Evaluation of chemical degradation of a trivalent recombinant protein vaccine against botulinum neurotoxin by LysC peptide mapping and MALDI-TOF mass spectrometry.
Vaccines utilizing recombinant protein antigens typically require an adjuvant to enhance immune response in the recipients. However, the consequences of antigen binding to adjuvant on both the short- and long-term stability of the protein remain poorly defined. In our companion paper (Vessely et al., in press, J Pharm Sci), we characterized the effects of binding to adjuvant on the conformation and thermodynamic stability of three antigen variants for botulinum vaccines: rBoNTA(H(c)), rBoNTB(H(c)), and rBoNTE(H(c)). In the current study, we evaluated the effect of binding to adjuvant (Alhydrogel, aluminum hydroxide) on chemical stability of these antigens during long-term storage in aqueous suspension. We developed methods that employ LysC peptide mapping in conjunction with MALDI-TOF mass spectrometry. Peptide maps were developed for the proteins for a vaccine formulation of rBoNTE(H(c)) as well as a trivalent rBoNT(H(c)) vaccine formulation. This method provided high sequence coverage for the proteins in part due to the implementation of a postdigestion elution fractionation method during sample preparation, and was also successfully utilized to evaluate the chemical integrity of adjuvant-bound rBoNT(H(c)) protein antigens. We found that all three of the rBoNT(H(c)) proteins were susceptible to degradation via both oxidation and deamidation. In many cases, such reactions occurred earlier with the adjuvant-bound protein formulations when compared to the proteins in control samples that were not bound to adjuvant. Additionally, some chemical modifications were found in the adjuvant-bound protein formulations but were not detected in the unbound solution controls. Our studies indicate that binding to aluminum-based adjuvants can impact the chemical stability and/or the chemical degradation pathways of protein during long-term storage in aqueous suspension. Furthermore, the methods we developed should be widely useful for assessing chemical stability of adjuvant-bound recombinant protein antigens.
Estey
Tia
T
Department of Pharmaceutical Sciences, School of Pharmacy, University of Colorado Health Sciences Center, SOP-215, Campus Box C238, Denver, Colorado 80262, USA.
Vessely
Christina
C
Randolph
Theodore W
TW
Henderson
Ian
I
Braun
Latoya Jones
LJ
Nayar
Rajiv
R
Carpenter
John F
JF
D000276:Adjuvants, Immunologic / Q000378:metabolism*; D000595:Amino Acid Sequence; D001428:Bacterial Vaccines / Q000032:analysis / Q000276:immunology / Q000378:metabolism*; D001905:Botulinum Toxins / Q000276:immunology*; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D011485:Protein Binding; D055550:Protein Stability; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D014614:Vaccines, Synthetic / Q000032:analysis / Q000276:immunology / Q000378:metabolism*
D000276:Adjuvants, Immunologic; D001428:Bacterial Vaccines; D014614:Vaccines, Synthetic; D001905:Botulinum Toxins
Vaccine. 2004 Mar 12;22(9-10):1172-6
15003645
Protein Sci. 2002 Feb;11(2):342-9
11790844
Biotechniques. 2006 Jun;40(6):790-8
16774123
Pharm Res. 1989 Nov;6(11):903-18
2687836
J Colloid Interface Sci. 1998 Jan 1;197(1):48-56
9466843
Pharm Biotechnol. 1995;6:229-48
7551219
Vaccine. 1999 Mar 17;17(11-12):1359-68
10195771
Nat Biotechnol. 1999 Nov;17(11):1075-81
10545912
J Biol Chem. 2005 Apr 8;280(14):13406-14
15684430
Vaccine. 2004 Sep 9;22(27-28):3658-68
15315845
Biotechnol Annu Rev. 2002;8:55-84
12436915
J Pharm Sci. 2007 Sep;96(9):2375-89
17518359
Pharm Biotechnol. 2002;14:1-46
12189723
Free Radic Biol Med. 2006 Nov 15;41(10):1507-20
17045919
J Biol Chem. 2006 Oct 13;281(41):30782-93
16891314
Immunol Invest. 1997 Jun;26(4):491-504
9246568
Vaccine. 2003 Jun 20;21(21-22):3011-8
12798645
Crit Rev Ther Drug Carrier Syst. 1993;10(4):307-77
8124728
Infect Immun. 1991 Sep;59(9):2978-86
1879922
Protein Sci. 2000 Nov;9(11):2260-8
11152137
Appl Environ Microbiol. 1997 Apr;63(4):1214-8
9097417
Biochem Biophys Res Commun. 2003 Jun 6;305(3):761-70
12763058
Pharm Biotechnol. 2002;13:1-25
11987748
Annu Rev Pharmacol Toxicol. 2004;44:167-93
14744243
Biochimie. 2000 Sep-Oct;82(9-10):955-66
11086225
Proc Natl Acad Sci U S A. 2007 Jan 2;104(1):359-64
17185412
Biomol Eng. 2001 Oct 15;18(3):69-85
11566599
Biochemistry. 2007 May 29;46(21):6213-24
17480058
Biochim Biophys Acta. 2005 Jan 17;1703(2):135-40
15680221
Vaccine. 2005 Jul 1;23(31):4029-35
15963360