Novel Bioconjugation Strategy Using Elevated Hydrostatic Pressure: A

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A Novel Bioconjugation Strategy Using Elevated Hydrostatic Pressure: Case Study for Site Specific PEGylation of rhCNTF Qi Wang, Chun Zhang, Fangxia Guo, Zenglan Li, Yong Dong Liu, and Zhiguo Su Bioconjugate Chem., Just Accepted Manuscript • Publication Date (Web): 20 Oct 2017 Downloaded from http://pubs.acs.org on October 22, 2017

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Bioconjugate Chemistry

A Novel Bioconjugation Strategy Using Elevated Hydrostatic Pressure: Case Study for Site Specific PEGylation of rhCNTF

Qi Wang,†,‡ Chun Zhang,† Fangxia Guo,†,‡ Zenglan Li,†,‡ Yongdong Liu,*,† and Zhiguo Su*,†,§

†

State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese

Academy of Sciences, No.1 North Second Street, Zhong-Guan Village, Beijing 100190, PR China ‡

University of Chinese Academy of Sciences, Beijing 100049, PR China

§

Jiangsu National Synergetic Innovation Center for Advanced Materials (SICAM), Nanjing, 210023,

PR China *

To whom correspondences should be addressed; Yongdong Liu, E-mail: [email protected], Tel/Fax:

+86-010-82545028; Zhiguo Su, E-mail: [email protected], Tel/Fax: +86-010-82545027

1

ACS Paragon Plus Environment


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Abstract In this paper, we reported a novel strategy for site specific PEGylation of proteins using elevated hydrostatic pressure. The process was similar to the conventional one except the reactor was under elevated hydrostatic pressure. The model protein was recombinant human ciliary neurotrophic factor (rhCNTF), and the reagent was mono-methoxy-polyethylene glycol-maleimide (mPEG-MAL). PEGylation with mPEG-40kDa-MAL at pH 7.0 under normal pressure for 5 hours achieved less than 5% yield. In comparison, when the pressure was elevated, the PEGylation yield was increased dramatically, reaching nearly 90% at 250 MPa. Furthermore, the following phenomena were observed: 1) high hydrostatic pressure PEGylation (HHPP) could operate at a low reactant ratio of 1:1.2 (rhCNTF to mPEG-MAL), while the conventional process needs a much higher ratio. 2) short and long chains of PEG gave a similar yield of 90% in HHPP, while the conventional yield for the short chain of the PEG was higher than that of the long chain. 3) the reaction pH in the range of 7.0 to 8.0 had almost no influence upon the yield of HHPP, while the PEGylation yield was significantly increased by three times from pH 7.0 to pH 8.0 at normal pressure. Surface accessibility analysis was performed using GRASP2 software and found that Cys17 of rhCNTF was located at the concave patches, which may have steric hindrance for the PEG to approach. The speculated benefit of HHPP was facilitation of target site exposure, reducing the steric hindrance and making the reaction much easier. Structure and activity analysis demonstrated the HHPP product was comparable to the PEGylated rhCNTF prepared through conventional method. Overall, this work demonstrated that HHPP, as we proposed, may have application potentials in various conjugations of biomacromolecules. 2


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Introduction PEGylation has currently become a well-established and highly refined strategy for ameliorating the medicinal properties of therapeutic proteins including extending circulation half-life and reducing immunogenicity. However, this technique still suffers several drawbacks such as product heterogeneity, loss of bioactivity and low reaction yields.1, 2 To overcome these problems, current researches are focused on methods of site-specific PEGylation, in which PEGylation via free cysteine residue was mostly exploited because of its excellent specificity and good reactivity with Michael acceptors. However, low PEGylation yields still happen despite the functional group of PEG reagent such as mPEG-MAL is highly reactive to the thiol group.3, 4 Many parameters affect the process. The surface accessibility of the reactive site directly influences its conjugation with the functional group of PEGs.5 Generally, there exist hydrophobic patches and concaves on the protein surface when forming their spatial conformations, which would keep off water and other hydrophilic molecules like PEGs. During the formation of hydrophobic patches and concaves, free cysteine residues are prone to be embedded for a relatively stable conformation. In such cases, less exposure and hydrophobic microenvironment make hydrophilic PEGs hardly contact the free thiols, resulting in low PEGylation efficiency. Improving the surface accessibility of free cysteine residues buried in hydrophobic patches is expected to favor the PEGylation process. Varied pH values, ions strength, and external stress could change proteins’ conformation and thus be utilized to increase the exposure of free thiols.7 Through reversible denaturation, an increased PEGylation yield for rhG-CSF could be obtained by increasing the exposure of its free thiol.6 3



