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.
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Abbreviations
- BRE-Luc:
-
BMP responsive element luciferase
- CHO:
-
Chinese hamster ovary
- DLS:
-
Dynamic light scattering
- E. coli :
-
Escherichia coli
- Gdn-HCl:
-
Guanidine hydrochloride
- nRFU:
-
normalized relative fluorescence units
- nRLU:
-
normalized relative light units
- pI:
-
Isoelectric point
- rhBMP-2:
-
Recombinant human bone morphogenetic protein 2
- RLU:
-
Relative light units
- TGF-β:
-
Transforming growth factor β
- Tm:
-
Denaturation (melting) temperature
References
Celeste AJ, Iannazzi JA, Taylor RC, Hewick RM, Rosen V, Wang EA, et al. Identification of transforming growth factor beta family members present in bone-inductive protein purified from bovine bone. Proc Natl Acad Sci USA. 1990;87:9843–7.
Scheufler C, Sebald W, Hülsmeyer M. Crystal structure of human bone morphogenetic protein-2 at 2.7 Å resolution. J Mol Biol. 1999;287:103–15.
Uludag H, D'Augusta D, Golden J, Li J, Timony G, Riedel R, et al. Implantation of recombinant human bone morphogenetic proteins with biomaterial carriers: a correlation between protein pharmacokinetics and osteoinduction in the rat ectopic model. J Biomed Mater Res. 2000;50:227–38.
McKay WF, Peckham SM, Badura JM. A comprehensive clinical review of recombinant human bone morphogenetic protein-2 (INFUSE bone graft). Int Orthop. 2007;31:729–34.
Granjeiro JM, Oliveira RC, Bustos-Valenzuela JC, Sogayar MC, Taga R. Bone morphogenetic proteins: from structure to clinical use. Braz J Med Biol Res. 2005;38:1463–73.
El Bialy I, Jiskoot W, Reza NM. Formulation, delivery and stability of bone morphogenetic proteins for effective bone regeneration. Pharm Res. 2017;34:1152–70.
Wozney JM, Rosen V, Celeste AJ, Mitsock LM, Whitters MJ, Kriz RW, et al. Novel regulators of bone formation: molecular clones and activities. Science. 1988;242:1528–34.
Walsh G. Biopharmaceutical benchmarks 2010. Nat Biotechnol. 2010;28:917–24.
Vallejo LF, Rinas U. Optimized procedure for renaturation of recombinant human bone morphogenetic protein-2 at high protein concentration. Biotechnol Bioeng. 2004;85:601–9.
Quaas B, Burmeister L, Li Z, Nimtz M, Hoffmann A, Rinas U. Properties of dimeric, disulfide-linked rhBMP-2 recovered from E. coli derived inclusion bodies by mild extraction or chaotropic solubilisation and subsequent refolding. Process Biochem. 2018;67:80–7.
van de Watering FC, van den Beucken JJ, van der Woning SP, Briest A, Eek A, Qureshi H, et al. Non-glycosylated BMP-2 can induce ectopic bone formation at lower concentrations compared to glycosylated BMP-2. J Control Release. 2012;159:69–77.
Nakashima M, Reddi AH. The application of bone morphogenetic proteins to dental tissue engineering. Nat Biotechnol. 2003;21:1025–32.
Katagiri T, Yamaguchi A, Ikeda T, Yoshiki S, Wozney JM, Rosen V, et al. The non-osteogenic mouse pluripotent cell line, C3H10T1/2, is induced to differentiate into osteoblastic cells by recombinant human bone morphogenetic protein-2. Biochem Biophys Res Commun. 1990;172:295–9.
Kisiel M, Ventura M, Oommen OP, George A, Walboomers XF, Hilborn J, et al. Critical assessment of rhBMP-2 mediated bone induction: an in vitro and in vivo evaluation. J Control Release. 2012;162:646–53.
Schwartz D, Sofia S, Friess W. Integrity and stability studies of precipitated rhBMP-2 microparticles with a focus on ATR-FTIR measurements. Eur J Pharm Biopharm. 2006;63:241–8.
Sofia SJ, Schwartz D, Friess W. Formulation comprising bioactive agents and method of using them. US Patent 7,897,174 B2. 2011.
Sampath TK, Reddi AH. Dissociative extraction and reconstitution of extracellular matrix components involved in local bone differentiation. Proc Natl Acad Sci USA. 1981;78:7599–603.
Luca L, Capelle MA, Machaidze G, Arvinte T, Jordan O, Gurny R. Physical instability, aggregation and conformational changes of recombinant human bone morphogenetic protein-2 (rhBMP-2). Int J Pharm. 2010;391:48–54.
Abbatiello SE, Porter TJ. Anion-mediated precipitation of recombinant human bone morphogenetic protein (rhBMP-2) is dependent upon the heparin binding N-terminal region. Poster presented at the Protein Society Meeting, Boston MA, July 13–16. 1997.
Gilde F, Maniti O, Guillot R, Mano JF, Logeart-Avramoglou D, Sailhan F, et al. Secondary structure of rhBMP-2 in a protective biopolymeric carrier material. Biomacromolecules. 2012;13:3620–6.
