NOTES. Zenix SEC-80. Sepax Zenix SEC-80 provides a valuable solution

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1 Having trouble applying size exclusion chromatography to peptides under 1, Da? What if there was a size exclusion column specifically designed for small protein and peptide separations? Sepax Zenix SEC-8 provides a valuable solution Highlighted FACTS: Zenix SEC-8 3um and 8Å: high efficiency 3um, 8Å pore for high resolution separation of peptides with molecular weights in the range of 1kD to 6kD. Mobile Phase Compatibility: compatible with different mobile phases, including TFA/acetonitrile and methanol additives for peptide separation. Peptides: insulin from porcine pancreas, glucagon, Angiotensin I and Bradykinin are separated on Zenix SEC-8 with good resolution. Complex Peptide Mixtures: E. coli tryptic digests are fractionated successfully on Zenix SEC-8. Effect of Acetonitrile Mobile Phase Concentration Separation parameters for peptide mixture using 75% acetonitrile with.1% TFA in water Peak Protein MW (Da) Retention time () NOTES Column: Zenix SEC-8 (3 m, 7.8x3 mm) Mobile phase:.1% TFA with the indicated percentage of acetonitrile Flow rate:.8 ml/; Injection volume: 5 µl Detection: UV214 nm; Temperature: Ambient (~25 ) Sample: 1. Insulin (.5mg/mL) 2. Glucagon (.5mg/mL) 3. Angiotensin I (.5mg/mL) 4. Bradykinin (.5mg/mL) Absorbance at 214 nm % ACN 65% ACN 75% ACN 85% ACN Resolution Plate counts 1 Insulin (porcine) Glucagon Angiotensin I Bradykinin Toll Free: SEPAX-US 1

2 Zenix SEC-8 NOTES What is Zenix SEC-8 5mM phosphate, 3% MeOH +.1% TFA Sepax Zenix SEC-8 (Size Exclusion): Ultra-high efficiency and resolution SEC column. Zenix -8 SEC 3um is specifically designed for small protein and peptide separations. Delivering unrivaled resolving power and reproducibility. Made of uniform, hydrophilic, and neutral nanometer thick proprietary surface coating chemically bonded on silica, offers long column lifespan and negligible non-specific interactions. Absorbance at 214 nm 25mM sodium acetate, 3mM NaCl, ph 4.5.1% TFA, 75% ACN 5mM phosphate, 3% MeOH +.1% TFA Technical Specifications: Phase Zenix SEC-8 Material Neutral, hydrophilic film bonded silica Particle size ( m) 3 Pore size (Å) ~ 8 Protein MW range (native) ph stability Backpressure (psi) for 7.8x3 mm (1. ml/) Maximum backpressure Salt concentration range Maximum temperature Mobile phase compatibility Up to 5, (ph can be tolerated temporarily) ~ 1,5 ~ 4,5 2 mm - 2. M ~ 8 o C Aqueous and organic Separation of E. coli digests on Zenix SEC-8 (7.8x3mm) For complex E. coli tryptic digest, a mobile phase of 25 mm sodium acetate/3 mm NaCl gave the best separation. The bottom chromatogram was run with 5mM phosphate/3%meoh/.1% TFA. The middle one was run with.1% TFA/75% ACN. The top chromatogram represents the best degree of peptide separation and was run with 25mM sodium acetate/3mm NaCl, ph 4.5. Effect of Salt and Organic Additives on Peptide Separation Absorbance at 214 nm A B mM sodium acetate/3mm NaCl, ph 4.5 5mM phosphate, 3% MeOH +.1% TFA 3 4 Sepax Technologies, Inc. 5 Innovation Way Newark, Delaware 19711, USA Tel: (32) Fax: (32) info@sepax-tech.com Peptide separation on Zenix SEC-8 (7.8 x 3mm). Panel A was with mobile phase 25mM sodium acetate and 3mM NaCl, ph 4.5. Panel B was with mobile phase 5mM phosphate, 3% MeOH and.1% TFA. Peak elution order: 1. Insulin, 2. Bradykinin, 3. Angiotensin I, 4. Glucagon. The flow rate was.8 ml/. Toll Free: SEPAX-US 2