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However, the simultaneously exposed hydrophobic amino acids would decrease protein’s solubility, resulting in aggregates as well as precipitates during the PEGylation process.7, 8 The aim of the present study was to investigate whether the elevated hydrostatic pressure could be effective in assisting site-specific PEGylation. It has been known that high hydrostatic pressure may induce the structure change of a protein through disrupting the inter/intra-molecular hydrophobic and electrostatic interactions which are necessary to maintain the protein’s quaternary and the tertiary structures.9-11 In the range between 100 and 300 MPa, water molecules could be forced into the hydrophobic regions, disrupting the hydrophobic patches and loosing protein’s spatial conformation.12-14 These conformational or structural changes would be easily recovered with pressure removed and there is less even no aggregate formation during the high pressure treatment.15-17 Thus, it’s reasonable to explore the pressure influence in improving the accessibility of the hydrophilic PEG to the target free cysteine residues while avoiding protein precipitation during PEGylation. The model protein we used was human ciliary neurotrophic factor (CNTF), which is a kind of non-glycosylated polypeptide hormone and nerve growth factor that promotes neurotransmitter synthesis and neurite outgrowth in certain neural populations including astrocytes. Recombinant human CNTF (rhCNTF) with a free cysteine (Cys17) was reported to exert neuroprotective effects in many types of neurodegenerative diseases such as Alzheimer's disease, Huntington's disease and Amyotrophic Lateral Sclerosis.18, 19 However, it is shown that rhCNTF bears an extremely short circulation half-life of less than 0.5 h, and thus needs to be PEGylated for improving pharmacokinetics behaviors.20 A relatively high yield was achieved for the PEGylation of rhCNTF with mPEG-MAL in our previous work, 4


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but high reaction pH values (pH 8.0), long reaction time (more than 12 h) and large consumption of PEG reagent (PEG: protein > 3:1) were necessary.21 However, maleimide reactions are specific for thiols in the pH range of 6.5–7.5. At pH 7.0, the reaction of the maleimide with sulfhydryl proceeds at a rate 1000 times greater than its reaction with amines. At higher pH values, other amino acid residues, especially lysine residues and terminal amines, may react with maleimide moieties more easily.22-24 Due to the hydrolytic instability of the maleimide group in aqueous phase, a shorter reaction time is also preferred.25 In this study, the PEGylation of rhCNTF with mPEG-MAL was carried out under high hydrostatic pressure. Process parameters under high hydrostatic pressure state were carefully studied and optimized. The efficiency and yield were also compared with that of atmospheric state to verify if there were any advantages. Also, the physiochemical properties and bioactivity of the PEGylated rhCNTF conjugates were analyzed and the possible mechanism for HHPP was proposed. Results Effect of elevating hydrostatic pressure on the PEGylation yield of rhCNTF Firstly, using rhCNTF as a model candidate, the PEGylation process under different hydrostatic pressure values was carefully studied. As shown in Fig. 1C, conventional PEGylation at normal pressure for 5 h gave less than 5% yield. In contrast, when the pressure was increased, the PEGylation yield was significantly increased. 150 MPa could raise the yield to 15%, while 250 MPa brought about a high yield of nearly 90%. Note that the comparison was carried out in the same reaction solution, with the same reagent of mPEG-40kDa-MAL, the same reaction pH of 7.0, and the same reactant ratio of 5



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1:1.2 (rhCNTF to mPEG-MAL) at room temperature. Fig. 1A is the result of RP-HPLC and Fig. 1B is the HP-SEC, both showing the products were mono-PEGylated, and negligible traces of multi-PEGylated rhCNTF were observed in the final reaction. Though the reaction of the maleimide with amines proceeds at a very low rate at pH 7.0, lysine residuals and terminal amines may have possibility to react with maleimide moieties, resulting in traces of multi-PEGylated rhCNTF.

Figure 1: RP-HPLC (A), HP-SEC (B) analysis and PEGylation yield (C) of rhCNTF reacted with 1.2 (molar ratio) fold of mPEG-40kDa-MAL under atmospheric pressure or elevated hydrostatic pressures for 5 h at pH 7.0 respectively.

