table 2 Adsorption density of γ-globulin on Alg-FF-160 at different pH values in 20 mmol·L?1 Na2 HPO4 -KH2 PO4

The q value of γ-globulin at pH 6.0 was slightly lower than that at pH 5.0,but an obvious decrease was found when the pH value was further increased to 7.0.It is considered that q mainly depends on the dissociation degree of ion exchange groups and the net charge of the protein[25],both of which are related to pH.The dissociation degree of carboxyl groups in the grafted alginate chains increases with the increase of pH,which can increase the electrostatic repulsion between the adjacent alginate chains,thus reducing available pore volume for γ-globulin adsorption.On the other hand,because the pI value of γ-globulin is about 6.9[14],the net charge of γ-globulin decreases with increasing pH from 5.0 to 7.0,which was not conducive to the binding of γ-globulin to the cation exchange groups.Both the factors are unfavorable for protein adsorption,so the q value of γ-globulin exhibited a declining trend with increasing pH.Thus,the subsequent experiments were conducted at pH 5.0.

According to the above results,some resins presented very low q values at each NaCl concentration(Fig.1),so no experiments of static γ-globulin adsorption onto these resins were done at the corresponding salt concentrations.The cation exchangers with high adsorption capacities under each salt concentration were selected for static adsorption and batch dynamic uptake experiments,and the results were compared with CM Sepharose FF.The adsorption isotherms of γ-globulin are presented in Fig.S2 and the fitted Langmuir parameters(qm,Kd)are listed in Table 3.It is well known that the adsorption ability was not suitable to be described when the adsorption isotherm did not reach a plateau,so the adsorbed protein density(qc)calculated by the Langmuir equation at an equilibrium liquid phase concentration of 2.5 mg·ml?1using the parameters listed in Table 3 was used to replace the protein adsorption capacity.The uptake kinetic curves of γ-globulin onto the cation exchangers are plotted in Fig.S3.The Devalues were obtained by fitting the PDM to the kinetic curves,and De/D0was defined as the protein uptake rate.Both the values of qcand De/D0are illustrated in Fig.2.

CM Sepharose FF shows a general behavior of decreasing qcvalue with increasing IS(Fig.2(a)to(d)).At 0 mmol·L?1NaCl,the qcvalue of CM Sepharose FF was about twofold higher than those of Alg-FF-160 and Alg-FF-110S230 (Fig.2(a)).Although the qcof Alg-FF-160 was similar to that of FF-PEI-L560 at 0 mmol·L?1NaCl,the Kdvalue of the former (0.097 mg·ml?1) was obviously smaller than the latter(0.60 mg·ml?1)[14],indicating that the interactions between the alginate ligands and γ-globulin were much stronger.However,the qcvalues of the resins in the IC range of 230–370 mmol·L?1were much higher than those of CM Sepharose FF at 50–100 mmol·L?1NaCl.For example,the qcvalue of Alg-FF-230(186 mg·ml?1)was about 1.4 times higher than that of CM Sepharose FF(135 mg·ml?1)(Fig.2(b)),which wasconsidered due to the effective interaction volume for protein binding offered by the three-dimensional structure of the grafted alginate[7].For Alg-FF-230S370,its qcvalue kept about 70 mg·ml?1at 100 mmol·L?1NaCl(Fig.2(d)),which was over 3.6 times higher than CM Sepharose FF at the same salt concentration.

Table 3 Equilibrium parameters qm and Km (both in mg·ml?1)for γ-globulin adsorption at different salt concentrations in 20 mmol·L?1 acetate buffer(pH 5.0)①