3 Zenix SEC-8 and Zenix -C SEC-8 for Peptide Analysis Having trouble applying size exclusion chromatography to peptides under 5, Da? What if there was a size exclusion column specifically designed for small protein and peptide separations? Sepax s Zenix and Zenix -C SEC-8 provide a valuable solution NOTES Overlay of Peptide Runs on Zenix SEC x 3 mm Column: Zenix SEC-8 7.8x3 mm, Flow rate: 1mL/, Detection: UV 214 nm, Mobile phase: 75% Acetonitrile with.1% TFA, Injection Volume: 5 µl Highlighted FACTS: Sepax s 8 Å pore size was developed for analyzing small peptides and proteins with molecular weights under 5, Daltons. Organics in the mobile phase are an alternative additive for the analysis of hydrophobic, or sticky, sample types. The Zenix -C SEC phase has a coating chemistry with a lay-down monolayer on porous silica which was developed for the analysis of hydrophobic, or sticky, sample types. A short, 2.1 x 5 mm, column is advantageous for use during a fast mass spec analysis of large biomolecules and small molecule drugs. Overlay of Peptide Runs on Zenix -C SEC x 3 mm Column: Zenix-C SEC-8 7.8x3 mm, Flow rate: 1mL/, Detection: UV 214 nm, Mobile phase: 75% Acetonitrile with.1% TFA, Injection Volume: 5 µl Differences between Zenix and Zenix -C SEC-8 Phases for the Analysis of Angiotensin I Acetate Column: Zenix SEC-8 7.8x3 mm, Flow rate: 1 ml/, Detection: UV 214 and 28 nm, Mobile phase: 15 mm Sodium Phosphate Buffer ph 7., Injection Volume: 5 µl Similarities between Zenix and Zenix -C SEC-8 Phases for the Analysis of Aprotinin Column: Zenix SEC-8 7.8x3 mm, Flow rate: 1 ml/, Detection: UV 214 and 28 nm, Mobile phase: 15 mm Sodium Phosphate Buffer ph 7., Injection Volume: 5 µl Toll Free: SEPAX-US 1

4 Zenix SEC-8 and Zenix -C SEC-8 for Peptide Analysis NOTES What are Zenix SEC-8 and Zenix -C SEC-8? Sepax Zenix and Zenix-C SEC-8 (Size Exclusion): Ultra-high efficiency and resolution SEC column. Zenix -8 SEC 3um is specifically designed for small protein and peptide separations. Delivering unrivaled resolving power and reproducibility. Made of uniform, hydrophilic, and neutral nanometer thick proprietary surface coating chemically bonded on silica, offers long column lifespan and negligible non-specific interactions. Calibration Curve for Zenix and Zenix -C SEC-8 Column: Zenix SEC-8 and Zenix -C SEC-8 7.8x3 mm, Flow rate: 1 ml/, Detection: UV 214 nm, Mobile phase: 75% Acetonitrile with.1% TFA Mobile Phase Comparison on Zenix SEC x 3 mm Column: Zenix SEC x3 mm, Flow rate: 1 ml/, Detection: UV 214 nm, Injection Volume: 5 µl, Sample: Insulin (1 mg/ml) Technical Specifications: Phase Material Particle size ( m) 3 Pore size (Å) 8 Zenix SEC-8 and Zenix-C SEC-8 Neutral, hydrophilic film bonded silica ph stability (ph can be tolerated temporarily) Backpressure (psi) ~ 1,5 Maximum backpressure ~ 4,5 Maximum temperature Mobile phase compatibility ~ 8 o C Aqueous and organics Column: Zenix SEC-8 7.8x3 mm, Flow rate:.2 ml/, Detection: UV 228 nm, Mobile phase: 5 mm NH 4 Ac : ACN = 7:3 (v/v), Injection Volume:.1 µl Sepax Technologies, Inc. 5 Innovation Way Newark, Delaware 19711, USA Tel: (32) Fax: (32) info@sepax-tech.com Toll Free: SEPAX-US 2

5 Peptide Separations Using Size Exclusion Chromatography Authors Haiying Chen Katherine McLaughlin Sepax Technologies, Inc. 5 Innovation Way Newark, DE USA Peptide Separation Abstract Size exclusion chromatography (SEC) has been widely applied in protein separation based on their molecular sizes. Here we present a peptide mixture from 1kD to 6 kd separation on size exclusion chromatography column Zenix -8. The effect of different mobile phases on the peptides separation performances was also explored. In the separation of insulin, glucagon, angiotensin I and bradykinin,.1% TFA/75% acetonitrile/h 2 O proved to be the optimum mobile phase. Peptide SEC can be applied to fractionate complex peptide mixtures as a first dimension chromatography in a multidimensional chromatography scheme. For complex E. coli tryptic digest, a mobile phase of 25 mm sodium acetate/3 mm NaCl gave the best separation.

