Documente Academic
Documente Profesional
Documente Cultură
ABSTRACT: Oxidation of methionine and tryptophan are common degradation pathways for monoclonal antibodies and present major
analytical challenges in biotechnology. Generally, protein oxidation is detectable in stability and/or stressed samples (e.g., exposed to
hydrogen peroxide, UV light, or metal ions). The induced chemical modifications may impact the biological activity of antibodies and may
have biological consequences. However, these effects and the contribution of individual protein modifications are difficult to delineate
as different amino acids are often oxidized simultaneously and accompanied by other degradants such as aggregates, especially in
forced degradation studies. Here, we report a new method to obtain selective oxidation of methionine or tryptophan by using oxidation
reagents combined with large excess of free tryptophan or methionine, correspondingly. More specifically, using hydrogen peroxide or
tert-butyl hydroperoxide in combination with addition of free tryptophan allowed for selective oxidation of methionine. Conversely, the
use of 2,2-azobis(2-amidinopropane) dihydrochloride in combination with free methionine resulted in selective tryptophan oxidation,
whereas methionine oxidation was not significantly altered. This novel stress model system may prove to be valuable tool in future
mechanistic studies of oxidative degradation of protein therapeutics. C 2015 Wiley Periodicals, Inc. and the American Pharmacists
Table 2. Liquid chromatography - mass spectrometry (LC-MS) Results for the Oxidation with H2 O2 or t-BHP with or Without Addition of Free
Tryptophan
The mAb was denatured and the disulfide bridges were reduced using DTT. Alkylation of free cysteines was performed using iodoacetic acid. After a buffer
exchange, the mAb was finally digested with trypsin. LC stands for light chain, HC stands for heavy chain. Methionine (M) and tryptophan (W) oxidation levels were
quantified.
ters were evaluated based on published studies and previous 2% t-BHP for 120 h at 25°C allowed for the nearly complete
in-house experiments (data not shown).12,14,22 A model IgG1 oxidation of the methionine residues from the Fc region.
was incubated with different oxidants while the reaction times The extent of methionine oxidation was also determined by
and incubation temperatures were varied as described in Ta- selected ion current chromatogram analysis of the oxidized pep-
ble 2. To curtail oxidation of the amino acids methionine or tides (Table 1). We found more than 91% oxidation for M364,
tryptophan, the corresponding free amino (L-methionine or L- 96% oxidation for M434, and 99% oxidation for M258 according
tryptophan) acids were added to the reaction mixtures. After to LC–MS results.
completion of all reactions, samples were screened for total ox- In order to assess the total tryptophan oxidation of the HC-
idation levels using protein A analytical chromatography and Fab, the oxidized samples were first digested with papain and
RP-HPLC. Selected samples were further analyzed using LC– then the disulfide bridges were reduced with DTT. The gen-
MS and LC–MS/MS in order to identify and semiquantify the erated LC, HC-Fc, and HC-Fab were then separated by RP-
methionine and tryptophan oxidation sites in the amino-acid HPLC (for details see Materials and Methods) as presented in
sequence. Figure 3a, and the tryptophan oxidation of the HC-Fab was
In the model IgG1 used in this study, several methionine quantified. The oxidized species because of tryptophan oxida-
residues were identified as susceptible to oxidation (M4, M34, tion elute before the HC-Fab peaks. The peak with a retention
M83, M258, M364, and M434). Basal levels of oxidation of time of 19.5 min represents in that case mainly oxidized tryp-
these susceptible residues were identified in the starting ma- tophan W108 of the HC-Fab (Figs. 3b and 3c; signal labeled as
terial (Table 1). From the oxidized tryptophan identified by “Trp-ox”).
