D'Hondt, Matthias

De Spiegeleer, Bart

Demaré, Wim

Ghent University Academic Bibliography

INTRODUCTION Dry heat stress stability of casein hydrolysate peptide mixtureMatthias D’Hondt1, Wim Demaré1 and Bart De Spiegeleer1,*1 Drug Quality and Registration (DruQuaR) group, Faculty of Pharmaceutical Sciences, Ghent University, Harelbekestraat 72, B-9000 Ghent, Belgium.* Corresponding author: bart.despiegeleer@ugent.be (O. Ref.: 2010-267c)DruQuaRHot melt extrusion (HME) uses a rotating screw and elevated temperature to pump dry, raw materials through a die and into a product of uniform shape. Itis currently one of the most widely used processing techniques within the plastics industry and has drawn the attention of the pharmaceutical industry.Several research groups have already demonstrated the possibility of HME in the processing of API’s e.g. nifedipine, theophyline, into variouspharmaceutical forms including tablets, granulates and implants [1].Peptides make up a promising class of new therapeutics. Recently, new manufacturing and delivery techniques, combined with intensified research anddevelopment programs, resulted in the approval of an anti-HIV peptide drug Fuzeon® and an estimated total of 200 peptides in various clinical stages [2].Depending on the therapeutic application, processing of certain peptides e.g. anti-cancer peptides, into implants with appropriate release rates, would bebeneficial for patient compliance and therapeutic effect. Due the to high temperatures reached, HME of peptides is surrounded by skepticism. In order toevaluate the HME processing possibilities of peptides, a number of selected peptides, present in a casein hydrolysate sample, were subjected to dry heatstress stability tests.Casein hydrolysate characterization: HPLC-UV/ESI Iontrap MS, peptide C18 fused-core column (75 mm × 3.0 mm, 2.7 µm; HALO; Achrom), linear gradient(MFA: 0.1 % (m/V) FA in H2O; MFB: 0.1 % (m/V) FA in acetonitrile) T0 min.: 95/5 (V/V) A/B; T40 min.: 71/29 A/B; 0.6 mL/min., 195 nm.Experimental design (duplicate): 100 °C – 140 °C – 180 °C1 min. – 3 min. – 5 min.EXPERIMENTAL [1] M. M. Crowley, F. Zhang, M. A. Repka, S. Thumma, S. B. Upadhye, S. Kumar, J. W. McGinity, C. Martin, Drug Development and Industrial Pharmacy, 2007, 33, 909.[2] P. McGee, Drug Discovery and Development, 2005, 4.RESULTS and DISCUSSION Hot-melt extrusion related dry heat stress stability study performed with a model peptide mixture set, originating from casein hydrolysate, showed peptide extrusion potential up toward 5 minutes at 140 °C and 3 minutes at 180 °C. However, significant degradation, ranging from 10 % to 40 %, was seen during 5 minutes incubation at 180 °C.CONCLUSIONSREFERENCES1. Model peptide mixture characterization (HPLC-ESI Iontrap MS)2. Dry heat stress stability tests (HPLC-UV)Peak information Relative amount 100 °C (%)(1,2) Relative amount 140 °C (%)(1,2) Relative amount 180 °C (%)(1,2)RT (min.) AA sequence 1 min. 3 min. 5 min. 1 min. 3 min. 5 min. 1 min. 3 min.(3) 5 min.(3)9.08 NKKIE 102.24 100.73 96.83 97.00 95.52 94.23 88.60 94.37 85.2310.17 YPVEP 105.06 101.99 102.29 98.55 101.41 99.67 94.63 97.51 78.5312.91 VLPVPQ 104.64 102.19 100.87 100.94 101.18 100.32 97.29 96.58 78.0015.50VAPFPENVPGEIVLEIVPNHLPLP104.04 99.58 98.23 99.56 100.36 99.89 94.57 101.44 75.5917.40 DIPNPI 109.59 105.16 102.50 105.86 105.42 98.03 96.49 101.15 94.2617.70 SDIPNPI 110.48 109.87 101.30 103.74 98.39 95.01 97.18 93.45 75.0419.19 RGPFPI 110.03 107.78 101.35 100.52 107.09 105.84 101.24 92.95 71.4320.88 PFPGPIPNAPFPEV 106.31 104.34 100.85 103.42 102.31 103.03 98.62 98.35 70.5224.78 YPFPGPIPN 101.90 99.24 102.35 96.53 98.82 98.53 100.65 93.76 62.7726.16 VVPPFLQPE 107.89 104.82 102.38 100.83 102.96 101.72 98.24 98.71 83.3227.00 VYPFPGPIPN 104.84 99.33 99.21 95.93 100.23 97.89 95.06 95.54 63.9628.73 TDAPSFSDIPNPI 105.49 102.37 102.39 99.35 101.15 100.36 97.38 99.03 76.9029.83 VYPFPGPI 108.17 103.55 101.16 98.83 99.18 96.42 93.72 91.24 89.69(1) Calculated using formula: ((∑peak area stressed) / (∑peak area unstressed))(2) Peptide abundance outside [90-110 %] interval relative to unstressed sample are considered significant and indicated in bold(3) Increase in degradation peaks: RRT: 0.983 (relative to RGPFPI) - [MH]+: 730.25; RRT: 0.986 (relative to VYPFPGPI) - [MH]+: 1315.40Typical degradation profile(TDAPSFSDIPNPI)020406080100120100 °C 140 °C 180 °CRelativeamount(%)1 min.3 min.5 min.Average peptide degradation (± SEM)TIC casein hydrolysate1234567891011 121314# Retention time (min.) Observed [MH]+ Amino acid sequence Origin1 9.66 631.26 NKKIE β-casein2 11.21 604.15 YPVEP β-casein3 14.08 652.15 VLPVPQ β-casein4 15.87 659.10727.15VAPFPENVPGEIVαS1-caseinβ-casein5 16.64 684.14576.10LEIVPNHLPLPαS1-caseinβ-casein6 18.53 668.10 DIPNPI αS1-casein7 18.89 755.18 SDIPNPI αS1-casein8 20.59 686.34 RGPFPI β-casein9 22.36 838.27659.18PFPGPIPNAPFPEVβ-caseinαS1-casein10 26.31 1001.28 YPFPGPIPN β-casein11 27.69 1025.33 VVPPFLQPE β-casein12 28.46 1100.36 VYPFPGPIPN β-casein13 30.11 1373.33 TDAPSFSDIPNPI αS1-casein14 31.34 889.25 VYPFPGPI β-casein707580859095100105110100 °C 140 °C 180 °CRelative amount (%)1 min.3 min.5 min.

Dry heat stress stability of casein hydrolysate peptide mixture

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Dry heat stress stability of casein hydrolysate peptide mixture

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