6 Introduction Size exclusion chromatography (SEC) has been applied successfully to separate different sizes of proteins under native conditions. Different pore sizes have been developed to accommodate different ranges of molecular weight of biological samples. In order to apply the size exclusion chromatography to peptides under 1, Da, a few limitations have to be overcome. Even very small peptides can exist in different conformations and exhibit secondary structures. 1 Therefore, peptides tend to adsorb to column matrixes by ionic and hydrophobic interactions. 2 High salt concentrations, denaturing agents, and organic additives will imize such interactions, thus enabling the separation of peptides according to their molecular weights. Sepax Zenix SEC columns are based on uniform, hydrophilic, and neutral nanometer thick films chemically bonded on high purity and mechanically stabilized silica. Zenix SEC-8 is specifically designed for small protein and peptide separations. Its phase is similar to the other Zenix SEC with the same particle size 3µm, but different pore size at 8 Å. In this application note, we present separation of four peptides bradykinin (1,6 Da), angiotensin I (1,297 Da), glucagon (3,483 Da) and insulin (5,778 Da) under different separation conditions. Separation of E. coli tryptic digest on Zenix - 8 is also investigated with different mobile phase conditions. Experimental HPLC system: Agilent 12 HPLC with binary pump SEC column and LC method: Zenix -8 (3 M, 8 Å, 7.8x3 mm) was used for the peptide size exclusion separations. Mobile phases include different percentages of acetonitrile with.1% TFA and water, high salt and methanol additives. The flow rate was at.8 ml/. Chemicals and Reagents: Bradykinin acetate salt, MW 1,6 Da Angiotensin I Acetate, MW 1,297 Da Glucagon, MW 3,483 Da Insulin from Porcine Pancreas, MW 5,778 Da All four peptide samples were purchased from Sigma-Aldrich. 5 mg/ml stock solutions were made with 5 mm acetic acid. For sample injections, stock solutions were further diluted with 5 mm acetic acid to desired concentration. Sequencing grade modified trypsin was purchased from Promega. E. coli lysate tryptic digestion: E. coli lysate tryptic digestion was performed according to the procedure described previously. 3, 4 Briefly.5 mg dried lysate were reconstituted in 1 l 6 M urea, 5 mm Tris- HCl, ph 8.. A.2 M dithiothreitol (DTT) stock solution was added to obtain 1 mm concentration and the protein mixture was incubated at room temperature for 1 hour. A final concentration of 3 mm iodoacetamide (IAM) was reached by adding a stock solution of.2 M IAM. The alkylation was performed at room temperature for 1 hour. The final urea concentration was reduced to.6 M with the addition of 5 mm Tris-HCl, 1 mm CaCl 2, ph 7.6. The protein mixture was then digested with trypsin overnight at 37 o C with a trypsin: protein ratio of 1:5 (w/w). 1 l of formic acid was added to stop the digestion. The digest was maintained at 4 o C in an auto sampler for SEC- HPLC runs or frozen at -2 o C for future analysis. Results Figure 1A showed the separation profile of four peptides with mobile phase 25 mm sodium acetate/3 mm NaCl, ph 4.5. Four peptides did not get separated with the addition of high salt. Based on the individual peptide injections (data not shown), the wide peak between 14.5 and 16.5 utes in figure 1A was glucagon and insulin. Interaction between the peptides and the solid phase was the reason that glucagon and insulin were eluted later than angiotensin I and bradykinin (the main peak at 1.2 utes). When the mobile phase was switched to 5 mm phosphate, 3% MeOH and.1% TFA, four peptides were separated, but not according to their molecular weight order. According to the molecular weights, the order of elution should be insulin, glucagon, angiotensin I and bradykinin. 2

