Overproduction of recombinant human hepatocyte growth factor in Chinese hamster ovary cells

Abstract

Hepatocyte growth factor (HGF) is a potent multi-functional protein that affects morphogenesis, cell migration, organ regeneration, and tumor invasion in various tissues, and has thus been considered to have potential as a therapeutic target in various diseases. In our current study, we established Chinese hamster ovary (CHO) cells overexpressing recombinant human HGF (rhHGF) protein and in a 5 day batch culture process using a 7.5 l bioreactor (5 l working volume) and serum-free medium these cells could produce over 13 mg/l of rhHGF protein. The recombinant protein was then purified to homogeneity from the culture supernatant using a two-step chromatographic procedure that resulted in about a 35% recovery rate. This purified rhHGF was found to be a mixture of inactive pro-HGF and an active heterodimeric form of this protein with a higher molecular weight than its counterpart expressed from insect cells. This finding suggests that the glycosylation of rhHGF protein in CHO cells differs from that in insect cells. Inactive pro-HGF was found to rapidly convert to the active heterodimeric form of HGF in the presence of FBS (Fetal Bovine Serum), suggesting that this process would occur also when injected into human body. We further demonstrate in cell proliferation and scattering activity assays that our purified rhHGF protein preparation is functionally active with a half-maximal effective concentration of 36.3 ng/ml.

Introduction

Hepatocyte growth factor (HGF)² was first identified as a serum growth factor that promotes liver regeneration [1], [2]. Human HGF is synthesized and secreted as a single chain inactive precursor protein, pro-HGF, and is cleaved by serine proteases into an active and mature heterodimer composed of a 69 kDa α-subunit and a 34 kDa β-subunit linked by a disulfide bond [2]. HGF is now recognized as a potent multi-functional factor that affects morphogenesis, cell migration, organ regeneration, and tumor invasion in various tissues including liver, lung, kidney, intestine, and cardiovascular system [3], [4]. HGF has also been postulated to have potential as a target for therapeutic angiogenesis based on its mitogenic and angiogenic activities [5], [6]. In addition, anti-HGF treatments have long been studied as possible cancer therapies [7]. Recent studies have also further revealed the therapeutic potential of anti-HGF agents in various diseases such as hepatic fibrosis/cirrhosis [8], renal fibrosis [9], lung fibrosis [10], arteriosclerosis obliterans [11], gastric ulcer [12], diabetes [13], osteoarthritic cartilage [14], and asthma [15]. Hence, the development of an industrially applicable manufacturing process for recombinant human HGF (rhHGF) will be essential for the development of future medical applications involving this protein.

Active HGF in vivo is a heterodimeric glycoprotein with one O-linked and two N-linked glycosylations on the α-subunit and two N-linked glycosylations on the β-subunit [16], [17]. There have been several earlier attempts to express biologically active rhHGF in both Escherichia coli and insect cell expression systems [18], [19]. However, although unglycosylated rhHGF produced from E. coli has been reported to be functionally equivalent to native form in biological activities [18], it still differs from the natural forms of HGF present in the human body. Furthermore, production of rhHGF in E. coli requires a refolding process because it is expressed in inclusion bodies [18]. Insect cells are not suitable hosts for expressing human glycoproteins because the glycosylation in insects differ markedly from those in human [20].

Although the correct glycosylation of rhHGF may not be critical for its functional activities [21], it is still desirable to use recombinant proteins with an equivalent structure to their native form in therapeutic applications. There have been attempts to express rhHGF in mammalian cells including COS-1 and rat hepatocytes, but industrially applicable expression levels of this protein have not so far been obtained [2], [22]. Chinese hamster ovary (CHO) cells have been the most widely used host system for recombinant protein production, not only because they are mammalian cells and thus provide appropriate post-translational modifications of the exogenous proteins, but also because they are a suitable cell system for industrial-scale manufacturing processes. For these reasons, we established a CHO cell line overexpressing biologically active rhHGF to be used for large-scale production.