Stability and Biological Activity of E. coli Derived Soluble and Precipitated Bone Morphogenetic Protein-2

Abstract

Purpose: There is a plethora of studies on recombinant human bone morphogenetic protein-2 (rhBMP-2) application and delivery systems, but surprisingly few reports address the biophysical properties of the protein which are of crucial importance to develop effective delivery systems or to solve general problems related to rhBMP-2 production, purification, analysis and application. Methods:The solubility, stability and bioactivity of rhBMP-2 obtained by renaturation of E. coli derived inclusion bodies was assessed at different pH and in different buffer systems using (dynamic) light scattering and thermal shift assays as well as intrinsic fluorescence measurements and luciferase based bioassays.Results: rhBMP-2 is poorly soluble at physiological pH and higher. The presence of divalent anions further decreases the solubility even under acidic conditions. Thermal stability analyses revealed that rhBMP-2 precipitates are more stable compared to the soluble protein. Moreover, correctly folded rhBMP-2 is also bioactive as precipitated protein and precipitates readily dissolve under appropriate buffer conditions. Once properly formed rhBMP-2 also retains biological activity after temporary exposure to high concentrations of chaotropic denaturants. However, care should be taken to discriminate bioactive rhBMP-2 precipitates from misfolded rhBMP-2 aggregates, e.g. resolvability in MES buffer (pH 5) and a discrete peak in thermoshift experiments are mandatory for correctly folded rhBMP-2.Conclusions: Our analysis revealed that E. coli derived rhBMP-2 precipitates are not only bioactive but are also more stable compared to the soluble dimeric molecules. Knowledge about these unusual properties will be helpful to design improved delivery systems requiring lower amounts of rhBMP-2 in clinical applications.

Varying the sustained release of BMP‐2 from chitosan nanogel‐functionalized polycaprolactone fiber mats by different polycaprolactone surface modifications

“…Some authors have reported differing BMP‐2 release rates in PBS compared to cell culture medium or serum (Ruhé et al, 2006; Strobel, Bormann, Kadow‐Romacker, Schmidmaier, & Wildemann, 2011). BMP‐2 has an isoelectric point of 8.5 (Uludag, D'Augusta, Palmer, Timony, & Wozney, 1999) and its solubility is extraordinarily dependent on ionic strength and pH (Quaas et al, 2019). Given the extremely poor solubility of BMP‐2 in PBS‐based buffers, there is a risk of release results being influenced by solubility‐ and aggregation‐dependent recovery effects.…”

“…Preparation, refolding, and purification of dimeric non‐glycosylated rhBMP‐2 was performed by refolding of rhBMP‐2 from inclusion bodies (IBs) produced in Escherchia coli and subsequent purification as previously described (Quaas et al, 2019).…”

“…For the quantification of BMP‐2 activity in loading solutions (cf. 3.5), the assay was performed in a 96 well‐plate format as described in (Quaas et al, 2019) and for the quantification of BMP‐2 activity after release (cf. 3.4) in a 24 well‐plate format as described in (Quaas et al, 2018).…”

ELISA- and Activity Assay-Based Quantification of BMP-2 Released In Vitro Can Be Biased by Solubility in “Physiological” Buffers and an Interfering Effect of Chitosan

“…2-(N-morpholino) ethanesulfonic acid (MES; ≥ 99%) was purchased from Carl Roth, Karlsruhe, Germany. E. coli derived recombinant human bone morphogenetic protein 2 (rhBMP-2), simply referred to as BMP-2 in the following, was produced at the Institute for Technical Chemistry, Leibniz Universität Hannover, Hannover, Germany, as previously described [22].…”

“…BMP-2 has an isoelectric point (pI) of about 8.5 after chaotropic denaturation [21] and the tendency to form redissolvable aggregate particles of native protein with a pI of 7.7-8.3 at physiologic ionic strength and pH [22,23]. We were interested in the potential influence of poor BMP-2 solubility in physiological buffers like PBS [24] on the outcome of in vitro release tests.…”