7 Figure 1B showed the separation profile with the elution in the order of insulin, bradykinin, angiotensin I and glucagon. The combination of methanol and TFA was not strong enough to disrupt the interactions between the column matrix and peptides. Thus, it did not achieve separation of four peptides according to their molecular sizes. When the mobile phase was switched to a TFA/acetonitrile system, all four peptides were eluted at earlier retention times, and separation resolutions were improved dramatically. The solid phase and peptide interaction was imized with the addition of high concentration acetonitrile. As seen in Figure 2, the separation of the four peptides was closely associated with the percentage of acetonitrile in the mobile phases. To achieve the best separation of the peptide mixture, the optimal acetonitrile concentration was at 75% in aqueous.1% TFA. Table 1 summarized the separation parameters for four peptides on Zenix -8. Angiotensin I and bradykinin achieved baseline separation. Furthermore, the elution order of the four peptides was related to their molecular weights or sizes under the denaturing.1% TFA/75% acetonitrile/h 2 O. Rarely, a complex protein digest can be resolved in one single reversed phase LC run. With Zenix -8 SEC, E. coli lysate can be prefractionated and the fractions can be further subjected to ion exchange or C18 reversed phase separation. SEC fractions can be directly applied to 2D LC/MS/MS as well for peptide mapping and identification purpose. Thus sample complexity can be greatly reduced with Zenix - 8 SEC pre-fractionation. For more information on Zenix TM - SEC products, please visit our website: or contact us at SEPAX-US. Size exclusion chromatography can be used as a pre-fractionation first dimension separation for complex peptide mixtures. Figure 3 showed the separation profiles of E. coli digest under different mobile phase conditions. Under acidic salt condition with 25 mm sodium acetate/.3 M NaCl, ph 4.5, the chromatogram exhibited a higher degree of peptide separation than the ones with the other two mobile phases -.1% TFA/75%acetonitrile/H 2 O and 5 mm phosphate/ 3% MeOH/.1% TFA. Conclusion Zenix -8 successfully separated four peptides whose molecular weights range from 1 kd to 6 kd. Organic additives help disrupt the columnpeptide interaction. There is an optimum concentration of acetonitrile at 75% in aqueous.1% TFA as mobile phase to give the best separation of the four peptides. Two 7.8x3 mm Zenix -8 columns in tandem will improve separation with wider retention time between peptides. 3

8 A 25 mm sodium acetate/3 mmnacl, ph 4.5 Absorbance at 214 nm B mm phosphate, 3% MeOH +.1% TFA Figure 1. Peptide separation (bradykinin, angiotensin I, glucagon and insulin) on Zenix -8 (7.8x3 mm). Panel A was with mobile phase 25 mm sodium acetate and 3 mm NaCl, ph 4.5. Panel B was with mobile phase 5 mm phosphate, 3% MeOH and.1% TFA. Peak elution order: 1. Insulin, 2. Bradykinin, 3. Angiotensin I, 4. Glucagon. The flow rate was.8 ml/ % ACN Absorbance at 214 nm % ACN 75% ACN 85% ACN Figure 2. Effect of mobile phase acetonitrile concentration on the separation of the four peptide mixtures on Zenix -8 (7.8x3 mm) (Peak 1, insulin, Peak 2, glucagon, Peak 3, angiotensin I, Peak 4, bradykinin). The mobile phases contained.1% TFA with the indicated percentage of acetonitrile. The flow rate was.8 ml/. 5 L of peptide mixture (.5 mg/ml concentration for each peptide) was injected. 4

9 Table 1. Separation parameters for the four peptide mixture with mobile phase 75% Acetonitrile in.1% TFA with water. Peak Protein MW (Da) Retention Resolution Plate counts time () 1 Insulin (porcine) 5, ,711 2 Glucagon 3, ,132 3 Angiotensin I 1, ,741 4 Bradykinin 1, ,6 5mM phosphate, 3% MeOH +.1% TFA 25 mm sodium acetate, 3 mm NaCl, ph 4.5 Absorbance at 214 nm.1% TFA, 75% ACN 5 mm phosphate, 3% MeOH +.1% TFA Figure 3. Separation of E. coli digests on Zenix -8 (7.8x3mm) under indicated mobile phases. Bottom chromatogram was for the run with 5mM phosphate/3%meoh/.1% TFA. The middle one was for the run with.1% TFA/75% acetonitrile. The top chromatogram represented the run with 25 mm sodium acetate/3 mm NaCl, ph 4.5. The inset was for the zoom view for the part of the chromatogram circled. Reference: 1. Irvine G. J. Biochem. Biophys. Methods, (23) Swergold G., and Rubin C. Anal. Biochem. (1983) 131, Tryptic digestion protocol: 4. Kinter, M., and Sherman, N. E. 2, Protein sequencing and identification using tandem mass spectrometry. JohnWiley & Sons, Inc. pp