Influences of the process parameters on the PEGylation yield of rhCNTF Parameters of reactant molar ratios, PEG molecular weights, as well as the pH values, buffer composition greatly affect the conventional PEGylation process, the influences of theses process parameters under high hydrostatic pressure state were then compared. In Fig. 2, the PEGylation yield of the conventional process increased from less than 5% to about 15% as the molar ratio of reactants (rhCNTF to mPEG-MAL) increased from 1:1.2 to 1:10. In comparison, the reaction at 250 MPa proved to be much less sensitive to the molar ratio. Nearly 90% PEGylation yield could be achieved with the molar ratio of 1:1.2. When the molar ratio was raised to 1:2, 1:5 and 1:10, the yield of 92.2%, 95.5% and 95.4% was

6


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acquired respectively, indicating that the PEGylation yield was only marginally influenced by the increased PEG reagent ratio.

Figure 2: The PEGylation yield of rhCNTF with different molar ratios of mPEG-40kDa-MAL under 250 MPa or atmospheric pressure for 5 h at pH 7.0 respectively.

The molecular mass of the PEG had a profound influence for the conventional PEGylation process. As shown in Fig. 3, the yield of mPEG-10kDa-MAL, a relatively short chain of PEG, was higher than that of mPEG-MAL 40 kDa, a much long chain PEG, under the same reaction parameters at atmospheric pressure, although both were much less than the new process. The yield of HHPP, however, did not change much for mPEG-MAL 10 kDa and 40 kDa at 250 MPa, both yields being about 90%.

7



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Figure 3: RP-HPLC (A), HP-SEC (B) analysis and PEGylation yield (C) of rhCNTF reacted with 1.2 (molar ratio) fold of 10 kDa and 40 kDa mPEG-MAL under 250 MPa or atmospheric pressure for 5 h at pH 7.0 respectively.

Further comparison is pH effect. Under normal pressure, the solution pH played important role. As indicated in Fig. 4, pH increasing from 7.0 to slight alkaline condition of pH 8.0 could change the yield from less than 5% to about 20%, a significant rise by the conventional method. Meanwhile, the buffer of Tris-HCl seemed to be more efficient than the buffer of phosphate at the same pH value as the yield of mPEG40k-MAL-rhCNTF in buffer of Tris-HCl at pH 7.5 (10%) was relatively higher than that in buffer of PB at pH 7.5 (5%). In contrast, the yield of the new process was very high at pH 7.0, and increased slightly as the elevated pH values, with much less degree of variation than the conventional strategy.

Figure 4: The effects of pH values and different buffers on the PEGylation yield of rhCNTF with 1.2 (molar ratio) fold of mPEG-40kDa-MAL under 250 MPa or atmospheric pressure for 5 h respectively.

The productivity of the high pressure process for rhCNTF was subsequently compared with the common PEGylation at atmospheric pressure in Table 1. The new process, high 8


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hydrostatic pressure PEGylation (HHPP), finally achieved a PEGylation yield of 87.5%, while the optimized yield was only 38.2% for the traditional strategy that requires more PEG reagents and longer reaction time. Table 1. A summary chart for the preparation of PEGylated rhCNTF by HHPP compared with that of traditional method at optimized condition.

Method

Pressure/Time

Buffer

PEG

Molar ratio

PEGylation

( mPEG-MAL)

(protein/PEG)

yield (%)

HHPP

250 MPa, 5 h

pH 7.0 PB

40 kDa

1:1.2

87.5%

Traditional method

Atmoa, 20 h

pH 7.0 PB

40 kDa

1:3

38.2%

a

Atmospheric pressure

Structural and in vitro bioactivity characterization of mPEG-MAL-rhCNTF To obtain the high hydrostatic pressure PEGylated rhCNTF, the reaction mixture for mPEG-40kDa-MAL was quenched and purified by one step of Q Sepharose FF (Fig. 5A). The purity for mPEG40k-MAL-rhCNTF was about 99% determined by SDS-PAGE (Fig. 5B).

Figure 5. Purification of PEGylated rhCNTF through Q Sepharose FF Chromatogram (A) and 15% 9



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SDS-PAGE analysis of process pools (B) Lane 1: HHPP sample; Lane 2: P1 (mPEG40k-MAL-rhCNTF); Lane 3: P2 (rhCNTF).

