Purification of Insulin with YMC-Triart Prep C8. Increased Output at Reduced Costs

Purification of Insulin with YMC-Triart Prep C8 Increased Output at Reduced Costs

2 Introduction YMC bulk material along with phases from Daiso and Kromasil took part in a study by a major peptide manufacturer. The aim of the study was to determine whether the present process of insulin purification by HPLC could be improved. 2 YMC phases, 1 Kromasil material and 1 were compared to the originally used (SP-120-10-C8-BIO), each packed in steel columns of 250 x 10 mm: YMC-basic Kromasil 100-13-C8 SP-200-10-C8-BIO Cover picture: Insulin from human being, PDB-Code - 4EY9, Software: Pymol

Contents 3 Sorbent and overall costs... page 04 Previous insulin purification method... page 05 Requirement for a new method... page 05 Comparison of insulin fractions... page 06 Direct insulin output... page 07 System backpressure... page 08 Mechanical stability... page 09 Possible production cycles... page 10 Insulin output per kg solvent... page 11 Stationary phase costs... page 12 Previous insulin purification method... page 13 Optimised method and advantages of YMC-Triart Prep C8... page 13 Materials and methods... page 14 Ordering information... page 14

4 Lowest sorbent and overall costs 1 kg insulin, purity 98.5% 19% cost reduction SP-120-10-C8-BIO total cost 35,000 sorbent cost 2,310 Kromasil 100-13-C8 total cost 28,900 sorbent cost 640 YMC-Triart Prep C8-L total cost 28,900 sorbent cost 490 Considering the overall costs, the proposed cost to obtain 1 kg insulin (purity 98.5%, starting from an initial purity 96.5%) with and appropriate solvents are 19% lower than with SP-120-10-C8-BIO. In terms of process economy YMC Triart Prep C8-L scores with regards to lowest overall purification costs and lowest absolute costs of the stationary phase. The amount of sorbent costs is only about 1.7% of the overall costs, which is also the lowest percentage of all phases used.

Previous insulin purification method 5 Previous method 50-70 cycles SP-120-20-C8-BIO (pre-treatment) 15 mm ammonium sulphate 60 mm glycine SP-120-10-C8-BIO (polishing) 250 mm acetic acid 11-17% isopropyl alcohol These process figures are not acceptable due to low insulin output of only 70.0 % (without re-chromatography) high loss of insulin because of low sorption reversibility very short operation period of the sorbent high sorbent and overall costs 98.5% insulin purity 70.0% output Requirement for a new method The efficacy of a preparative purification depends on the selectivity of the stationary phase and its ability to separate possible impurities from the main product. An increase in selectivity would not only improve the degree of separation of the peaks, but also reduce the cleaning costs by increasing the load on the column. Furthermore, the amount of sorbent and consumption of mobile phase should be reduced to increase the yield for a given capacity or improve the product quality. YMC bulk material along with phases from Daiso and Kromasil took part in a study by a major peptide manufacturer. The aim of the study was to determine whether the present process of insulin purification by HPLC could be improved. 2 YMC phases, 1 Kromasil material and 1 were compared to the originally used (SP-120-10-C8-BIO), each packed in steel columns of 250 x 10 mm: YMC-basic Kromasil 100-13-C8 SP-200-10-C8-BIO

6 No re-chromatography with YMC-Triart Prep C8 Analysed insulin fractions Insulin purification on (A) and YMC-basic (B) separate insulin from the side product desamido insulin and other impurities with good selectivities. However, shows an even better separation of insulin from impurities in fraction 1-3. Kromasil 100-13-C8 (C) also separated insulin from its impurities, but desamido insulin co-eluted with insulin in fractions 7-10. On the Daiso SP-200-10-C8-BIO (D) material insulin is not separated from impurities or from desamido insulin. In contrast to re-chromatography of side fractions is needed when using the Kromasil phase, the Daiso material and YMC-basic. Analysed insulin fractions Insulin purification on YMCbasic re-chromatography Insulin purification on Kromasil 100-13-C8 re-chromatography Insulin purification on Daiso SP-200-10-C8-Bio re-chromatography

Highest insulin output 7 100% 90% Direct insulin output (without re-chromatography; purity 98.5%) 80% 70% Insulin output 60% 50% 40% 30% 20% 10% 0% SP-120-10-C8-BIO SP-200-10-C8-BIO Kromasil 100-13-C8 YMCbasic The use of YMC material, either or YMCbasic, results in insulin yields of 92.6% and 92.5% with purities of 98.5%. The output of Kromasil 100-13-C8 is 89.6%, while SP-200-10-C8-BIO and the original SP-120-100-C8-BIO show results of only 82.1% and 78.0%. Again, the yields mirror the superior performance of the YMC-phases.