10 Order information Part Number Particle Size Pore Size IDxLength [1] 3 µm 8 Å 2.1x5mm µm 8 Å 2.1x3mm [1] 3 µm 8 Å 4.6x5mm 2138P-465 [1][2] 3 µm 8 Å 4.6x5mm µm 8 Å 4.6x15mm µm 8 Å 4.6x25mm µm 8 Å 4.6x3mm 2138P-463 [2] 3 µm 8 Å 4.6x3mm [1] 3 µm 8 Å 7.8x5mm µm 8 Å 7.8x15mm µm 8 Å 7.8x2mm µm 8 Å 7.8x3mm [1] 3 µm 8 Å 1.x5mm µm 8 Å 1.x1mm µm 8 Å 1.x15mm µm 8 Å 1.x25mm µm 8 Å 1.x3mm [1] 3 µm 8 Å 21.2x5mm µm 8 Å 21.2x25mm µm 8 Å 21.2x3mm [1] Guard column [2] Column packed with PEEK tubing 6

11 AE11 Analysis of Peptide on Antibodix NP1 (1 µm, 4.6x25 mm) Gradient:% - 5% B (2 ) Gradient:% - 2% B (2 ) 2%B after Min Columns: Antibodix NP1 (1 µm,4.6x25 mm) Mobile Phases: A, 3 mm phosphate buffer, ph 5.; B, 2mM phosphate buffer, ph 7.5 Gradient: as indicated on the chromatogram Flow Rate: 1. ml/ Detection: UV 214 nm Temperature: Ambient Concentration: 2. mg/ml Injection Volume: 5 µl Sample: a peptide drug (pi=7~8) Keywords: Weak cation exchange, Antibodix, peptide Sepax Technologies, Inc.

12 BE11 Analysis of a Peptide on Bio-C18 (5µm, 3 Å, 4.6x25mm) Column: Mobile Phase: Flow Rate: Temperature: Detection: UV 214 nm Injection Volume: 2 µl Samples: 1. Peptide Bio-C18, 5 m, 3 Å, mm A: 1% ACN (.1% TFA); B: 9% ACN (.1% TFA) Time () B (%) ml/ Ambient Keywords: Bio-C18, reverse phase, peptide, biochemistry Sepax Technologies, Inc.

13 BE12 Analysis of Microcystin on Bio-C18 (5µm, 3 Å, 4.6x25mm) Column: Bio-C18, 5 m, 3 Å, 4.6x25 mm Mobile phase: CH 3 OH: Buffer (.5 M KH 2 PO 4, ph 3.) =57: 43 Flow rate:.8 ml/ Temperature: Ambient Detection: 238nm Injection Volume: 2 µl Sample: 1. Microcystin Keywords: Bio-C18, reverse phase, microcystin, environmental Sepax Technologies, Inc.

14 EE11 Analysis of mpeg and mpeg-peptide on Zenix and Zenix-C SEC Phases 12 Zenix-3 SEC Zenix-C 3 SEC-3 2xmPEG-peptide mpeg-peptide Column: Zenix SEC-3, 3 m, 7.8x3mm and Zenix-C SEC-3, 3 m, 7.8x3mm Mobile Phase: 15 mm Sodium Phosphate Buffer, ph 7. Flow Rate: 1. ml/ Temperature: 25 o C Detection: UV 214nm Injection Volume: 2 µl Sample: 4kD peptide and 2kD mpeg-mal (methoxy-peg-maleimide) (6mg/mL) methoxy-peg-maleimide Sepax Technologies, Inc.

15 14 Zenix SEC-3 Zenix Zenix-C SEC-3 Zenix-C Column: Zenix SEC-3, 3 m, 7.8x3mm and Zenix-C SEC-3, 3 m, 7.8x3mm Mobile Phase: 15 mm Sodium Phosphate Buffer, ph 7. Flow Rate: 1. ml/ Temperature: 25 o C Detection: UV 214nm Injection Volume: 2 µl Sample: 2kD mpeg-mal (methoxy-peg-maleimide) (1mg/mL)

16 OH13 Analysis of Valsartan on HP-C18 Column: HP-C18, 5 µm, 12 Å, mm Mobile phase: ACN: H 2 O: HAC = 5: 5: 1 (v/v) Flow rate: 1. ml/ Temperature: Ambient Detection: 273 nm Injection Volume: 1 µl Sample: Valsartan Keywords: HP-C18, reverse phase, valsartan, antihypertensive drugs, pharmaceuticals Sepax Technologies, Inc.