To validate the conjugation site, mPEG40k-MAL-rhCNTF and native rhCNTF were separately digested with trypsin and the resulted fragments were analyzed by HPLC-MS. The theoretically expected peptide fragments and the experimentally identified fragments were outlined in Table 2. The tryptic peptide fragment 15-19 that containing Cys17 was present in the digested mixture of the native rhCNTF, while it almost completely disappeared for PEGylated rhCNTF, confirming the conjugation site was at Cys17 through HHPP. Table 2. Tryptic peptides of PEGylated and native rhCNTF with molecular masses determined by HPLC-MS. Molecular mass (Da) Tryptic peptide

Peptide Sequence

rhCNTFa

PEGylated-rhCNTFa

Calculatedb

1-13

MAFTEHSPLTPHR

1523.71

1523.72

1523.7423

15-19

DLCSR

593.26

Not foundc

593.2712

20-25

SIWLAR

745.45

745.45

745.4355

29-40

SDLTALTESYVK

1326.67

1326.66

1326.6787

41-46

HQGLNK

696.37

696.37

696.3787

47-72

NINLDSADGMPVASTDQWSE

2849.28

2849.29

2849.2894

LTEAER 73-81

LQENLQAYR

1134.60

1134.59

1134.5902

82-89

TFHVLLAR

956.58

956.55

956.5676

90-133

LLEDQQVHFTPTEGDFHQAIHT

5155.63

5155.61

5155.6488

LLLQVAAFAYQIEELMIL LEYK 137-154

NEADGMPINVGDGGLFEK

1862.88

1862.81

1862.8588

156-160

LWGLK

616.40

616.36

616.3817

161-171

VLQELSQWTVR

1358.76

1358.79

1358.7426

172-177

SIHDLR

740.40

740.40

740.4049

10


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178-185 a

876.39

876.41

876.421

Experimental molecular mass determined by HPLC-MS.

b

c

FISSHQTG

Molecular mass calculated by ExPASy.

Fragment 15-19 was not detected in the mass spectrum of the tryptic digest of PEGylated rhCNTF.

In Fig. 6A, the intrinsic fluorescence emission profile of tryptophan excitation was similar between native rhCNTF and mPEG40k-MAL-rhCNTF, and a slight blue shift for PEGylated rhCNTF indicated a compact packing of the protein structure, which could be attributed to the tiny interactions between PEG chain and protein surface. The secondary structure was not undermined according to the far-UV CD spectra of the PEGylated rhCNTF, which almost overlaps with that of the native protein (Fig. 6B). Subsequently, HP-SEC and RP-HPLC

analysis

also

showed

the

mono-dispersed

peak

of

purified

mPEG40k-MAL-rhCNTF (Fig. 6C and D). Additionally, the PEGylated rhCNTF through the new process showed similar advanced structure to that through conventional method.21 In order to investigate the bioactivity change of mPEG40k-MAL-rhCNTF prepared by HHP strategy, the TF-1(CN5α-1) cells assay was carried out. As shown in Fig. 6E, the PEGylation reduced the in vitro activity to 35% compared with that of native rhCNTF. The attached PEG of PEGylated rhCNTF might shield the target site of rhCNTF for receptor, resulting in lower uptake of rhCNTF and thus decreasing the in vitro bioactivity.26 However, the degree of such a decrease is similar to the literature report where PEGylation of rhCNTF by conventional way reduced its in vitro bioactivity. Furthermore, the literature also demonstrated that although the in vitro activity was decreased, the PEGylated product showed extended in vivo circulation half-life and efficiently induced weight loss compared with the native rhCNTF.27 11



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Figure 6: Biophysical characterization of purified mPEG40k-MAL-rhCNTF by Fluorescence (A), far-UV CD (B), Superdex200 (C) and RP-HPLC (D). In vitro bioactivity comparison of mPEG40k-MAL-rhCNTF and native rhCNTF by the TF-1(CN5α-1) cells assay (E).

