High Resolution Silica Hydrophobic Interaction Chromatography (HIC) Column for Protein/Peptide Separations with Improved Hydrolytic Stability

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1 High Resolution Silica Hydrophobic Interaction Chromatography (HIC) Column for Protein/Peptide Separations with Improved Hydrolytic Stability S. Rao, A. Bordunov, and C. Pohl, Corporation, Sunnyvale, CA, USA ABSTRACT HIC is a powerful technique and is routinely used for protein purification. We have developed a new silica based HIC stationary phase with multifunctional amide group attachments. The surface chemistry of this phase was optimized by inclusion of hydrophobic and hydrophilic ligands which were designed to improve the hydrolytic stability of silica support in the aqueous media. These modifications resulted in superior resolution and peak efficiencies of proteins/ peptides and provided a versatile column with sufficient longevity. HIC separates proteins, peptides and other biomolecules by taking advantage of surface exposed hydrophobicities. HIC complements ionexchange chromatography and gel filtration chromatography for separation of various bio molecules. In this poster, we have demonstrated the use of ProPac HIC- column for various applications. Some examples include ) Mixture of proteins consisting of cytochrome c, myoglobin, ribonuclease A, lysozyme and chymotrypsinogen ) Mixture of peptides ) Trypsin digests of proteins ) Monoclonal antibodies ) Snake venom proteins 6) Serum proteins 7) Pancreatin. We have determined the loading capacity for proteins and for monoclonal antibodies. Also, the effect of addition of solvent to the eluent on the peak shape was evaluated. ProPac HIC- column exhibited excellent reproducibility and ruggedness. We have compared the ProPac HIC- column with other leading HIC columns for efficiency, peak shape, resolution, and capacity. The new ProPac HIC- column displayed high capacity and superior overall performance. INTRODUCTION Hydrophobic interaction chromatography (HIC) is a technique for separation of proteins, peptides, and other biomolecules by their degree of hydrophobicity. The mobile phase consists of a salting-out agent (e.g., ammonium sulfate), which at high concentration retains the protein by increasing the hydrophobic interaction between the solute and the stationary phase. The bound proteins are eluted by decreasing the salt concentration. The effects of salts in HIC can be accounted for:. The precipitation of proteins or. Their positive influence in increasing the molal surface tension of water. Increasing precipitation (salting - out) effect Anions: PO -, SO -, CH COO -, Cl -, Br -, NO -, CLO -, I -, SCN - Cations: NH +, Rb +, K +, Na +, Cs +, Li +, Mg +, Ca +, Ba + Increasing chaotropic (salting - in) effect Relative effects of salts on the molal surface tension of water are as follows. Sodium, potassium and ammonium sulphates produce relatively higher salting out or molal surface tension increment effects than sodium or lithium chloride. Na SO > K SO > (NH ) SO >Na HPO > NaCl> LiCl >KSCN HIC differs from reverse-phase chromatography in the selection of solvent conditions. HIC does not denature proteins during purification as compared to reverse-phase HPLC and preserves biological activity. HIC is ideal after salt precipitations and ion-exchange chromatography. HIC has been used extensively for purifying a variety of biomolecules, including serum proteins,8, membrane bound proteins, nuclear proteins 6, recombinant proteins 7, and receptors 9. Several recent applications reported analysis of human α-thrombin, and purifications of various isotypes of caseins from milk. HIC has also been used in the purification of monoclonal antibodies and purification of enzymes. These and various other applications have shown the general applicability of HIC as an important purification step at the initial, intermediate, and/or final steps in the purification schemes of proteins. HPLC 6 Presentation HPLC 6 Presentation