17 PE11 Separation of Peptide on Proteomix SCX-NP3 (3 µm, 4.6x5 mm) volatile buffer Pressure C1 C2 C3 C4 Column: Proteomix SX-NP3 (3 m, 4.6x5mm) Mobile Phases: A, 5 mm CH 3 COONH 4 ; B,.5 M CH 3 COONH 4 :ACN=4:1 (v/v) Gradient: -5% B (2 ) Flow Rate:.6 ml/ Detection: ELSD SofTA & UV 28 nm Injection Volume: 5 µl Concentration:.1 mg/ml Samples: Peptide Sequence Net Charge C1 Ac-Gly-Gly-Gly-Leu-Gly-Gly-Ala-Gly-Gly-Leu-Lys-amide +1 C2 Ac-Lys-Tyr-Gly-Leu-Gly-Gly-Ala-Gly-Gly-Leu-Lys-amide +2 C3 Ac-Gly-Gly-Ala-Leu-Lys-Ala-Leu-Lys-Gly-Leu-Lys-amide +3 C4 Ac-Lys-Tyr-Ala-Leu-Lys-Ala-Leu-Lys-Gly-Leu-Lys-amide + 4 (Courtesy of Miyako Kawakatsu, M&S Instruments Inc.) Keywords: Ion-exchange, Proteomix, strong cation exchange, peptide, volatile buffer Sepax Technologies, Inc.

18 ZE12 Analysis of Peptide Mixture on Zenix -1 (7.8x3) Column: Zenix -1 (3 µm, 1 Å, 7.8x3 mm) Mobile Phase: ACN:H2O:TFA=65:35:.1(v/v) Flow Rate: 1. ml/ Temperature: Ambient Detection: UV 214 nm Injection Volume: 1 µl Samples: Ribonuclease A (5.931 ), human insulin (6.936 ), thymosin α1 (7.252 ), somatostatin (8.89 ) Keywords: Size exclusion, Zenix, peptide, thymosin α1, human insulin, Somatostatin Sepax Technologies, Inc.

19 ZE14 SEC Resolution Comparison for Peptide Separation Brand X (5 µm, 15 Å, 4.6x3mm) Zenix SEC-15 (3 µm, 15 Å, 4.6x3mm) Zenix SEC-15 (3 µm, 15 Å, 7.8x3mm) Min Mobile Phase: 15 mm Phosphate Buffer, ph 7.; Flow Rates:.35 ml/ for 4.6x3 mm & 1. ml/ for 7.8x3 mm Temperature: Ambient Detection: UV 214 nm Injection Volumes: 5 µl for 4.6x3 mm & 2 µl for 7.8x3 mm Sample: Peptide mixture (provided by a customer) Keywords: Size exclusion, Zenix, peptide, high resolution Sepax Technologies, Inc.

20 ZE15 Analysis of 5kD and 8kD Peptides by Zenix SEC AU Min Column: Zenix SEC-15 (3 µm, 15 Å, 7.8x3 mm) Mobile Phase: 15 mm Phosphate Buffer, ph 7.; Flow Rate: 1. ml/ Temperature: Ambient Detection: UV 22 nm Injection Volume: 1 µl Sample: Salcatonin-type peptide mixture with MW of 8 kd and 5kD Keywords: Size exclusion, Zenix, peptide, salcatonin Sepax Technologies, Inc.

21 ZE16 Analysis of Four Peptide Mixture from 1kD to 6kD on Zenix -8 (783) Zoom Column: Zenix SEC-8 (3 µm, 8 Å, 7.8x3 mm) Mobile Phase: ACN:H 2 O:TFA=75:25:.1(v/v) Flow Rate:.8 ml/ Temperature: Ambient Detection: UV 214 nm Injection Volume: 5 µl Sample: Insulin from Porcine pancreas (7.749, MW 5,778), Glucagon (8.34, MW 3,483), Angiotensin I (8.45, MW 1,297), Bradykinin (8.858, MW 1,6) Keywords: Size exclusion, Zenix, peptide, 8 Å pore size, insulin, bradykinin, glucagon, angiotensin I Sepax Technologies, Inc.

22 Z Analysis of an mpeg-peptide on Zenix SEC Phases 12 Zenix Zenix-C 3 mpeg-peptide 2 1 2xmPEG-peptide Column: Zenix SEC-3, 3 m, 7.8x3mm and Zenix-C SEC-3, 3 m, 7.8x3mm Mobile Phase: 15 mm Sodium Phosphate Buffer, ph 7. Flow Rate: 1. ml/ Temperature: 25 o C Detection: UV 214nm Injection Volume: 2 µl Sample: 4kD peptide and 2kD mpeg-mal (methoxy-peg-maleimide) (6mg/mL) methoxy-peg-maleimide Sepax Technologies, Inc.