LC–MS/MS (W35, W50, W96, W108, W283, and W319), only As previously reported, the oxidation of mAb with H2 O2 or
surface-exposed W50 and W108 showed significant changes be- t-BHP induced relatively low tryptophan oxidation levels (see
tween unstressed and stressed samples. Figs. 3b and 3c).22 The well-resolved signal with a retention
time of 19.5 min (corresponding to tryptophan oxidation prod-
ucts) increased not only with increasing H2 O2 or t-BHP con-
Selective Methionine Oxidation centrations, but also when increasing the reaction time and
Oxidation levels of Fc methionine were first quantified by the temperature of incubation (data not shown). In order to
protein A chromatography. The nonoxidized antibody species exclude the possibility of metal ion contamination resulting in
eluted at 23 min (Fig. 2). The oxidized species formed upon in- Fenton-like reactions, protein oxidation reactions were carried
cubation with oxidant eluted earlier (before 19.8 min) because out in the presence and absence of EDTA. As no differences
of the lower affinity of oxidized Fc methionine to protein A.7,12 in the oxidation levels of Trp were detected (data not shown),
As depicted in Figures 2a and 2b, the incubation of the mAb the mechanism of Trp oxidation in these reactions remains un-
with 1% H2 O2 for 24 h at 5°C as well as the incubation with clear. To avoid this unwanted tryptophan oxidation, reaction
oxidation sites by subsequent LC–MS analyses. Upon incuba- still present in the sample containing free methionine during
tion at 40°C for 120 h with AAPH (0%–5%), a considerable incubation with 5% AAPH for 120 h at 40°C. The content of
tryptophan oxidation (between 12% and 95%) was observed, as soluble fragments remained below 0.3% for the control and the
seen in Supplementary Tables 2 and 3. stressed samples in all studies. The correlation between Met
As reported in previous studies, the reaction of proteins oxidation and protein aggregation observed in this study was
with alkyperoxyl radicals (generated by AAPH that reacts not unexpected, as such correlation has been reported previ-
with oxygen) also induced methionine oxidation.22,32 As seen in ously by others and studied in some detail.11,33 These studies
Figures 2c and 4a, oxidation with 5% AAPH lead to almost have demonstrated that Met oxidation in mAbs may result in
complete oxidation of Fc methionine measured by protein A changes of the secondary and tertiary structure and in turn
chromatography and LC–MS peptide mapping. significantly affect the physical stability of the protein result-
The addition of a large excess of free methionine into solu- ing in increased aggregation propensity. Thus, the decrease of
tion before oxidation at 40°C for 120 h allowed protection of Fc aggregation upon the addition of free Met can be attributed to
methionine as seen in the protein A chromatogram (Fig. 2c). As the oxidation protective effect of Met.
depicted in Supplementary Figures 1 and 2, the low levels or The experimental approach of using combinations of oxida-
the absence of peptide signal in the TIC plots and extracted ion tive reagents and antioxidants presented here provided an ef-
chromatograms for M258ox (RT = 12.65 min), M364ox (RT = ficient strategy for selective oxidation of Met and Trp residues
1.78 min), and M434ox (RT = 18.45 min) when adding free me- in therapeutic IgG molecules. Although residual low levels of
thionine into the mAb solution before oxidation with 5% AAPH undesirable oxidation were detected in some cases (which pre-
(at 40°C for 120 h) is a clear confirmation of the protective effect sumably would need to be accounted for in follow-up biological
of free methionine against methionine oxidation. studies), we believe that this approach will find broad applica-
The results from LC–MS analyses of AAPH-oxidized sam- tion in studying the biological effects of protein oxidation.