At 0 mmol·L?1NaCl,the De/D0value of CM Sepharose FF was obviously higher than the other two resins(Fig.2(a)).Although the qcvalue of Alg-FF-160 was similar to that of Alg-FF-110S230,its De/D0value was about 4.5 times higher than Alg-FF-110S230(Fig.2(a)),and more than twice of SP Sepharose FF(De/D0～0.2)reported in the literature[26].Increase of the alginate-grafting density seemed to have a positive effect on the increase of De/D0,namely the occurrence of“chain delivery”,possibly because of the decreased distance between adjacent polymer chains and the higher flexibility of alginate chains[7],thus the De/D0value of Alg-FF-160 was higher than that of Alg-FF-110S230 (Fig.2(a)).Nevertheless,it is likely that the steric hindrance of the alginate chains reduced the number of successful collisions between the γglobulin and ion exchange groups,thus resulting in the lower uptake rates on Alg-FF-160 and Alg-FF-110S230 as compared to CM Sepharose FF at 0 mmol·L?1NaCl(Fig.2(a)).For the resins with IC values of 230 and 340 mmol·L?1(Alg-FF-110S230,Alg-FF-230 and Alg-FF-340),the De/D0values increased with increasing salt concentration(Fig.2(a)to(d)).The increase of De/D0was mainly attributed to the enhanced happening of“chain delivery”effect caused by the decreased binding strength of the protein and the smaller steric and electrostatic hindrances for protein transfer [27].The uptake rate of Alg-FF-230 was higher than the other resins in the salt concentration range of 50–75 mmol·L?1(Fig.2(b)to(c)).Especially at 75 mmol·L?1NaCl,the De/D0value on Alg-FF-230 was about twice that on CM Sepharose FF.For Alg-FF-160S340,the De/D0value increased to 0.282 when NaCl concentration was increased to 100 mmol·L?1,which was about 6-fold increase over that at 75 mmol·L?1NaCl.

Fig.2.Calculated adsorption density of γ-globulin at an equilibrium liquid-phase concentration of 2.5 mg·ml?1 and effective pore diffusivity of γ-globulin on the cation exchangers at(a)0,(b)50,(c)75 and(d)100 mmol·L?1 NaCl in the equilibration buffer(20 mmol·L?1 acetate buffer,pH 5.0).

In summary,Alg-FF-230 displayed preferable γ-globulin adsorption performance at 50 and 75 mmol·L?1,so it could be chosen to be the typical resin for the separation of γ-globulin.

4.Conclusions

The purpose of this study is to investigate γ-globulin adsorption and transport on Alg-grafted Sepharose FF and their sulfonate derivatives.It was found that alginate-grafting procedure had negative effect on γ-globulin adsorption with the IC value of 230 mmol·L?1at 0 mmol·L?1NaCl.Furthermore,ionic strength played an important part in protein adsorption capacity,which could be reflected in the specific IC value that offered the maximum equilibrium capacity at each salt concentration.The phenomena mainly resulted from the comprehensive effects of the decreased steric and electrostatic exclusions and the increased electrostatic screening effect caused by the salt ions.For the resins with IC ≥230 mmol·L?1,the qcwas much higher than CM Sepharose FF at 50–100 mmol·L?1NaCl.The uptake rate of γ-globulin was enhanced by alginate-grafting at salt concentrations over 50 mmol·L?1,and the De/D0value of Alg-FF-230 was much higher than that of CM Sepharose FF at 75 mmol·L?1NaCl.

This work has also indicated that the adsorption behavior of γ-globulin was different from that of lysozyme[12]on the resins due to the difference of protein characteristics.The findings in this work have contributed to revealing the potential of Alg-grafted resins and their sulfonate derivatives for purifications of multiple proteins and optimizing practical chromatographic processes of therapeutic proteins with the resins developed in this work.In consideration of exploring more details about the practical protein purification,however,further work should direct toward examining other proteins of different sizes and net charges to deeply understand the effects of IC and salt concentration on different protein adsorption and investigating the potential applications of the resins on protein purification.

Nomenclature

DeEffective pore diffusivity of the protein,m2·s?1

D0Free solution diffusivity of γ-globulin(4.4×10?11m2·s?1at 25°C)

KdDissociation constant,mg·ml?1

q Equilibrium protein adsorption density in the solid phase,mg·ml?1

qcProtein adsorption density at an equilibrium liquid-phase concentration,mg·ml?1

qmAdsorption capacity of protein,mg·ml?1

Acknowledgements

This work was supported by the National Natural Science Foundation of China(Nos.21878222 and 21621004).

Supplementary Material

Supplementary data to this article can be found online at https://doi.org/10.1016/j.cjche.2020.06.017.