Yano K, Hoshino M, Ohta Y, Manaka T, Naka Y, Imai Y, et al. Osteoinductive capacity and heat stability of recombinant human bone morphogenetic protein-2 produced by Escherichia coli and dimerized by biochemical processing. J Bone Miner Metab. 2009;27:355–63.
Ruppert R, Hoffmann E, Sebald W. Human bone morphogenetic protein 2 contains a heparin-binding site which modifies its biological activity. Eur J Biochem. 1996;237:295–302.
Vallejo LF, Brokelmann M, Marten S, Trappe S, Cabrera-Crespo J, Hoffmann A, et al. Renaturation and purification of bone morphogenetic protein-2 produced as inclusion bodies in high-cell-density cultures of recombinant Escherichia coli. J Biotechnol. 2002;94:185–94.
Georgiou CD, Grintzalis K, Zervoudakis G, Papapostolou I. Mechanism of Coomassie brilliant blue G-250 binding to proteins: a hydrophobic assay for nanogram quantities of proteins. Anal Bioanal Chem. 2008;391:391–403.
Demeule B, Gurny R, Arvinte T. Detection and characterization of protein aggregates by fluorescence microscopy. Int J Pharm. 2007;329:37–45.
Schindelin J, Rueden CT, Hiner MC, Eliceiri KW. The ImageJ ecosystem: an open platform for biomedical image analysis. Mol Reprod Dev. 2015;82:518–29.
Boivin S, Kozak S, Meijers R. Optimization of protein purification and characterization using Thermofluor screens. Protein Expr Purif. 2013;91:192–206.
Lorenz C, Hoffmann A, Gross G, Windhagen H, Dellinger P, Mohwald K, et al. Coating of titanium implant materials with thin polymeric films for binding the signaling protein BMP2. Macromol Biosci. 2011;11:234–44.
Korchynskyi O, ten Dijke P. Identification and functional characterization of distinct critically important bone morphogenetic protein-specific response elements in the Id1 promoter. J Biol Chem. 2002;277:4883–91.
Martinez-Rosell G, Giorgino T, De FG. PlayMolecule ProteinPrepare: a web application for protein preparation for molecular dynamics simulations. J Chem Inf Model. 2017;57:1511–6.
Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, et al. UCSF chimera - a visualization system for exploratory research and analysis. J Comput Chem. 2004;25:1605–12.
Bolten SN, Rinas U, Scheper T. Heparin: role in protein purification and substitution with animal-component free material. Appl Microbiol Biotechnol. 2018;102:8647–60.
Zhou C, Qi W, Lewis EN, Carpenter JF. Characterization of sizes of aggregates of insulin analogs and the conformations of the constituent protein molecules: a concomitant dynamic light scattering and raman spectroscopy study. J Pharm Sci. 2016;105:551–8.
Urist MR, Huo YK, Brownell AG, Hohl WM, Buyske J, Lietze A, et al. Purification of bovine bone morphogenetic protein by hydroxyapatite chromatography. Proc Natl Acad Sci USA. 1984;81:371–5.
Rao MT, Bhuyan AK, Venu K, Sastry VS. Nonlinear effect of GdnHCl on hydration dynamics of proteins: a 1H magnetic relaxation dispersion study. J Phys Chem B. 2009;113:6994–7002.
Kumar R, Bhuyan AK. Entropic stabilization of myoglobin by subdenaturing concentrations of guanidine hydrochloride. J Biol Inorg Chem. 2009;14:11–21.
Sole M, Brandt W, Arnold U. Striking stabilization of Rana catesbeiana ribonuclease 3 by guanidine hydrochloride. FEBS Lett. 2013;587:737–42.
Mayr LM, Schmid FX. Stabilization of a protein by guanidinium chloride. Biochemistry. 1993;32:7994–8.
Vallejo LF, Rinas U. Folding and dimerization kinetics of bone morphogenetic protein-2, a member of the transforming growth factor-β family. FEBS J. 2013;280:83–92.
Roberts D, Keeling R, Tracka M, van der Walle CF, Uddin S, Warwicker J, et al. Specific ion and buffer effects on protein-protein interactions of a monoclonal antibody. Mol Pharm. 2015;12:179–93.
Roberts CJ. Therapeutic protein aggregation: mechanisms, design, and control. Trends Biotechnol. 2014;32:372–80.
Schmoekel H, Schense JC, Weber FE, Gratz KW, Gnagi D, Muller R, et al. Bone healing in the rat and dog with nonglycosylated BMP-2 demonstrating low solubility in fibrin matrices. J Orthop Res. 2004;22:376–81.
ACKNOWLEDGMENTS AND DISCLOSURES
The authors gratefully acknowledge funding through the Forschergruppe “Gradierte Implantate” FOR2180 and the Exzellenzcluster “Rebirth” EXC62, both Deutsche Forschungsgemeinschaft (DFG), and excellent technical assistance by Anika Hamm (bioactivity measurements), Graded Implants and Regenerative Strategies. We also want to thank the reviewers for their careful and critical reading which helped a lot to improve the manuscript.
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Quaas, B., Burmeister, L., Li, Z. et al. Stability and Biological Activity of E. coli Derived Soluble and Precipitated Bone Morphogenetic Protein-2. Pharm Res 36, 184 (2019). https://doi.org/10.1007/s11095-019-2705-5
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DOI: https://doi.org/10.1007/s11095-019-2705-5