ples are presented in Tables 1 and 2 of the Supplementary
Material. These results clearly demonstrate that methionine
residues otherwise susceptible to oxidation by AAPH can be se- CONCLUSIONS
lectively protected by addition of free methionine, even in the
Free methionine and/or tryptophan are used as pharmaceuti-
harshest reaction conditions used in this study [longest incu-
cal antioxidants because of their protective action against ox-
bation time (120 h), highest oxidant concentration (5%), and
idation of amino acids in protein therapeutics. In the present
highest temperature of incubation (40°C)]. This specific sample
report, we explore the potential of these antioxidants to selec-
showed 10.3% oxidation for M258, whereas the same sample
tively generate methionine- or tryptophan-oxidized residues. A
without methionine addition showed oxidation of 99.7%. Simi-
combination of oxidative (H2 O2 , t-BHP, and AAPH) and protec-
larly, 6.3% versus 98.8% M434ox was verified by LC–MS. The
tive (free tryptophan or methionine) components will serve as a
oxidation of M364 was lower than 1% in the protected sample
strategy to address questions related to the impact of oxidation-
(99% oxidation in the sample without addition of free methio-
induced changes on the biological properties of protein thera-
nine). It has to be mentioned that 4.5% M258ox, 0.4% M364ox,
peutics (e.g., bioactivity, PK/PD, immunogenicity, etc.). Such
and 1.1% M434ox were found in the nonstressed reference ma-
mechanistic studies will be instrumental to better understand
terial (Fig. 4a).
and control oxidative degradation of antibodies.
Figures 4a–4c show a comparison of the methionine (M258,
M364, and M434) and tryptophan (W50 and W108) distribution
between AAPH-stressed samples with and without addition of
ACKNOWLEDGMENT
free methionine. Over and above the decrease of methionine
oxidation, the presence of free methionine in the mAb solution We thank Y. John Wang, Ulla Grauschopf, and Kishore Ravuri
for AAPH incubation slightly changed the oxidation profile of for insightful discussions and comments on the manuscript.
tryptophan residues as seen in Figures 4b and 4c. As already
observed in the RP-HPLC data, the total amount of trypto-
phan oxidation (as quantified by LC–MS) slightly decreased REFERENCES
but remained considerable when adding free methionine to the
1. Mohammadian-Mosaabadi J, Naderi-Manesh H, Maghsoudi N,
solution (Figs. 4 and 5). On the one hand, a higher percentage
Khalilzadeh R, Shojaosadati SA, Ebrahimi M. 2005. Effect of oxida-
of hydroxytryptophan (+16) for W50 and W108 was measured tive stress on the production of recombinant human interferon-gamma
in the samples containing free methionine; on the other hand, in Escherichia coli. Biotechnol Appl Biochem 41(Pt 1):37–42.
a lower percentage of N-formylkynurenine (+32) was found for 2. Krishnamurthy R, Madurawe RD, Bush KD, Lumpkin JA. 1995.
W50 and W108 when adding free methionine (Supplementary Conditions promoting metal-catalyzed oxidations during immobilized
Figs. 3 and 4). Presumably, this effect is related to shift in the cu-iminodiacetic acid metal affinity-chromatography. Biotechnol Progr
equilibrium between reaction intermediates caused by the ad- 11(6):643–650.
dition of the antioxidant. Further elucidation of this mechanism 3. Kerwin BA. 2008. Polysorbates 20 and 80 used in the formulation of
may be of interest as a topic of future studies. protein biotherapeutics: Structure and degradation pathways. J Pharm
Sci 97(8):2924–2935.
Significant formation of soluble aggregates was detected by
4. Takenawa T, Yokota A, Oda M, Takahashi H, Iwakura M. 2009.
size SEC correlating well with the increase in AAPH concentra-
Protein oxidation during long storage: Identification of the oxidation
tion. The addition of free methionine, however, had a protective sites in dihydrofolate reductase from Escherichia coli through LC-MS
effect against aggregation as seen in Figure 5b. This effect cor- and fragment studies. J Biochem 145(4):517–523.
relates well with overall reduction on oxidation. The monomer 5. Torosantucci R, Schoneich C, Jiskoot W. 2014. Oxidation of thera-
content represented 30% of the total area in the AAPH sample peutic proteins and peptides: Structural and biological consequences.
without methionine addition, whereas 65% of monomers were Pharm Res 31(3):541–553.