Discussion Site specific PEGylation via free cysteine residue is mostly exploited for protein's PEGylation. The common principle is to carry out the reaction in phosphate buffer (PB) around pH 7.0 to guarantee a homogeneous PEGylated product. Modification at alkaline pH values would bring the risk of PEGylation happening at some other residues besides thiols.3, 28

However the PEGylation yield of rhCNTF at this condition was very low. Increasing

reagent ratio, prolonging the reaction time, and raising pH values always result in increased PEGylation yield, but concomitantly, a great waste of expensive PEG reagents and the increased risk of heterogamous products cannot be avoided. Surface accessibility is very important for an efficient PEGylation reaction. For the conventional PEGylation of rhCNTF via mPEG-MAL, the yield for 10 kDa was obviously

12


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higher than that for 40 kDa, indicating that the free thiol of Cys17 for rhCNTF was not easily contacted. To demonstrate this opinion, the surface accessibility of the free thiol in rhCNTF was simulated by using GRASP2 software.29 As shown in Fig. 7A, there are concave patches with different depth distributed on the simulated protein surface that means different degrees of accessibility. Cys17 of rhCNTF is found to be located at the concave patches. The low spatial accessibility of the conjugation site accounted for the low yield of the PEGylated rhCNTF even at the preferential conditions for the maleimide reactions. Therefore, increasing the accessibility of the specific site should be given the priority for a PEGylation process. High hydrostatic pressure was explored for this purpose and showed outstanding capacity for enhancing the PEGylation efficiency in a pressure-dependent manner. To demonstrate whether this increased efficiency was resulted from the increased exposure of Cys17 under elevated pressure, rhCNTF itself was treated at 100-300 MPa in the same buffer as HHPP. Obvious precipitate was discovered when the pressure was increased beyond 250 MPa and non-reducing SDS-PAGE indicated that some dimer with disulfide bond was involved in the precipitate (Fig. 7B and C). Theoretically, high pressure would irreversibly disrupt protein's native structures once above particular values, in spite that high pressure below the critical point is mostly used to recover protein's natural structure from inclusion bodies or other kinds of aggregates.30, 31 The formation of precipitate in Fig. 7B indicated such critical value for rhCNTF is about 250 MPa. The pKa for thiol is 8.3, the easily formed disulfide bond between rhCNTF at neutral pH should own to an increased exposure of the free thiol of Cys17. For HHPP at neutral pH, the reaction speed for mPEG-MAL with free Cys17 is much faster than that of disulfide bond formation in the case of no steric hindrance. 13



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As a result, the PEGylation efficiency was greatly improved as the more exposure of Cys17 under pressure. Moreover, the coupled hydrophilic PEG chain could veil the hydrophobic patches which were simultaneously exposed under high pressure, thus preventing precipitate during HHPP otherwise protein precipitated at the same pressure.

Figure 7: Surface accessibility analysis of free cysteine residue in rhCNTF from pdb file 1CNT by using GRASP2 software and the coloring scheme indicates deeply concave (gray) and highly convex (green) respectively (A). Protein recovery (B) and SDS-PAGE analysis (C) of rhCNTF itself after high hydrostatic pressure treatment. Lane 1: Marker; Lane 2: rhCNTF (pH 7.0) (non-reducing); Lane 3: soluble fraction of rhCNTF treated by 300 MPa for 5 h (non-reducing); Lane 4: precipitates of rhCNTF treated by 300 MPa for 5 h (non-reducing); Lane 5: rhCNTF (pH 7.0) (reducing); Lane 6: soluble fraction of rhCNTF treated by 300 MPa for 5 h (reducing); Lane 7: precipitates of rhCNTF treated by 300 MPa for 5 h (reducing).

Parameters of reactant molar ratios, PEG molecular weights, buffer composition, and the pH values greatly affect the conventional PEGylation process. For an acceptable PEGylation yield, there is always an absolute overdose of PEG reagents to the proteins for the traditional PEGylation strategy, resulting in great waste of PEG reagents. High PEGylation yield could be achieved for HHPP strategy with nearly 1:1 ratio of protein to PEG reagent and a much short reaction time, greatly reducing the reagents and time consumption and especially benefiting the scale-up production. The mPEG-MAL reagents with larger molecular weights often encountered low PEGylation efficiency at atmospheric pressure because of the strong