2 In this study, we have presented results from a newly developed silicabased HIC stationary phase with multifunctional attachment of amide groups. The surface chemistry of this phase was optimized by inclusion of hydrophobic and hydrophilic ligands that were designed to improve the hydrolytic stability of silica support in the aqueous media. These modifications resulted in superior resolution and peak efficiencies of proteins/peptides and provided a versatile column with sufficient longevity. We have compared our new ProPac HIC- with columns from leading vendors and the results indicated that the new HIC column showed high-capacity and superior overall performance. MATERIALS Chromatographic Components P68 HPG pump, UVD-U detector, GINA autosampler were from Corporation Column oven TCC- from Corporation and L-6 is from SSI. Chromatography was controlled by Chromeleon Chromatography Management Software from Corporation. Proteins, Peptides, Biomaterials, and Chemicals Cytochrome c, myoglobin, ribonuclease A, chymotrypsinogen, bovine serum albumin, β-casein, bovine serum, thrombin, cobra venom, and HPLC peptide standard mixture were obtained from Sigma. MAb was a gift from Protein Design Labs, Fremont, CA. Ammonium sulfate and all other analytical-grade chemicals were also obtained from Sigma. Lysozyme is from Calbiochem. Immobilized trypsin was from Pierce. Columns from Corporation ProPac HIC- are Silica columns based on proprietary ethyl/amide based chemistry P/N Description Dimensions 66 ProPac HIC- µm, Å. mm 66 ProPac HIC- µm, Å.6 mm 666 ProPac HIC- µm, Å mm Columns from Commercial Vendors for Comparison Biosuite Phenyl (Polymer, µm, Å, 7. 7 mm) from Waters Corp. Column-Phenyl PW ( Polymer, µm, Å, 7. 7 mm) from Corp. Ethyl (Silica, µm, Å,.6 mm) from PolyLC Inc. METHODS Capacity test by breakthrough experiment After equilibrating the column with eluent A, either lysozyme (. mg/ml, Figure ) or MAb (.8 mg/ml, Figure ) was added to eluent A with mixing. Continue pumping the column with eluent A (containing the protein) and monitor the UV trace to record the capacity. Stability of the ProPac HIC- bonded phase and mechanical ruggedness An isocratic small molecule test was performed between the repeated gradient runs to access the ruggedness of the ProPac HIC- column. The test mixture contained cytosine, -butyl benzoic acid, amitriptyline, and phenanthrene; each of µg/ml. This small molecule mixture was injected ( µl) interspersed by blank methanol gradient runs (% 9%). The total run time for blank is 6 min. The total run time for the small molecule mixture is min including min pre-injection equilibration. A total of cycles were performed including runs of small molecule injections to test the ruggedness of the ProPac HIC- column. The data was plotted for one of the small molecules (phenanthrene) as injection number vs. retention time, asymmetry, peak width (%) and efficiency (plates). Gradients. mm format: %B in min.6 mm format: %B in min 7. 7 mm format: %B in 6 min mm format: %B in 6 min Or, as described in figures. Dual Gradients Dual gradients include an initital aqueous gradient followed by an organic solvent gradient. High Resolution Silica Hydrophobic Interaction Chromatography (HIC) Column for Protein/Peptide Separations with Improved Hydrolytic Stability