6. Khor HK, Jacoby ME, Squier TC, Chu GC, Chelius D. 2010. Iden- 18. Li S, Schoneich C, Borchardt RT. 1995. Chemical instability of pro-
tification of methionine sulfoxide diastereomers in immunoglobulin tein pharmaceuticals: Mechanisms of oxidation and strategies for sta-
gamma antibodies using methionine sulfoxide reductase enzymes. bilization. Biotechnol Bioeng 48(5):490–500.
MAbs 2(3):299–308. 19. Wu Y, Levons J, Narang AS, Raghavan K, Rao VM. 2011. Reac-
7. Gaza-Bulseco G, Faldu S, Hurkmans K, Chumsae C, Liu H. 2008. tive impurities in excipients: Profiling, identification and mitigation of
Effect of methionine oxidation of a recombinant monoclonal antibody drug–excipient incompatibility. AAPS PharmSciTech 12(4):1248–1263.
on the binding affinity to protein A and protein G. J Chromatogr B 20. Wang W. 1999. Instability, stabilization, and formulation
Analyt Technol Biomed Life Sci 870(1):55–62. of liquid protein pharmaceuticals. Int J Pharm 185(2):129–
8. Pan H, Chen K, Chu L, Kinderman F, Apostol I, Huang G. 188.
2009. Methionine oxidation in human IgG2 Fc decreases bind- 21. Mozziconacci O, Ji JA, Wang YJ, Schoneich C. 2013. Metal-
ing affinities to protein A and FcRn. Protein Sci 18(2):424– catalyzed oxidation of protein methionine residues in human parathy-
433. roid hormone (1-34): Formation of homocysteine and a novel
9. Bertolotti-Ciarlet A, Wang W, Lownes R, Pristatsky P, Fang Y, methionine-dependent hydrolysis reaction. Mol Pharm 10(2):739–755.
McKelvey T, Li Y, Drummond J, Prueksaritanont T, Vlasak J. 22. Ji JA, Zhang B, Cheng W, Wang YJ. 2009. Methionine, tryptophan,
2009. Impact of methionine oxidation on the binding of human IgG1 and histidine oxidation in a model protein, PTH: Mechanisms and sta-
to Fc Rn and Fc gamma receptors. Mol Immunol 46(8–9):1878– bilization. J Pharm Sci 98(12):4485–4500.
1882. 23. Lam XM, Yang JY, Cleland JL. 1997. Antioxidants for prevention
10. Wang W, Vlasak J, Li Y, Pristatsky P, Fang Y, Pittman T, Roman J, of methionine oxidation in recombinant monoclonal antibody HER2. J
Wang Y, Prueksaritanont T, Ionescu R. 2011. Impact of methionine ox- Pharm Sci 86(11):1250–1255.
idation in human IgG1 Fc on serum half-life of monoclonal antibodies. 24. Keck RG. 1996. The use of t-butyl hydroperoxide as a probe for
Mol Immunol 48(6-7):860–866. methionine oxidation in proteins. Anal Biochem 236(1):56–62.
11. Liu D, Ren D, Huang H, Dankberg J, Rosenfeld R, Cocco MJ, Li L, 25. Wasylaschuk WR, Harmon PA, Wagner G, Harman AB, Templeton
Brems DN, Remmele RL Jr. 2008. Structure and stability changes of AC, Xu H, Reed RA. 2007. Evaluation of hydroperoxides in common
human IgG1 Fc as a consequence of methionine oxidation. Biochemistry pharmaceutical excipients. J Pharm Sci 96(1):106–116.