14


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steric hindrance between PEG reagents and free thiols. However, the longer chain of PEG reagents could acquire similar PEGylation efficiency to the short one through HHPP because of the improved surface accessibility of free thiol. The PEGylation reaction of free thiols with mPEG-MAL always becomes more active as the elevated pH values at atmospheric pressure, whereas the high pressure enables the thiol to be more exposed to the mPEG-MAL, accelerating the PEGylation reaction rate and partly counteracting the influence of the lower pH values. On the basis of the improved surface accessibility for the target PEGylation site, the similarly high yields of PEGylated rhCNTF could be observed for HHPP with various parameters, suggesting that elevating the solution’s hydrostatic pressure could weaken the impacts of other factors and overwhelmingly control the PEGylation process. After one step purification of Q Sepharose FF, the mPEG-40k-MAL-rhCNTF with a purity above 95% was obtained. The conjugation site of PEGylated rhCNTF through HHPP was then confirmed at Cys17 by HPLC-MS. Moreover, PEGylated rhCNTF prepared through HHPP exhibited similar advanced structures and in vitro bioactivity to that prepared through conventional method. These results demonstrated that high hydrostatic pressure PEGylation (HHPP) would be an alternative method to enhance the PEGylation efficiency of proteins by improving the spatially accessibility of potential conjugation sites. It’s worth noting that HHPP requires extra high hydrostatic pressure equipment compared with the conventional strategy, thus increasing the capital cost. Besides, this method particularly works to the proteins with reversible pressure-induced denaturation while other proteins might not be applicable. One 15



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should consider the pros and cons. Conclusions Elevation of the hydrostatic pressure in PEGylation process could increase the reaction yield as demonstrated by the conjugation of mPEG-MAL with rhCNTF. A high PEGylation yield up to nearly 90% was obtained at 250 MPa in 5 hours while the conventional process with the same reaction parameters received less than 5% yield. High hydrostatic pressure PEGylation (HHPP) could be operated with low or almost equal molar ratio of the reactants, thus greatly reducing the reagent cost and post separation cost. Furthermore, it also reduces the waste disposal. The solution pH exerts much less influence for HHPP strategy, allowing the reaction to carry out at the ideal pH for protecting the protein’s bioactivity. Similar PEGylation efficiency could be gained for both high and low molecular weights of PEG reagents. The increased spatial accessibility with the elevated pressure was suggested as the foundational mechanism for HHPP, which predominantly benefits the conjugated reaction. This work provides a new sight for bioconjugation of macromolecules via the reactive groups with low spatial accessibility. Experimental procedures Materials and equipment The rhCNTF was prepared by our laboratory.18 Reagents for experiment were obtained from the following vendors: The mPEG-10kDa-MAL and mPEG-40kDa-MAL were purchased from the JK Co.td (China). DL-Dithiothreitol (DTT) (Merck, USA); Trypsin (Sigma); all the other reagents used in experiment were analytical grade. The high-pressure equipment HPP.L3 600/0.6 (Hua Tai Sen Miao, Tianjin, China) was 16


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utilized for the PEGylation process, which could provide pressure up to 600 MPa with an effect reaction volume of 600 mL. Elevated hydrostatic pressure PEGylation of rhCNTF by mPEG-MAL The rhCNTF (1 mg/mL) was reacted separately with 10 kDa and 40 kDa mPEG-MAL at various molar ratios ranging from 1:1.2 to 1:10 (protein : PEG) in 1 mL of 20 mM PB (pH 7.0). After being mixed well, the mixtures were set in disposable syringes and then placed into pressure chamber at room temperature for pressurization. The reactions were conducted under different pressure values ranging from atmospheric pressure to 250 MPa for 5 h respectively. After depressurization, a DTT stock solution was added into the mixture to quench the reaction with a final concentration of 5 mM. Then, the solution was centrifuged at 12,000 rpm for 15 min and the supernatants were collected for further analysis. Yields of all PEGylation were determined by RP-HPLC method. In order to study the effects of different buffers and pH values on the new process, the rhCNTF (1 mg/mL) was reacted separately with 40 kDa mPEG-MAL at a 1:1.2 molar ratio in different buffers, including 20 mM PB (pH 7.0), 20 mM PB (pH 7.5), 20 mM Tris-HCl (pH 7.5), and 20 mM Tris-HCl (pH 8.0). The following procedure was carried out according to the method mentioned above and the PEGylation was respectively conducted at atmospheric pressure and 250 MPa for 5 h. Determination of the PEGylation yield The PEGylation yield was calculated according to Eq. (1), based on the RP-HPLC analysis. The column was Proteonavi C4 (4.6 mm × 250 mm, 5 µm, SHISEIDO, Japan), equilibrated in 50% acetonitrile including 0.1% TFA at 0.5 mL/min. The absorbance was 17



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monitored at 280 nm by use of an Agilent 1100 HPLC Workstation. 50 µL of PEGylated sample was injected and then eluted using a linear gradient of 50%-80% acetonitrile containing 0.1% TFA in 30 min.

