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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

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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.

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Abbreviations

AAA:

Amino acid analysis

ICT:

Icatibant acetate

HSOH:

Head space oven hydrolysis

MAH:

Microwave-assisted hydrolysis

RAAMR:

Relative amino acid mole ratios

R s :

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

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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.

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Authors and Affiliations

  1. Analytical Research and development, IPDO, Dr. Reddy’s Laboratories, Hyderabad, 500090, India

    Yagnakirankumar Komaravolu & Venugopala Rao Dama

  2. Jawaharlal Nehru Technological University, Kukatpally, Hyderabad, 500085, India

    Thirumala Chary Maringanti

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  1. Yagnakirankumar Komaravolu
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  2. Venugopala Rao Dama
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  3. Thirumala Chary Maringanti
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Correspondence to Yagnakirankumar Komaravolu.

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

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Komaravolu, Y., Dama, V.R. & Maringanti, T.C. 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. Amino Acids 51, 295–309 (2019). https://doi.org/10.1007/s00726-018-2665-9

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