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Identification of D-Amino Acids in Light Exposed mAb Formulations

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Abstract

Purpose

We previously demonstrated that D-amino acids can form as a result of photo-irradiation of a monoclonal antibody (mAb) at both λ = 254 nm and λ > 295 nm (λmax = 305 nm), likely via reversible hydrogen transfer reactions of intermediary thiyl radicals. Here, we investigate the role of various excipients (sucrose, glucose, L-Arg, L-Met and L-Leu) on D-amino acid formation, and specifically the distribution of D-amino acids in mAb monomers and aggregates present after light exposure.

Methods

The mAb-containing formulations were photo-irradiated at λ = 254 nm and λmax = 305 nm, followed by fractionation of aggregate and monomer fractions using size exclusion chromatography. These aggregate and monomer fractions were subjected to hydrolysis and subsequent amino acid analysis.

Results

Both aggregate and monomer fractions collected from all formulations showed the formation of D-Glu and D-Val, whereas the formation of D-Ala was limited to the aggregate fraction collected from an L-Arg-containing formulation. Interestingly, quantitative analysis revealed higher yields of D-amino acids in the L-Arg-containing formulation.

Conclusions

Generally, D-amino acids accumulated to similar extents in monomers and aggregates.

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Abbreviations

BMS:

Bis-(2-mercaptoethyl) sulfone

DAAO:

D-amino acid oxidase

HPLC:

High pressure liquid chromatography

IAA:

Iodoacetamide

IgG:

Immunoglobulin G

mAb:

Monoclonal antibody

Q-TOF:

Quadrupole time-of-flight

SEC:

Size exclusion chromatography

UPLC-MS:

Ultra pressure liquid chromatography- mass spectrometry

UV:

Ultraviolet

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Author notes

  1. Rupesh Bommana

    Present address: MedImmune, Gaithersburg, Maryland, 20878, USA

  2. Olivier Mozziconacci

    Present address: Merck Research Labs, Rahway, New Jersey, 07065, USA

Authors and Affiliations

  1. Department of Pharmaceutical Chemistry, University of Kansas, 2095 Constant Avenue, Lawrence, Kansas, 66047, USA

    Rupesh Bommana, Natalia Subelzu, Olivier Mozziconacci & Christian Schöneich

  2. Late Stage Pharmaceutical Development, Genentech, ,1 DNA Way, South San Francisco, California, 94080, USA

    Alavattam Sreedhara

Authors
  1. Rupesh Bommana
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  2. Natalia Subelzu
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  3. Olivier Mozziconacci
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  4. Alavattam Sreedhara
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  5. Christian Schöneich
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Corresponding author

Correspondence to Christian Schöneich.

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Bommana, R., Subelzu, N., Mozziconacci, O. et al. Identification of D-Amino Acids in Light Exposed mAb Formulations. Pharm Res 35, 238 (2018). https://doi.org/10.1007/s11095-018-2520-4

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