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Purification and Identification of High Molecular Weight Products Formed During Storage of Neutral Formulation of Human Insulin

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Abstract

Purpose

To identify High Molecular Weight Products (HMWP) formed in human insulin formulation during storage.

Methods

Commercial formulation of human insulin was stored at 37°C for 1 year and HMWP was isolated using preparative size exclusion chromatography (SEC) and reverse phase (RP) chromatography. The primary structure of the isolated species was analysed using liquid chromatography mass spectrometry (LC-MS) and tandem mass spectrometry (MS/MS). To test the hypothesis that amino groups of insulin are involved in HMWP formation, the HMWP content of various formulations spiked with amine compounds or formulations of insulin with modified amino groups was measured.

Results

More than 20 species of HMWP were observed and 16 species were identified using LC-MS. All identified species were covalent dimers of human insulin linked via A21Asn and B29Lys, formed via the formation of an anhydride intermediate at A21Asn. Two types of HMWP were identified, with the covalent link in the open or closed (succinimidyl) form. Some species also contained single deamidation at B3 or the desPhe(B1)-N-oxalyl-Val(B2) modification. Reduced rate of HMWP formation was observed after addition of L-lysine, L-arginine or piperazine or when insulin analogues with methylated N-terminals and side chain amines and A21Gly mutation were used. Formulations of human insulin without zinc and m-cresol were found to contain a different pool of HMWP.

Conclusions

HMWP formed in formulation of human insulin at pH 7.4 with zinc and m-cresol consists primarily of covalent dimers linked via A21Asn and B29Lys. Insulin formulation properties determine the amount and identity of formed HMWP.

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Abbreviations

HI:

Human insulin

HMWP:

High Molecular Weight Products

LC-MS:

Liquid chromatography mass spectrometry

MS:

Mass spectrometry

MS/MS:

Tandem mass spectrometry

RP:

Reverse phase (chromatography)

SEC:

Size exclusion chromatography

TCEP:

Tris(2-carboxy-ethyl)phosphine

V8:

Endoproteinase Glu-C V8 enzyme

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ACKNOWLEDGMENTS AND DISCLOSURES

This study was jointly funded by Innovation Fund Denmark and Novo Nordisk A/S as part of the Industrial Ph.D. programme (Ministry of Higher Education and Science). Laboratory facilities, equipment, and materials were provided by Novo Nordisk. The authors would like to thank Peter Madsen and Thomas Høeg-Jensen from Diabetes Protein Engineering (Novo Nordisk A/S) for providing the insulin analogues.

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Authors and Affiliations

  1. Diabetes Protein Engineering, Novo Nordisk A/S, Novo Nordisk Park, 2760, Måløv, Denmark

    Christian Fogt Hjorth, František Hubálek, Jonatan Andersson, Christian Poulsen & Helle Naver

  2. Interdisciplinary Nanoscience Center (iNANO), Aarhus University, Gustav Wieds Vej 14, 8000, Aarhus C, Denmark

    Christian Fogt Hjorth & Daniel Otzen

Authors
  1. Christian Fogt Hjorth
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  2. František Hubálek
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  3. Jonatan Andersson
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  4. Christian Poulsen
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  5. Daniel Otzen
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  6. Helle Naver
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Corresponding author

Correspondence to Christian Fogt Hjorth.

Electronic supplementary material

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Fig. S1

Synthesized insulin analogues. Analogue 1 (a), analogue 2 (b) (GIF 10 kb)

High resolution image (EPS 434 kb)

Fig. S2

Analytical RP-UPLC analysis of SEC fractions containing HMWP (12–95 min). HMWP isolated using analytical SEC method (Fig. 1, peak C) (red trace) and preparative SEC method (Fig. 1, peak F) (black trace) (GIF 9 kb)

High resolution image (EPS 594 kb)

Fig. S3

Preparative RP test run to evaluate the number of HMWP species in isolated preparative SEC fractions of HMWP. HMWP species eluted after residual human insulin monomer (A) until column wash gradient was initiated (B) (GIF 12 kb)

High resolution image (EPS 605 kb)

Fig. S4

Mass spectra of cross-linked peptide A-II + B-III obtained from samples of HMWP treated with V8 and TCEP. HMWP with intact mass 11590.31 Da (top), HMWP with intact mass 11572.31 Da (bottom) (GIF 17 kb)

High resolution image (EPS 814 kb)

Fig. S5

MS/MS of HMWP A-II+B-III peptide (a–b). HMWP 11,590 Da, parrent ion 537.87 m/z (top), HMWP 11,572 Da, parrent ion 531.91 m/z (bottom) (GIF 33 kb)

High resolution image (EPS 855 kb)

Fig. S6

MS of B-I peptide obtained from sample of HMWP treated with V8 and TCEP, mass range 440–780 m/z. HMWP B-I peptide with deamidation (+1 Da), deconvoluted mass of 1482.69 Da (top), human insulin B-I peptide, deconvoluted mass of 1481.71 Da (bottom) (GIF 14 kb)

High resolution image (EPS 572 kb)

Fig. S7

MS/MS of B-I peptide (a–e) obtained from sample of HMWP treated with V8 and TCEP (top) and human insulin B-I peptide (bottom) (GIF 116 kb)

High resolution image (EPS 1.45 mb)

Fig. S8

MS of B-I peptide obtained from sample of HMWP treated with V8 and TCEP. HMWP B-I peptide with desPhe(B1)-N-oxalyl-Val(B2) modification, 1406.62 Da (top), human insulin B-I peptide, 1481.71 Da (bottom) (GIF 14 kb)

High resolution image (EPS 630 kb)

Fig. S9

MS/MS of B-I peptide containing desPhe(B1)-N-oxalyl-Val(B2) modification (a–d) obtained from HMWP treated with V8 and TCEP (top) and human insulin reference (bottom) (GIF 72 kb)

High resolution image (EPS 1.41 mb)

Fig. S10

LC-MS of human insulin formulation spiked with L-arginine (top), lysine (middle) or piperazine (bottom). Calculated monoisotopic masses based on [M+6H]6+: HI+arginine: 5960.72 Da (994.4612 m/z), −HI+lysine: 5932.73 (989.7968 m/z), HI+piperazine 5872.69 Da (989.7968 m/z) (GIF 16 kb)

High resolution image (EPS 834 kb)

Fig. S11

Nano-spray MS/MS of human insulin linked to arginine or lysine (a-d) obtained from human insulin formulation spiked with arginine (top) or lysine (middle) and human insulin control (bottom) (GIF 51.2 kb)

High resolution image (EPS 2.26 mb)

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Hjorth, C.F., Hubálek, F., Andersson, J. et al. Purification and Identification of High Molecular Weight Products Formed During Storage of Neutral Formulation of Human Insulin. Pharm Res 32, 2072–2085 (2015). https://doi.org/10.1007/s11095-014-1600-3

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  • DOI: https://doi.org/10.1007/s11095-014-1600-3

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