8 Lowest system backpressure After every insulin treatment the sorbents were regenerated by cleaning in place (CIP) by: 1) flushing with 0.05 M solution of NaOH in 50% acetonitrile (5 CV) 2) flushing with mobile phase B (3 CV) 3) flushing with mobile phase A (3 CV) The stationary phase is only replaced when the required peptide quality cannot be achieved or the backpressure exceeds the limits of operation. Sorbent lifetime is a very important factor, especially regarding process costs. 80 System back pressure 70 60 Backpressure [bar] 50 40 30 SP-200-10-C8-Bio Kromasil 100-13-C8 YMCbasic 20 10 High mechanical stability of YMC-Triart 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Purification cycle A comparison of the system pressure for the different chromatography phases after several production cycles showed that, YMCbasic and gave an initial backpressure of 8 bar. However, the Kromasil material initially has a relatively high backpressure of 16 bar despite of its 13 μm particles. A pressure increase took place from the fifth cycle onwards, while the only lasted until cycle three before a steep pressure increase began. YMCbasic also showed a pressure increase. However, demonstrated only a slight increase starting in cycle 12, indicating a very high hydrolytic stability in contrast to the other phases. YMC Statement Out of consideration for the traditional silica phases 0.05 M NaOH was used for CIP instead of the more usual 0.1 M NaOH. is fully able to withstand 0.1 M NaOH for CIP.

Mechanical stability (dynamic) 9 Shearing and crushing of silica particles leads to the formation of fines, which results in an increased backpressure. By using mechanically stable, spherical particles the formation of fines can be reduced. Here, high mechanical stability of YMC-Triart Prep is demonstrated by means of repeated packing of a DAC column (DAU-50-700; bed length 100 mm; packing pressure 65 bar). Even after more than 10 repacking cycles for the same material the pressure does not increase. The absence of fines is proven by a constant backpressure. This makes YMC-Triart Prep a valuable asset for method development and especially for process optimisation! Psi/m 2000 Backpressure 1500 1000 500 Less backpressure = higher throughput possible 0 1 5 10 Packing number YMC-Triart Prep (10 µm, 20 nm) (Hybrid silica) YMC HG-series (10 µm, 20 nm) (High grade silica) Traditional Silica (10 µm, 20 nm) after 1 st packing after 11 th packing

10 Highest number of production cycles Possible production cycles (insulin purity 98.5%) 12 Production cycles 10 8 6 12 times more cycles 4 2 0 SP-120-10-C8-BIO SP-200-10-C8-BIO Kromasil 100-13-C8 YMCbasic A phase replacement was required on the basis of increasing back pressure for all sorbents except for, which had to be exchanged only on the basis of reducing product purity. However, also showed the highest amounts of production cycles before insulin purity fell below 98.5%. Process cycles with re-chromatography of side fractions 450 400 Production cycles 350 300 250 200 150 350-370 more cycles 100 50 0 SP-120-10-C8-BIO SP-200-10-C8-BIO Kromasil 100-13-C8 YMCbasic When accepting a lower initial insulin quality and re-chromatography of the side fractions, a much greater number of process cycles can be performed before material replacement is required. Again, showed the greatest number of cycles, namely 420, which is about 8-times more than with the original and even double that of the second best, the Kromasil material.

Maximum output 11 Purified insulin per kg sorbent 12 10 Purified insulin [kg] 8 6 4 10 times more 2 0 SP-120-10-C8-BIO SP-200-10-C8-BIO Kromasil 100-13-C8 YMCbasic Because of its superior stability is more robust towards production processing compared to other phases and therefore shows a much higher efficiency. This results in an increased amount of purified insulin when using, which is 10 times higher than with the original system.

12 Lowest sorbent costs Stationary phase costs for producing 1 kg insulin (purity 98.5%) 2,500 2,000 20% reduction Costs for stationary phase [ ] 1,500 1,000 500 0 SP-120-10-C8-BIO SP-200-10-C8-BIO Kromasil 100-13-C8 YMCbasic In addition, the costs for production of 1 kg polished insulin are reduced by about 20%. None of the other materials are as cost effective as. However, the amount of insulin produced using 1 kg of sorbent does not fully reflect the economic feasibility of a sorbent use. For a correct choice all costs associated with HPLC purification should be taken into account (page 4): costs of the sorbent mobile phase costs for eluting and cleaning raw insulin.