23 14 Zenix Zenix-C Column: Zenix SEC-3, 3 m, 7.8x3mm and Zenix-C SEC-3, 3 m, 7.8x3mm Mobile Phase: 15 mm Sodium Phosphate Buffer, ph 7. Flow Rate: 1. ml/ Temperature: 25 o C Detection: UV 214nm Injection Volume: 2 µl Sample: 2kD mpeg-mal (methoxy-peg-maleimide) (1mg/mL)

24 ZE18 Peptide Separations on Zenix TM -8 (783) from 13.7 kd to 12 Da Column: Zenix SEC-8 (3 µm, 8 Å, 7.8x3 mm) Mobile Phase: ACN:H 2 O:TFA=75:25:.1(v/v) Flow Rate:.8 ml/ Temperature: Ambient Detection: UV 214 nm Injection Volume: 5 µl Sample: 1.Ribonuclease A (13.7 kd); 2. Insulin from Porcine pancreas (5,778 Da); 3. Glucagon (3,84 3); 4. Angiotensin I (1,297 Da); 5. Bradykinin (16 Da); 6. Uracil (12 Da) Keywords: Size exclusion, Zenix, peptide, 8 Å pore size, insulin, bradykinin, glucagon, angiotensin I, ribonuclease A, Uracil, small peptide Sepax Technologies, Inc.

25 ZE12 Analysis of Peptide Mixture on Zenix -1 (7.8x3) Column: Zenix -1 (3 µm, 1 Å, 7.8x3 mm) Mobile Phase: ACN:H2O:TFA=65:35:.1(v/v) Flow Rate: 1. ml/ Temperature: Ambient Detection: UV 214 nm Injection Volume: 1 µl Samples: Ribonuclease A (5.931 ), human insulin (6.936 ), thymosin α1 (7.252 ), somatostatin (8.89 ) Keywords: Size exclusion, Zenix, peptide, thymosin α1, human insulin, Somatostatin Sepax Technologies, Inc.

26 Exenatide Analysis on Zenix SEC-8 Column: Zenix SEC-8 (3 µm, 8 Å, 7.8 x 3 mm), Flow rate:.8 ml/, Detector: UV 214 nm, Injection volume: 1 µl, Column temperature: Room temperature, Samples: 1 mg/ml exenatide in water (MW= 4,187 Da) used for treatment of diabetes mellitus type 2 EE % ACN with.1% TFA 5 5% ACN with.1% TFA 8% ACN with.1% TFA Better Surface Chemistry for Better Separation Sepax Technologies, Inc.

27 Exenatide Analysis on Zenix SEC-8 Column: Zenix SEC-8 (3 µm, 8 Å, 7.8 x 3 mm), Mobile phase: ACN : H 2 O : TFA =8 : 2 :.1 ( v/v ), Flow rate:.8 ml/, Detector: UV 214 nm, Injection volume: 1 µl, Column temperature: Room temperature, Samples: 1 mg/ml exenatide in water EE Better Surface Chemistry for Better Separation Sepax Technologies, Inc.

28 Pegylated Exenatide on Zenix and Zenix-C Column: Zenix SEC-3 and Zenix-C SEC-3 (3 µm, 3 Å, 7.8 x 3 mm), Mobile phase: 5 mm CH 3 COONH 4 : ACN = 9 : 1 ( v/v ), Flow rate:.5 ml/, Detector: UV 214 nm, Column temperature: 25, Injection volume: 15 µl, Pressure: 42 bar, Sample: 3.3 mg/ml PEG-Exanatide in water (PEG 23 kd) EE Zenix-C SEC-3 Zenix SEC-3 Tailing 1.7 Tailing Better Surface Chemistry for Better Separation Sepax Technologies, Inc.

29 Transfer Factor Oral Solution SEC Separation Column: SRT SEC-1, Zenix SEC-8 and Zenix-C SEC x 3 mm, Mobile phase: 15 mm sodium phosphate buffer ph 7., Flow rate: 1. ml/, Detector: UV 251 nm, Sample: 1 µl 1mg/mL transfer factor oral solution (dietary supplement/peptide mixture, immune molecules to boost immune response) 25 Zenix-C SEC-1 Best separation Zenix SEC SRT SEC EE13 Better Surface Chemistry for Better Separation Sepax Technologies, Inc.

