Comprehensive Biochemical and Biophysical Characterization of

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A Comprehensive Biochemical and Biophysical Characterization of Hemoglobin-based Oxygen Carrier (HBOC) Therapeutics: All HBOCs Are Not Created Equally Fantao Meng, Tigist Kassa, Sirsendu Jana, Francine Wood, Xiaoyuan Zhang, Yiping Jia, Felice D'Agnillo, and Abdu I. Alayash Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00093 • Publication Date (Web): 23 Mar 2018 Downloaded from http://pubs.acs.org on March 28, 2018

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Bioconjugate Chemistry

A Comprehensive Biochemical and Biophysical Characterization of Hemoglobin-based Oxygen Carrier (HBOC) Therapeutics: All HBOCs Are Not Created Equally

Fantao Meng*, Tigist Kassa*, Sirsendu Jana, Francine Wood, Xiaoyuan Zhang, Yiping Jia, Felice D’Agnillo, and Abdu I. Alayash#

Laboratory of Biochemistry and Vascular Biology, Division of Blood Components and Devices, Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, MD 20993, USA

Key words: Hemoglobin Therapeutics, Oxidation Reactions, Toxicity

Running Title: Characterization of HBOCs

# Corresponding author: Abdu I. Alayash, PhD, DSc Laboratory of Biochemistry and Vascular Biology Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, Maryland 20993, United States E-mail: [email protected] *Equal contributions

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ABSTRACT The development of hemoglobin (Hb)-based oxygen carriers (HBOCs) has been hampered because of safety concerns in humans. Chemical and/or genetic modifications of the Hb introduce varied structural and conformational constraint on the molecule that resulted in proteins with diverse allosteric responses, nitrosative and oxidative side reactions. Here, we present for the first time a comprehensive biochemical and biophysical comparison of human, bovine and genetically engineered HBOCs that have been tested in humans. We evaluate oxygen equilibrium and ligand binding kinetics under different experimental conditions as well as their autoxidation kinetics, redox reactions, and heme release. We determined the effects of HBOCs on cellular redox states and mitochondrial respiration. Taken together, these experiments provide a better understanding of the relationship between the structure-function and oxidative reactivity of these proteins. One can therefore select independently among these diverse properties to engineer a safe and effective HBOC with improved biochemical/biophysical characteristics.


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INTRODUCTION Blood transfusion is a lifesaving intervention but despite the overall safety of donated blood in the United States, allogenic blood transfusion still carries some risks of exposure to blood borne pathogens (e.g., viral hepatitis and HIV). Moreover, numerous biochemical and metabolic changes occur within the red blood cells (RBCs) during storage, prior to and after transfusion including perturbations of RBC membranes that may adversely affect the safety of transfused blood. Subsequent hemolysis due to storage conditions triggers pathophysiological consequences collectively known as the “storage lesions”.1 In addition, hospitals and blood banks often experience shortages of donated blood and it may be especially challenging to fully meet the need for blood in a mass casualty situation or in combat casualty care.2 This and many other factors led researchers and developers to explore alternatives to blood donation. The concept of using acellular Hb (nature best engineered oxygen transporter) as a pharmacologic agent to deliver oxygen to tissues was developed in the late 70s and the reengineering of the Hb molecule to act as a temporary oxygen bridge was therefore required. Early designs of modified Hbs relied primarily on non-site specific modifications of the proteins i.e. polymerization and/or on intramolecular cross-linking reactions to increase the size, decrease oxygen affinity of Hb and to minimize rapid clearance by the kidney.3 Hb is isolated from outdated human blood or from bovine blood and a stroma free (SFH) or stroma “poor” Hb is then subjected to further purification and filtration and used as a starting material. In some cases chemical modifications are introduced while Hb still partially purified, retaining in some cases RBC proteins/enzymes that often are crosslinked to the Hb protein.4-7 One of the first Hb-based oxygen carriers (HBOCs) (initially referred to as blood substitutes) was diaspirin-crossed linked Hb (DCLHb) (HemAssist) also commonly known as DBBF-Hb developed independently by Baxter and the US Army (see Table 1). The reagent bis(3,5dibromosalicyl) fumarate was used to crosslink deoxy SFH between the two alpha subunits (Lys99 α1 and Lys99 α2).6,8 DCLHb was extensively tested in various preclinical and clinical studies.9-11 However, in Phase III studies; patients treated with DCLHb had significantly higher mortality rates than the control groups.12 Using recombinant technology, another manufacturer, Somatogen engineered a similarly crosslinked tetramer. The Hb was constructed in E. Coli expressing the low oxygen affinity mutant, Hb Presbyterian (βN108K) and incorporating the amino acid glycine to bridge the two α subunits (di-α-gly-α] (Optro).13,14 Some Phase I/II clinical trials in elective surgeries with Optro (5 g Hb/dL) were conducted but discontinued due to the hypertensive effects and other related adverse events.15 Unlike Optro, extensive biochemical analysis and animal studies were carried out on the chemically modified tetramers by several independent laboratories due to the availability of Army’s version of DCLHb, i.e. DBBF to several research groups.9 Other chemical strategies included the conjugation or “decoration” of human or bovine tetrameric Hbs by non-protein entities such as polyethylene glycol (PEG) or polyoxyethylene (POE) to increase their retention time in circulation and to maintain low oxygen affinity capabilities. Site-specific thiolation of amino groups on the protein surface with iminothiolane, followed by the reaction of the protein with PEG reagent was employed to produce MP4 or 3 ACS Paragon Plus Environment

