Purification of the human fibroblast growth factor 2 using novel animal-component free materials

Highlights

• Avoiding animal-based materials in protein purification.

• Replacement of animal-based ligand heparin in affinity chromatography.

• Alternative ligands can be mixed-mode and pseudo-biospecific ligands.

• Mixed-mode chromatography as an alternative to heparin affinity chromatography.

Abstract

This paper analyzes the use of animal-component free chromatographic materials for the efficient purification of the human fibroblast growth factor 2 (hFGF-2). hFGF-2 is produced in Escherichia coli and purified via three different chromatography steps, which include a strong cation exchange chromatography as a capture step, followed by heparin affinity chromatography and an anion exchange chromatography as a polishing step. The affinity chromatography step is based on the animal-derived material heparin. Chemically produced ligands provide a viable alternative to animal-derived components in production processes, since they are characterized by a defined structure which leads to reproducible results and a broad range of applications. The alternative ligands can be assigned to adsorber of the mixed-mode chromatography (MMC) and pseudo-affinity chromatography.

Eight different animal-component free materials used as adsorbers in MMC or pseudo-affinity chromatography were tested as a substitute for heparin. The MMCs were cation exchangers characterized with further functional residues. The ligands of the pseudo-affinity chromatography were heparin-like ligands which are based on heparin's molecular structure. The alternative methods were tested as a capture step and in combination with another chromatographic step in the purification procedure of hFGF-2. In each downstream step purity, recovery and yield were analysed and compared to the conventional downstream process.

Two types of MMC – the column Foresight^TM Nuvia^TM cPrime^TM from Bio-Rad Laboratories and the column HiTrap^TM Capto^TM MMC from GE Healthcare Life Sciences - can be regarded as effective animal-component free alternatives to the heparin - based adsorber.

Introduction

The glycosaminoglycan heparin is a highly sulphated polysaccharide which is important for various biological interactions [1], [2]. Heparin is known for its function as an inhibitor in the coagulation cascade to maintain blood flow in the vasculature system [3]. Based on this property, heparin has seen widespread clinical use [4], [5]. The biological activities of heparin are associated with its interaction with various proteins, which have led to its use in protein purification in biotechnological applications [6], [7]. Diverse biologically active heparin-binding proteins, such as the human fibroblast growth factor 1 (hFGF-1) [8], hFGF-2 [9], [10], the human bone morphogenic protein 2 (hBMP-2) [11], the serine protease thrombin [12], and the glycoprotein anti-thrombin III (ATIII) [13], can be successfully purified by heparin affinity chromatography in a very effective and simple process [7], [14], [15], [16]. However, heparin is an animal-derived material, which is most frequently obtained from bovine or porcine tissue. Animal-based components are ethically problematic and carry the risk of virus contaminations [14], requiring strict quality controls and the validation of effective good manufacturing practice (GMP) implementation. Adequate animal-free components are therefore of considerable interest.

There are two options to replace conventional heparin in affinity chromatography. One option is to use chemically synthesized heparin [17], [18], chemoenzymatically synthesized heparin [17], [19], or bioengineered recombinant heparin, e.g. from genetically modified Chinese Hamster Ovary (CHO) cells [20]. Such bioengineered heparins have been applied in pharmaceuticals and for structure analysis but are very expensive. To our knowledge, there have not been studies on the use of bioengineered heparin for purification methods to date.

The second option is the use of animal-component free chromatographic methods, applying MMC and pseudo-affinity chromatography as an alternative to heparin affinity chromatography. Ligands of MMCs are characterized by a multimodal functionality compared to traditional single-mode chromatographic ligands [21], [22]. These materials are characterized by the availability of different ligands in one material. Various functional groups can build up different types of interactions (ionic interactions, hydrogen bonds, Van-der-Waals interactions) which can lead to a high affinity to different proteins, enabling a broad range of applications [9], [23], [24], [25]. MMCs are salt-tolerant due to their hydrophobic functionalities. This behaviour shortens the purification process time through fewer desalting steps and thus reduces the purification costs. This is advantageous as each additional purification step is associated with protein loss and higher purification costs [22], [25].

The pseudo-affinity chromatography is related to the affinity chromatography but is based on pseudo-specific ligands which are chemically synthesized. In contrast to the ligands in the traditional affinity chromatography, the ligands of the pseudo-affinity chromatography are easier and cheaper to produce and are more robust to chemical or biochemical degradation [26].

Several groups have already tested animal-component free materials to purify heparin-binding proteins [25], [27], [28], [29]. They all have in common that the purified proteins are produced as inclusion bodies. These inclusion bodies have already a purity of 80 % which makes further purification steps easier.

In this study we produced the cytokine hFGF-2 in recombinant Escherichia coli (E. coli) in a soluble form. After cell disruption and centrifugation, the experiments were performed with the soluble cell fraction. Eight different animal-component free materials (different mixed-mode and pseudo-affinity chromatography materials) were tested in bead-based column chromatography or membrane adsorbers technology (Table S1).