3 SAMPLE PREPARATION Protein mixture Mixture of proteins ( mg/ml each final after : dilution with eluent A). () cytochrome c, () myoglobin, () ribonuclease A, () lysozyme, () chymotrypsinogen Peptide mixture Mixture of peptides : diluted with eluent A. () Gly-Tyr, () Val-Tyr-Val, () Methionine Enkephalin Acetate (Tyr-Gly-Gly-Phe- Met), () Leucine Enkephalin (Tyr-Gly-GLy-Phe-Leu), () Angiotensin II acetate (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe) Bovine serum Bovine serum (: diluted with D.I. H O and mixed with equal volume of M (NH ) SO in. M NaH PO (ph 7.) Cobra venom Naja venom mg/ml in PBS and diluted : with M (NH ) SO in. M NaH PO (ph 7.) Pancreatin mg + µl D.I. H O + µl eluent A, mixed well and spun at, rpm for min. Supernatant was used as Pancreatin sample. Proteomics applications; Separation of peptides (). mg of cytochrome c was digested with immobilized trypsin (Pierce). The digest was mixed with equal volume of eluent A before applying onto the column. See Figure 8. Monoclonal antibody (MAb) MAb was prepared as described in Figure 9 and was separated with % MeCN in Eluent B (Figure 9B) or without MeCN in eluent B (Figure 9A). Protein/Peptide Standards Column:.6 mm (PolyLC).6 mm ( ProPac HIC-) Flow:. ml/min Eluents: (A) M (NH ) SO in. M NaH PO (ph 7.) (B). M NaH PO (ph 7.) Sample: Mixture of proteins ( mg/ml each final after : dilution with eluent A) Inj. Volume: µl, Figure. Separation of a Mixture of Proteins PolyLC Order of elution:. Cytochrome c. Myoglobin. Ribonuclease A. Lysozyme. Chymotrypsinogen Figure. Separation of a Mixture of Proteins Column:. mm ( ProPac HIC-) Flow:. ml/min Eluents: (A) M (NH ) SO in. M NaH PO (ph 7.) (B). M NaH PO (ph 7.) Sample: Mixture of proteins ( mg/ml each final after : dilution with eluent A) Inj. Volume: µl Order of elution:. Cytochrome c. Myoglobin. Ribonuclease A. Lysozyme. Chymotrypsinogen 6 Figure. Separation of a Mixture of Proteins Column: 7. 7 mm (Waters and ); Sample: mm ( ProPac HIC-) Flow:. ml/min Eluents: (A) M (NH ) SO in. M NaH PO (ph 7.) (B). M NaH PO (ph 7.) Mixture of proteins (mg/ml each final after : dilution with eluent A) Inj. Volume: µl Order of elution:. Cytochrome c. Myoglobin. Ribonuclease A. Lysozyme. Chymotrypsinogen Waters HPLC 6 Presentation

4 Figure. Separation of Peptide Mixture Figure 6. Separation of Pancreatin 8 Column: 7. 7 mm (Waters and ) mm ( ProPac HIC-) Flow:. ml/min Eluents: (A) M (NH ) SO in. M NaH PO (ph 7.) (B). M NaH PO (ph 7.) Sample: Peptide mixture from Sigma (: diluted with eluent A) Inj. Volume: µl Column: 7. 7 mm (Waters and ); mm ( ProPac HIC-) Flow:. ml/min Eluents: See Figure Bound 7 9 Sample: Pancreatin 8 Inj. Volume: µl 6 6 Unbound Waters, Waters APPLICATIONS 6 Figure. Separation of Bovine Serum Proteins Column: 7. 7 mm (Waters and ) mm ( ProPac HIC-) Flow:. ml/min Eluents: See Figure Sample: Bovine serum (: dilited with D.I. H O and mixed with equal volume of M (NH ) SO in. M NaH PO (ph 7.) Inj. Volume: µl BSA Waters IGG Figure 7. Separation of Snake Venom Proteins/Peptides Column: 7. 7 mm (Waters and ) mm ( ProPac HIC-) Flow:. ml/min Eluents: (A) M (NH ) SO in. M NaH PO (ph 7.) (B). M NaH PO (ph 7.) Sample: Naja venom from Sigma [: diluted with M (NH ) SO in. M NaH PO (ph 7.)] Inj. Volume: µl Waters High Resolution Silica Hydrophobic Interaction Chromatography (HIC) Column for Protein/Peptide Separations with Improved Hydrolytic Stability