PEGylation yield =

! "

(1)

Purification of PEGylated rhCNTF The reaction mixture for mPEG-40kDa-MAL (20 mL) was loaded onto a Q Sepharose FF column (16 mm × 100 mm, GE Healthcare, USA) column that was equilibrated in a running buffer of 20 mM PB (pH 7.0). The PEGylated rhCNTF were eluted from the column using a 0-30% gradient of 20 mM PB (pH 7.0), containing 1 M NaCl. The target peak was pooled and subjected to SDS-PAGE analysis.32 Biophysical characterization of PEGylated rhCNTF Size exclusion chromatography High performance size exclusion chromatography was performed on a Superdex200 10/300 column (GE Healthcare, USA), equilibrated with buffer (20 mM PB, 0.15 M Na2SO4, pH 7.0). ӒKTA purifier 1100 FPLC (GE Healthcare, USA) was used with an UV detector and the signals were recorded at 280 nm. The amount of sample injected was 500 μL and the flow rate was 0.5 mL/min. HPLC-MS analysis The rhCNTF and PEGylated rhCNTF were desalted into 50 mM NH4HCO3 using a Sephadex G25 column that was already equilibrated by the same buffer. Trypsin was then added to digest the sample with an enzyme/substrate ratio of 1:100 (w/w). The digestion was subsequently incubated for 20 h at 37℃. Afterwards, the samples were reduced by 20 mM 18


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DTT for 10 min and centrifuged at 12,000 rpm for 10 min prior to applying the supernatants to the HPLC-MS equipment (Orbitrap Fusion, Thermo Scientific). RP-HPLC was performed on an EASY-spray C18 column (75 µm × 15 cm, 5 µm particle size, 120 Å pore size) in the HPLC system (Thermo Scientific Easy-nLC 1000). Mobile phase A (0.1% formic acid in water) and mobile phase B (0.1% formic acid in acetonitrile) were used to establish the 100 min gradient comprising 3 min of 3%–6%B, 72 min of 6%–23%B, 15 min of 23%–35%B, and then 5 min of 35%–90%B, finally maintained at 90%B for 5 min. In total, 6 µL of each sample was loaded onto the column, and the flow rate was 0.2 mL/min. The mass spectrometer was operated with a full mass scan from m/z 300 to 2000 and electronic spray ion voltage was 4.5 kV. Circular dichroism (CD) measurement The far-UV CD spectra of PEGylated and native rhCNTF (0.1 mg/mL) were recorded at room temperature with a Jasco J-810 spectropolarimeter (Tokyo, Japan). The spectra of the samples were obtained at 190 nm to 260 nm in a 0.1 cm quartz cuvette. Samples were firstly desalted with 20 mM PB at pH 7.0 before detection and the background spectrum of buffer was recorded and subtracted from subsequent readings. The software package Spectra Manager (Jasco, Japan) was used for data collection and analysis. Intrinsic emission fluorescence F-4500 fluorescence spectrophotometer (Hitachi, Japan) was used for analyzing the intrinsic emission fluorescence spectra of native and PEGylated rhCNTF (0.2 mg/mL). Spectra were recorded on a 1 cm path-length cuvette with an excitation slit width of 5 nm and an emission slit width of 5 nm. The excitation wavelength was set at 295 nm and the emission 19



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spectra were recorded from 300 to 400 nm.