Previous insulin purification method 13 Previous method 50-70 cycles SP-120-20-C8-BIO (pre-treatment) 15 mm ammonium sulphate 60 mm glycine SP-120-10-C8-BIO (polishing) 250 mm acetic acid 11-17% isopropyl alcohol These process figures are not acceptable due to low insulin output of only 70.0 % (without re-chromatography) high loss of insulin because of low sorption reversibility very short operation period of the sorbent high sorbent and overall costs 98.5% insulin purity 70.0% output Increased output at reduced costs with YMC-Triart Prep C8 Optimised method ca. 420 cycles 200 mm ammonium acetate 500 Mm acetic acid ph = 4.3 10% acetontrile [solvent A] (50% acetonitrile [solvent B]) 98.5% insulin purity 92.6% output Using results in highest insulin output of 92.6% (without re-chromatography) increase in insulin output by 23% (without re-chromatography) superior phase stability constant backpressure less stationary phase replacement one step method instead of two step method reduction of costs by 19%

14 Materials and methods Preparative HPLC: Gilson: pumps 305, 306, manometer 808, dynamic mixer 811S, UV/Vis-detector 112 Analytical HPLC: Agilent 1200 Mobile phase: A: buffer/acetonitrile 90/10 B: buffer/acetonitrile 50/50 (Buffer: 0.2 M ammonium acetate, 0.5 M acetic acid) Flow rate: Gradient: Sample: Fractions: 1.96 m/min 0.1 % per minute 330 mg of recombinant human insulin, with a purity of 96.5 % (pre-cleaned by IEX) dissolved in 25 ml HCl (15 mm)/acetonitrile (90/10), filtered through a membrane filter of 0.2 microns 2-5 ml Ordering information YMC-Triart Prep, preparative bulk media YMC-Triart Prep C18-S YMC-Triart Prep C8-S Pore size (nm) Particle size (µm) Product Code Pore size (nm) Particle size (µm) Product Code 10 TAS12S11 10 TOS20S11 12 15 TAS12S16 20 15 TOS20S16 20 TAS12S21 20 TOS20S21 Typical pack sizes: Laboratory scale: 100 g - 5 kg PE bottle Industrial scale: 5 kg - 50 kg in double lined PE bags inside metal drum Larger pack sizes on request Other particle and pore size combinations on request. Examples of existing customised material: TAS08S11, TOS12S11 We fully appreciate that further approaches of method optimisation with every material might have led to a better output. Yet, in this specific case, the customer felt confident enough to select YMC-Triart C8-L and perform further optimisation.

Other available brochures 15 YMC*Gel HG-series High grade silica phases for preparative HPLC The Selectivity Company YMC*Gel HG-series brochure General purpose preparative phases on high grade silica base Available as silica or with C18, C8, C4, C1, phenyl, cyano, amino or diol bonding Particle size: 10 µm, 15 µm, 20 µm, 50 µm Pore size: 8 nm, 12 nm, 20 nm, 30 nm www.ymc.de YMC-BioPro prep brochure YMC-BioPro IEX materials Polymeric ion exchange material for biochromatography Available as strong anion- or cation-exchanger Excellent flow properties and high dynamic binding capacity Prep Bulk media Extremely economic in industrial scale applications YMC-Triart prep brochure Robust and ph stable hybrid silica process material Robust, flexible and economic Increased chromatographic performance ph 1-12 is a trademark of Daiso Co., Ltd. Kromasil ia a trademark of Akzo Nobel, Separation Products.

Your local distributor: Co., Ltd. YMC Karasuma-Gojo Bld. 284 Daigo-cho, Karasuma Nisiiru Gojo-dori Shimogyo-ku, Kyoto 600-8106 Japan TEL. +81(0)75-342-4515, FAX +81(0)75-342-4550 www.ymc.co.jp Europe GmbH Schöttmannshof 19 D-46539 Dinslaken Germany TEL. +49(0)2064/427-0, FAX +49(0)2064/427-222 www.ymc.de Taiwan Co., Ltd. 3F, No. 1353, Zhongzheng Rd., Taoyuan City, Taoyuan Country 330, Taiwan (R.O.C.) TEL. +886-3-2150-630, FAX +886-3-2150-286 www.ymctaiwan.com America, Inc. 941 Marcon Boulevard Suite 201 Allentown, PA18109 USA TEL. +1-610-266-8650, FAX +1-610-266-8652 www.ymcamerica.com Korea Co., Ltd. #208, Owners Tower, 16-5, Sunae-dong, Bundang-gu, Seongnam-si, Gyeonggi-do, 463-825 Korea TEL. +82-31-716-1631, FAX +82-31-716-1630 www.ymckorea.com India Ltd. CX - 07, 3rd Floor, Lobe - 1, A-Towers, The Corenthum, Plot No- A-41, Sector - 62, Noida - 201301 (U.P.) India. TEL. +91(0)120-4276020 - 25, FAX +91(0)120-4276026 www.ymcindia.com Co., Ltd. Shanghai Rep. Office Far East International Plaza A2404 No. 319 Xianxia Road, Shanghai 200051 P.R. China TEL: +86-21-6235-1388, FAX: +86-21-6235-1398 www.ymcchina.com 16_03/2014