30 Analysis of Peptide Digest on Proteomix SCX NP1.7 Column: Proteomix SCX NP x 3 mm without guard, Flow rate:.35 ml/, Detection: UV 214 nm, Mobile phase: A: 1 mm phosphate buffer, ph 2.5, B: A + 1 M NaCl, C: 1% acetonitrile, Sample: trypsin digested BSA, Pressure: 38 bar PE µg digested BSA 1 µg digested BSA Time () %A %B %C

31 Gradient Comparisons for the Analysis of Peptides on Proteomix SCX NP1.7 Column: Proteomix SCX NP5 4.6 x 1 mm without guard, Flow rate:.35 ml/, Detection: UV 214 nm, mobile phase: A: 1 mm phosphate buffer, ph 2.5, B: A + 1 M NaCl, C: 1% acetonitrile, Sample: Peptide mixture, Pressure: 31 bar, Gradient: as indicated PE AAAAL (1 mg/ml) 2. AAAKL (2mg/mL) 3. AAKKL (2mg/mL) 4. AKKKL (2mg/mL) 5. KKKKL (2mg/mL) % B 3-9% B % B - 9% B

32 Peptide Separation Comparison on Proteomix SCX NP5 and Proteomix SCX NP1.7 Column: Proteomix SCX NP5 4.6 x 1 mm without guard and NP x 3 mm, Detection: UV 214 nm, mobile phase: A: 1 mm phosphate buffer, ph 2.5, B: A + 1 M NaCl, C: 1% acetonitrile, Sample: 2 L Peptide mixture, Pressure: 74 bar for NP5, 54 bar for NP1.7 PE Time () %A %B %C µm.6 ml/ 5 µm 1. ml/

33 Zoomed View of Peptide Separation Comparison on Proteomix SCX NP5 and Proteomix SCX NP1.7 Column: Proteomix SCX NP5 4.6 x 1 mm without guard and NP x 3 mm, Detection: UV 214 nm, mobile phase: A: 1 mm phosphate buffer, ph 2.5, B: A + 1 M NaCl, C: 1% acetonitrile, Sample: 2 L Peptide mixture, Pressure: 74 bar for NP5, 54 bar for NP1.7 7 PE Time () %A %B %C µm.6 ml/ 5 µm 1. ml/

34 Exenatide Analysis on Zenix SEC-8 Column: Zenix SEC-8 (3 µm, 8 Å, 7.8 x 3 mm), Flow rate:.8 ml/, Detector: UV 214 nm, Injection volume: 1 µl, Column temperature: Room temperature, Samples: 1 mg/ml exenatide in water, MW= 4,187 Da, used for treatment of diabetes mellitus type 2 ZE19 Mobile phase 1: ACN : H 2 O : TFA =5 : 5 :.1 ( v/v ) Mobile phase 2: ACN : H 2 O : TFA =7 : 3 :.1 ( v/v ) Mobile phase 3: ACN : H 2 O : TFA =8 : 2 :.1 ( v/v ) Mobile 2 5 Mobile 1 Mobile Better Surface Chemistry for Better Separation Sepax Technologies, Inc.

35 Exenatide Analysis on Zenix SEC-8 Column: Zenix SEC-8 (3 µm, 8 Å, 7.8 x 3 mm), Mobile phase: ACN : H 2 O : TFA =8 : 2 :.1 ( v/v ), Flow rate:.8 ml/, Detector: UV 214 nm, Injection volume: 1 µl, Column temperature: Room temperature, Samples: 1 mg/ml exenatide in water Better Surface Chemistry for Better Separation Sepax Technologies, Inc.

36 Pegylated Exenatide on Zenix and Zenix-C Column: Zenix SEC-3 and Zenix-C SEC-3 (3 µm, 3 Å, 7.8 x 3 mm), Mobile phase: 5 mm CH 3 COONH 4 : ACN = 9 : 1 ( v/v ), Flow rate:.5 ml/, Detector: UV 214 nm, Column temperature: 25, Injection volume: 15 µl, Pressure: 42 bar, Sample: 3.3 mg/ml PEG-Exanatide in water (PEG 23 kd) Zenix-C SEC-3 Zenix SEC-3 Tailing 1.7 Tailing Better Surface Chemistry for Better Separation Sepax Technologies, Inc.