In vitro biological activity assay The bioactivity of PEGylated rhCNTF was tested in comparison with the native rhCNTF according to the method of Wang Q et. al.18 The TF-1(CN5α-1) cells were obtained from the National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, and Chinese Academy of Sciences. Apoptosis of TF-1(CN5α-1) cells would quickly appear in culture medium without the cytokine (GM-CSF) while CNTF could maintain the cell's viability as receptors of CNTF are expressed on the membrane of the cells. These cells were then cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS), 1% penicillin streptomycin, 0.4 mg/mL of G-418 sulfate and 2 ng/mL of GM-CSF. One day after, the TF-1(CN5α-1) cells were collected by centrifugation and suspended in complete medium without GM-CSF. TF-1(CN5α-1) cells were then diluted to 4.0 × 105 cells/mL in complete medium and distributed in 96-well microliter plate with 50 µL for each well. Subsequently, 100 µL of serially diluted native rhCNTF and mPEG40k-MAL-rhCNTF were added to each well and incubated at 37°C, 5% CO2 for 72 h. After that, 10 µL of CCk-8 reagent (Sigma-Aldrich, St Louis, MO, USA) was added in each well and the plates were incubated for further 2 h at 37°C. The percent of cell viability was estimated by analyzing the values obtained at 450 nm by a VersaMax micro-plate reader (Molecular Devices, USA). All samples were analyzed in triplicate on each plate. Statistical analysis The experiments were carried out in triplicate (3 independent runs) and the standard deviations of the results were calculated and reported. The analysis of variance test for 20


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significant effects of treatments and assay samples were determined using the SPSS Statistics software. Acknowledgements This research was financially supported by the National Natural Science Foundation of China (Grant no. 21576267), Beijing Natural Science Foundation (Grant no. 2162041), the Major State Basic Research Development Program of China (Grant no. 2013CB733604), and the Open Funding Project of the National Key Laboratory of Biochemical Engineering (Grant no. 2014KF-05). Conflict of Interest Statement We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its out-come.

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Table of Contents Graphic

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Table of Contents Graphic 150x88mm (300 x 300 DPI)



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Figure 1: RP-HPLC (A), HP-SEC (B) analysis and PEGylation yield (C) of rhCNTF reacted with 1.2 (molar ratio) fold of mPEG-40kDa-MAL under atmospheric pressure or elevated hydrostatic pressures for 5 h at pH 7.0 respectively. 161x50mm (300 x 300 DPI)


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Figure 2: The PEGylation yield of rhCNTF with different molar ratios of mPEG-40kDa-MAL under 250 MPa or atmospheric pressure for 5 h at pH 7.0 respectively. 150x114mm (300 x 300 DPI)



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Figure 3: RP-HPLC (A), HP-SEC (B) analysis and PEGylation yield (C) of rhCNTF reacted with 1.2 (molar ratio) fold of 10 kDa and 40 kDa mPEG-MAL under 250 MPa or atmospheric pressure for 5 h at pH 7.0 respectively. 160x41mm (300 x 300 DPI)


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Figure 4: The effects of pH values and different buffers on the PEGylation yield of rhCNTF with 1.2 (molar ratio) fold of mPEG-40kDa-MAL under 250 MPa or atmospheric pressure for 5 h respectively. 113x86mm (300 x 300 DPI)



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Figure 5. Purification of PEGylated rhCNTF through Q Sepharose FF Chromatogram (A) and 15% SDS-PAGE analysis of process pools (B) Lane 1: HHPP sample; Lane 2: P1 (mPEG40k-MAL-rhCNTF); Lane 3: P2 (rhCNTF). 150x62mm (300 x 300 DPI)


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Figure 6: Biophysical characterization of purified mPEG40k-MAL-rhCNTF by Fluorescence (A), far-UV CD (B), Superdex200 (C) and RP-HPLC (D). In vitro bioactivity comparison of mPEG40k-MAL-rhCNTF and native rhCNTF by the TF-1(CN5α-1) cells assay (E). 150x83mm (300 x 300 DPI)



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Figure 7: Surface accessibility analysis of free cysteine residue in rhCNTF from pdb file 1CNT by using GRASP2 software and the coloring scheme indicates deeply concave (gray) and highly convex (green) respectively (A). Protein recovery (B) and SDS-PAGE analysis (C) of rhCNTF itself after high hydrostatic pressure treatment. Lane 1: Marker; Lane 2: rhCNTF (pH 7.0) (non-reducing); Lane 3: soluble fraction of rhCNTF treated by 300 MPa for 5 h (non-reducing); Lane 4: precipitates of rhCNTF treated by 300 MPa for 5 h (non-reducing); Lane 5: rhCNTF (pH 7.0) (reducing); Lane 6: soluble fraction of rhCNTF treated by 300 MPa for 5 h (reducing); Lane 7: precipitates of rhCNTF treated by 300 MPa for 5 h (reducing). 189x50mm (300 x 300 DPI)


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Novel Bioconjugation Strategy Using Elevated Hydrostatic Pressure: A

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