5 Figure 8. Separation of Peptides Proteomics Application Column:.6 mm ( ProPac HIC-) Flow:. ml/min Eluents: (A) M (NH ) SO in. M NaH PO, ph 7. (B). M NaH PO, ph 7. Eluents: (C) D.I. H O (D) 7% MeCN Sample: Trypsin digest (. mg) of Cytochrome c (Immobilized trypsin was used) Inj. Volume: µl Dual gradient: % B in min. After equilibration: % D in min,6 %B %D %C Figure 9. Separation of Monoclonal Antibody (MAb) Column: ProPac HIC- (.6 mm) Flow: ml/min WVL: 8 nm Sample: MAb µl ( mg/ml) + µl Eluent A Eluents: (A). M (NH ) SO in.m NaH PO (ph 7.) (B). M NaH PO (ph 7.) Inj. Volume: µl ( µg) Gradient: 7 % B in min A. No MeCN in eluent B 7 %B COLUMN capacity,,8 Figure. Capacity of HIC Columns, Lysozyme Column: 7. 7 mm, (Waters) mm, ( ProPac HIC-) Flow:. ml/min Eluents: (A) M (NH ) SO in. M NaH PO (ph 7.) (For loading eluent A +. mg/ml lysozyme) (B). M NaH PO (ph 7.) Waters column ProPac HIC- Capacity mg mg Waters HIC column HIC column 6 8,,,,6,8 97 Figure. Capacity of HIC Columns, MAb Column:.6 mm ( ProPac HIC-) Flow:. ml/min Eluents: (A) M (NH ) SO in. M NaH PO (ph 7.) (for loading eluent A +.8 mg/ml MAb) (B). M NaH PO (ph 7.) Breakthrough capacity of MAb = ~mg, Sample: Inj. Volume: Gradient: MAb µl ( µg) 7 % B in 7 min. B. % MeCN in eluent B 7 %B HPLC 6 Presentation

6 COLUMN STABILITY Figure. Stability of the Bonded Phase and Mechanical Ruggedness Peak Width/ Asymmetry/ Retention Time Column:.6 mm, ( ProPac HIC-) Flow: ml/min Eluent (Isocratic ): % CH CN/. M Amm. Acetate ph. Eluents (Gradient): Eluent A: mm Ammonium acetate ph. Eluent B: % Methanol Sample: Small molecule mixture was injected every th cycle Cycle Number Retention Time Asymmetry CONCLUSION Peak Width (% Height) Efficiency (Plates) Efficiency (Plates) 9 We introduced a new Hydrophobic Interaction Chromatography column (ProPac HIC-) for the separation of proteins and peptides. We used our proprietary silica bonding technology for improved hydrolytic stability. The new silica ProPac HIC- column displayed excellent resolution and stability, and high capacity. Breakthrough capacity for lysozyme is mg (7.8 7 mm) and for MAb is mg (.6 mm). ACKNOWLEDGEMENTS We thank A. Heckenberg for her contributions to the development of ProPac HIC-. REFERENCES. Shaltiel, S.; Er-el, Z. Proc. Nat. Acad. Sci. US. 97, 7, Er-el, Z.; Zaidenzaig, Y.; Shaltiel, S. Biochem. Biophys. Res. Commun. 97, 9, 8.. Alpert, A. J. J. Chromatogr. 986, 9, 8.. Hrkal, Z.; Rejnkova, J. J. Chromatogr. 98,, 8.. McNair, R. D.; Kenny, A. J. Biochem. J. 979, 79, Comings, D. E. ; Miguel, A. G.; Lesser, H. H. Biochim. Biophys. Acta 979, 6,. 7. Belew, M.; Yafang, M.; Bin, L.; Berglof, J.; Janson, J.-C. Bioseparation 99,, Goheen, S. C.; Matson, R. S. J. Chromatogr 98, 6,. 9. Warren, B. S.; Kusk, P.; Wolford, R.G.; Hager, G. L. J. Biol. Chem. 996, 7,.. Karlsson, G. Prot. Exp. and Purif., 7, 7.. Bramanti, E.; Sortino, C.; Onor, M.; Beni, F.; Raspi, G. J. Chromatogr., A, 99, 9.. Yamasaki, Y.; Kitamura, T.; Kato, Y. Poster # presented at the 9th Intl. Symposium on HPLC of Proteins, Peptides, and Polynucleotides, Philadelphia, 6 9 Nov (989). Chromeleon and ProPac are registered trademarks of Corporation. We compared the performance of the ProPac HIC- column with other HIC columns for various applications. The ProPac HIC-, under the same conditions exhibited higher resolution and better overall performance. Passion. Power. Productivity. Corporation 8 Titan Way P.O. Box 6 Sunnyvale, CA North America Sunnyvale, CA (8) 77-8 Westmont, IL (6) Houston, TX (8) 87-6 Atlanta, GA (77) -8 Marlton, NJ (86) 96-6 Canada (9) 8-96 (8) Switzerland () High Resolution Silica Hydrophobic Interaction United Kingdom Chromatography () (HIC) Column for Protein/Peptide Separations with Improved Hydrolytic Stability Europe Austria () 66 Belgium () 9 Denmark () France () 9 Germany (9) Italy (9) 6 66 The Netherlands () 6 Asia Pacific Australia (6) 9 China (8) 8 8 India (9) 87 Japan (8) Korea (8) 6 8 LPN 86-6/6 6 Corporation