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Amino Acids

, Volume 51, Issue 2, pp 295–309 | Cite as

Novel, efficient, facile, and comprehensive protocol for post-column amino acid analysis of icatibant acetate containing natural and unnatural amino acids using the QbD approach

  • Yagnakirankumar KomaravoluEmail author
  • Venugopala Rao Dama
  • Thirumala Chary Maringanti
Original Article

Abstract

Qualitative and quantitative determination of amino acid composition using amino acid analysis (AAA) is an important quality attribute and considered an identity of therapeutic peptide drugs by the regulatory agencies. Although huge literature is available on pre- and post- column derivatization AAA methods, arriving at an appropriate hydrolysis protocol coupled with adequate separation of the derivatized/underivatized amino acids is always challenging. Towards achieving a facile and comprehensive protocol for AAA, the present work is geared towards developing a deeper understanding of the extent of hydrolysis of peptide, and the nature and stability of amino acids present in the peptide backbone. This defines the suitability of the method in meeting the end goals and the regulatory requirement. Analysis of historical data generated during the method optimization of AAA for icatibant acetate (ICT) using head space oven hydrolysis (HSOH) and microwave-assisted hydrolysis (MAH) methods helped in arriving at fast (< 1 h) and efficient hydrolysis (0.9–1.1 of theoretical residue) conditions. Better separations for the natural and unnatural amino acids were achieved using 3.45 ≤ pH ≤ 10.85, and a column oven gradient program. This approach was useful in meeting the method quality attributes [resolution (Rs) > 2.0; plate count (N) > 5600; and USP tailing factor < 1.2] with a target analytical method profile of relative amino acid mole ratios (RAAMR) in the range of 0.9–1.1 for Ser, Oic, Tic, Hyp, Ala (Thi), Gly and Pro, and between 2.7 and 3.3 for Arg. The developed method was validated as per the ICH guidelines and is precise, accurate, linear and robust.

Keywords

Post-column derivatization Amino acid analysis Quality by design ICT Ninhydrin Unnatural amino acids 

Abbreviations

AAA

Amino acid analysis

ICT

Icatibant acetate

HSOH

Head space oven hydrolysis

MAH

Microwave-assisted hydrolysis

RAAMR

Relative amino acid mole ratios

Rs

Resolution

USP

United States pharmacopeia

Ser

Serine

Gly

Glycine

Ala(thi)

Thienylalanine

Arg

Arginine

Hyp

Hydroxy-proline

Pro

Proline

Oic

Octahydro-1H-indole-2-carboxylic acid

Tic

D-1, 2, 3, 4-Tetrahydroisoquinoline-3-carboxylic acid

TP

Therapeutic peptides

NMR

Nuclear magnetic resonance spectroscopy

IUPAC

International Union of Pure and Applied Chemists

CHMP

Committee for Medicinal Products for Human Use

Pr-CDT

Pre-column derivatization

Po-CDT

Post-column derivatization

RSM

Response surface methodology

F-moc

9-Fluorenylmethyl-chloroformate

OPA

Orthophthalaldehyde

PITC

Phenylisothiocyanate

Tf

Peak tailing

Rs

Resolution

N

Theoretical plate count

FOM

Figure of merit

EOH

Extent of hydrolysis

HSGC

Head space gas chromatography

GC

Gas chromatography

ICH

International Conference on Harmonization

RSD

Relative standard deviation

Notes

Acknowledgements

Two of the authors (KYKK and DVGR) thank the management of Dr. Reddy’s Laboratories Ltd., Hyderabad, India, for supporting this work. Support from colleagues in Research and Development, and Analytical Research and Development of Dr. Reddy’s Laboratories Ltd. is appreciated.

Compliance with ethical standards

Conflict of interest

The authors Yagnakirankumar Komaravolu, Venugopala Rao Dama, and Thirumala Chary Maringanti declare that they have no conflicts of interest.

Ethical statement

This article does not contain any studies with human participants or animals performed by any of the authors.

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Copyright information

© Springer-Verlag GmbH Austria, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Analytical Research and developmentIPDO, Dr. Reddy’s LaboratoriesHyderabadIndia
  2. 2.Jawaharlal Nehru Technological UniversityHyderabadIndia

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