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Hemospan by Sangart Inc.5,16 Unlike other HBOCs this Hb had a very high oxygen affinity (P50 = 4.0 mmHg) and was introduced specifically to counter the autoregulatory responses (due to the premature unloading of oxygen) associated with first generation HBOCs. MP4 was indicated for use in elective surgery and went through early clinical trials in orthopedic patients in Sweden.17 Sangart failed to secure new funding and had to terminate development operations.18 Table 1. Biochemical and molecular characteristics of HBOCsa Productsb HemAssist/ DCLHb (αXLHb)

Company

Chemical Modifier(s) O Br

Baxter Br

H C

C C H

O C

O

Br

O

C HO

OH

64

30

500: Tyr apomyoglobin as a reagent for measuring rates of hemin dissociation. Journal of Biological Chemistry 269, 4207-4214. (73) Kassa, T., Jana, S., Meng, F., and Alayash, A. I. (2016) Differential heme release from various hemoglobin redox states and the upregulation of cellular heme oxygenase-1. FEBS Open Bio 6, 876-884. (74) Hayashi, A., Suzuki, T., and Shin, M. (1973) An enzymic reduction system for metmyoglobin and methemoglobin, and its application to functional studies of oxygen carriers. Biochimica et Biophysica Acta (BBA) - Protein Structure 310, 309-316.


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(75) Jia, Y., and Alayash, A. I. (2009) Effects of cross-linking and zero-link polymerization on oxygen transport and redox chemistry of bovine hemoglobin. Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics 1794, 1234-1242. (76) Elmer, J., Buehler, P. W., Jia, Y., Wood, F., Harris, D. R., Alayash, A. I., and Palmer, A. F. (2010) Functional comparison of hemoglobin purified by different methods and their biophysical implications. Biotechnology and Bioengineering 106, 76-85. (77) Meng, F., Manjula, B. N., Tsai, A. G., Cabrales, P., Intaglietta, M., Smith, P. K., Prabhakaran, M., and Acharya, S. A. (2009) Hexa-thiocarbamoyl Phenyl PEG5K Hb: Vasoactivity and structure. The Protein Journal 28, 199-212. (78) Grassetti, M. J., Murray, J. F. (1967) Determination of sulfhydryl groups with 2,2'- or 4,4'dithiodipyridine. Arch Biochem Biophys 119, 41-49. (79) Eich, R. F., Li, T., Lemon, D. D., Doherty, D. H., Curry, S. R., Aitken, J. F., Mathews, A. J., Johnson, K. A., Smith, R. D., Phillips, G. N., and Olson, J. S. (1996) Mechanism of NO-induced oxidation of myoglobin and hemoglobin. Biochemistry 35, 6976-6983.



TABLE OF CONTENTS GRAPHIC

100

% Oxygen Saturation

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

50

HbA O-R-PolyHb0 PolyHHb αXLHb PEGHHb AARBC

0

0 40 80 120 Partial Pressure of O2 (mmHg)


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Comprehensive Biochemical and Biophysical Characterization of

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