Separation of pegylated recombinant proteins and isoforms on CIM ion exchangers

https://doi.org/10.1016/j.jchromb.2016.07.020Get rights and content

Highlights

  • Development of fast screening method for optimal separation of pegylated biopharmaceuticals.

  • Separation of monomer SHLA from renatured inclusion bodies.

  • Differentiation between different mono-pegylated isoforms of SMLA.

Abstract

Protein pegylation is a process of covalent attachment of a polyethylene glycol (PEG) group to the protein tertiary structure that can “mask” the agent from the immune system and also increases the hydrodynamic size of the agent. Usually the pegylation prolongs the protein stability in the organism due to reduced renal clearance and provides superior water solubility to hydrophobic molecules. The mono-pegylated form of protein is usually prefered for medical applications. Different conditions with different PEG reagents have to be tested to find optimal pegylation procedure with specific protein.

The goal of this study was to prepare screening method for separation of random mono-pegylated protein. Cytochrome C and beta lactoglobulin were pegylated with four reagents and a complete screening of several chromatographic monoliths in ion exchange mode with different buffers was performed to optimaly separate each mono-pegylated protein.

The screening method was developed that produces optimal separation of target pegylated protein on CIM monoliths. Because of short chromatographic run time, CIM monoliths are perfect candidates to test alot of parameters.

The results obtained show that each protein has its own unique separation parameters (pH, ionexchange ligand, buffer type). Two biopharmaceuticals were isolated using protocol: super human leptin antagonist (SHLA) was purified from inclusion bodies and mono-pegylated super mouse leptin antagonist (SMLA) from pegylated mixture. During study it was observed that the convective interaction media (CIM) monoliths additionally discriminate between protein isoforms pegylated on different sites in 3D structure of the protein.

Introduction

The technology of polyethylene glycol (PEG) attachment to protein backbone holds significant promise in maintaining effective plasma concentrations of systemically administered drugs that might otherwise be hampered in vivo by a number of factors, such as rapid elimination by the kidneys [1]. With the control of the size and complexity of PEG molecules, the attachment site can be manipulated and steric hindrance can be avoided [1]. Ion exchangers are preferred method for isolation of mono-pegylated proteins on porous beads [2], [3], [4], [5], [6].

The structure of chromatographic monoliths enables convective mass transport, which was shown to be optimal for separation of large molecules, such as proteins, viruses, DNA [7], [8], [9].

Consequently, chromatographic monoliths have already been tested as alternatives to porous beads for isolation of mono-pegylated proteins [4], [10], [11]. Low back pressure on monolith itself gives flexibility in terms of varying flow rates and extending column lifetime. Chromatographic runs on CIM monolithic columns are shorter compared to runs on traditionally bead packed columns, because of length of the monolithic columns and their flow independent properties. Short runs contribute to lower buffer consumption that leads to better economics of the process.

The principle of separation on ion exchanger is based on the difference in net charge. With pegylation, part of the protein charge is masked with large PEG molecule. Because of that PEG molecules bind with different strength onto ion exchange support and elute at different salt concentration. Target amino acid residues that can be specifically pegylated are: lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, tyrosine. The N-terminal amino group and c- terminal group can also be conjugated in site specific way [12].

One of the important biopharmaceutical targets for pegylation is Leptin, which is a 16-kDa protein hormone that plays a key role in regulating body energy level, its expenditure, appetite/hunger and metabolism [13], [14], [15], [16]. The protein, designated super-active mouse leptin antagonist (SMLA) (mutated at D23L, L39A, D40L, F41A) has 64 fold higher binding affinity toward human Leptin binding domain than its wild type counterpart [17], [18]. Its biological inhibitory activities in vitro (BAF/3 or H-49 cell bioassays) were increased by 14 fold [17] in vivo it has profound weight gain effect (biopharmaceutical effect), resulting mainly from increased food intake. Similar results were obtained for super-active human leptin antagonist (SHLA) (mutated on the same sites as SMLA). The biological activities of SHLA and SMLA in H-49 cell bioassays and in binding assays were identical. Published purification protocol for SHLA uses traditional porous sepharose columns [17] − for preparation of monomeric form of SHLA (Q sepharose) as well as for isolation of mono-pegylated form of SHLA (SP sepharose).

We describe here a successful method development − screening of monolithic ion exchangers for separation of mono-pegylated proteins. The method was developed on pegylated cytochrome C (12 kDa) and beta lactoglobulin (18.4 kDa) protein using CIM monolithic ion exchange columns. Methods for separation of monomeric SHLA and mono-pegylated SMLA pharmaceuticals were optimized using fast screening protocol with CIM monoliths. The screening protocol is the necessary step to find optimal separation condition. Developed screening on CIM disk is rapid compared to particle based columns. This means fast method development and later fast separation of target molecule. The columns can also discriminate mono-pegylated isoforms of the protein, so they can potentially be used to separate active from inactive mono-pegylated form [19].

Section snippets

Protein pegylation

Proteins were pegylated with four different reagents. All proteins Cytochrome C (12 kDa), soybean trypsin inhibitor (20.1 kDa); myoglobin (16.7 kDa); beta lactoglobulin (18.4 kDa) were bought from Sigma.

Pegylation reagents were from Thermo Scientific (Waltham, MA) (MSPEG24 (1.2 kDa, NHS ester reactive group) and MMPEG24 (1.2 kDa, maleimide reactive group)), and NOF America Corporation (White Plains, New York) Sunbright ME120MA (12 kDa, maleimide reactive group) and Sunbright ME100TS (12 kDa, NHS

Development of screening method for separation of pegylated proteins

Four proteins (cytochrome C, myoglobin, beta-lactoglobulin and trypsin inhibitor) were pegylated by different reagents from different manufacturers/suppliers: Sunbright ME120MA, ME100TS PEG (24 h reaction); Thermo scientific MSPEG24, MMPEG24 (30 min at room temperature) and PEG reagent with molecular weight of 4.48 kDa that was not commercially acquired. Maleimide reactive group reacts with cysteine and NHS group reacts with primary amines that are accessible on the surface of the protein. Thermo

Discussion

Separation of pegylated forms of proteins is highly dependent on the protein structure, especially its properties like total charge, surface charge, pI and binding sites of pegylation reagent. Electrostatic charge of pegylated proteins differs from non-pegylated proteins, therefore they elute at different salt concentration [2].

Pretesting of different PEG reagents and reaction time is necessary to achieve high yields of mono-pegylated protein. It’s necessary to choose high molecular weight

Conclusions

A fast screening protocol for isolation of mono-pegylated protein of choice was developed on CIM monolithic columns. Each protein has its own characteristics that contribute to specific optimization parameters like pH value and ligand on ion exchanger. It was observed that under optimal conditions ion exchangers separate pegylated proteins with ease from its non-pegylated counterparts. If fractions containing mono-pegylated protein are slightly contaminated with multi-pegylated forms,

Acknowledgements

ME120MA, ME100TS and PEG 4.48 kDa were kind gift of Laboratory for molecular biology and nanobiotechnology, National institute of Chemistry. SMLA pegylated mixture and SHLA inclusion bodies were kind gift from Protein Laboratories Rehovot Ltd, 11 Hagefen Str. Rehovot, 76349 Israel. Miloš Barut and Matjaž Peterka from BIA Separations are acknowledged for their help on the project. Urh Černigoj is acknowledged for proof reading of the manuscript. This work was partially financially supported by

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