47(18):5088–5100. 26. Luo Q, Joubert MK, Stevenson R, Ketchem RR, Narhi LO, Wypych
12. Loew C, Knoblich C, Fichtl J, Alt N, Diepold K, Bulau P, J. 2011. Chemical modifications in therapeutic protein aggregates gen-
Goldbach P, Adler M, Mahler HC, Grauschopf U. 2012. Analytical pro- erated under different stress conditions. J Biol Chem 286(28):25134–
tein a chromatography as a quantitative tool for the screening of me- 25144.
thionine oxidation in monoclonal antibodies. J Pharm Sci 101(11):4248– 27. Qi P, Volkin DB, Zhao H, Nedved ML, Hughes R, Bass R, Yi SC,
4257. Panek ME, Wang D, Dalmonte P, Bond MD. 2009. Characterization of
13. Schlothauer T, Rueger P, Stracke JO, Hertenberger H, Fin- the photodegradation of a human IgG1 monoclonal antibody formulated
gas F, Kling L, Emrich T, Drabner G, Seeber S, Auer J, Koch S, as a high-concentration liquid dosage form. J Pharm Sci 98(9):3117–
Papadimitriou A. 2013. Analytical FcRn affinity chromatography for 3130.
functional characterization of monoclonal antibodies. MAbs 5(4):576– 28. Liu H, Gaza-Bulseco G, Zhou L. 2009. Mass spectrometry analysis
586. of photo-induced methionine oxidation of a recombinant human mono-
14. Hensel M, Steurer R, Fichtl J, Elger C, Wedekind F, Petzold A, clonal antibody. J Am Soc Mass Spectrom 20(3):525–528.
Schlothauer T, Molhoj M, Reusch D, Bulau P. 2011. Identification 29. Rao PE, Kroon DJ. 1993. Orthoclone OKT3. Chemical mechanisms
of potential sites for tryptophan oxidation in recombinant antibod- and functional effects of degradation of a therapeutic monoclonal anti-
ies using tert-butylhydroperoxide and quantitative LC–MS. PloS One body. Pharm Biotechnol 5:135–158.
6(3):e17708. 30. Steinmann D, Ji JA, Wang YJ, Schoneich C. 2012. Oxidation of
15. Wei ZP, Feng JH, Lin HY, Mullapudi S, Bishop E, Tous GI, Casas- human growth hormone by oxygen-centered radicals: Formation of
Finet J, Hakki F, Strouse R, Schenerman MA. 2007. Identification of a Leu-101 hydroperoxide and Tyr-103 oxidation products. Mol Pharm
single tryptophan residue as critical for binding activity in a humanized 9(4):803–814.
monoclonal antibody against respiratory syncytial virus. Anal Chem 31. Grewal P, Mallaney M, Lau K, Sreedhara A. 2014. Screening
79(7):2797–2805. methods to identify indole derivatives that protect against reactive
16. Haberger M, Bomans K, Diepold K, Hook M, Gassner J, Schlothauer oxygen species induced tryptophan oxidation in proteins. Mol Pharm
T, Zwick A, Spick C, Kepert JF, Hienz B, Wiedmann M, Beck H, Metzger 11(4):1259–1272.
P, Molhoj M, Knoblich C, Grauschopf U, Reusch D, Bulau P. 2014. As- 32. Werber J, Wang YJ, Milligan M, Li X, Ji JA. 2011. Anal-
sessment of chemical modifications of sites in the CDRs of recombinant ysis of 2,2 -azobis (2-amidinopropane) dihydrochloride degradation
antibodies: Susceptibility vs. functionality of critical quality attributes. and hydrolysis in aqueous solutions. J Pharm Sci 100(8):3307–
MAbs 6(2):327–339. 3315.
17. Stracke J, Emrich T, Rueger P, Schlothauer T, Kling L, Knaupp A, 33. Zhang A, Hu P, MacGregor P, Xue Y, Fan H, Suchecki P, Olszewski
Hertenberger H, Wolfert A, Spick C, Lau W, Drabner G, Reiff U, Koll L, Liu A. 2014. Understanding the conformational impact of chemical
H, Papadimitriou A. 2014. A novel approach to investigate the effect modifications on monoclonal antibodies with diverse sequence variation
of methionine oxidation on pharmacokinetic properties of therapeutic using hydrogen/deuterium exchange mass spectrometry and structural
antibodies. MAbs 6(5):1229–1242. modeling. Anal Chem 86(7):3468–3475.