Switch to BioSep GFC Columns for Protein Analysis

Transcription

1 Switch to BioSep GFC Columns for Protein Analysis Expanded Resolution Windows vs. Other GFC Columns Batch-to-Batch Reproducibility Highly Inert Material for Better Recovery and Quantitation Designed for Biogenerics and Peptide/Protein Therapeutics Phenomenex India Laxmi Cyber City, Ground Floor, B Block, Survey no: Kondapur, Hyderabad 8, India Tel: - Fax: - indiainfo@phenomenex.com Web:

2 Columns for Gel Filtration Chromatography (GFC) GFC is used for the analysis and/or characterization of proteins, peptides and other biomolecules; including antibodies, immunoglobulins, protein complexes, protein aggregates, and desalting. BioSep GFC columns offer many important benefits to keep your research, method development/validation, and ongoing size exclusion separations SIMPLE: High Performance: Analytical and preparative BioSep columns offer high resolution, maximum efficiency, and exceptional peak asymmetry. Easy Column Selection: Simply choose the right phase based on the MW of your sample, recommended application, currently used GFC column, or contact us for assistance! Higher Value Solution: BioSep is a high quality gel filtration media that comes with an affordable price tag. Method Development and Optimization Services: Phenomenex Services offers method development and optimization support for new methods, as well as transferring your current methods from other GFC media to BioSep-SEC-S phases. If BioSep analytical columns do not provide you with at least an equivalent separation as any other GFC column of similar porosity, type, and dimensions, return column with comparative data within days for a FulL REFUND.

3 The BioSep Advantage Higher Efficiency for Greater Resolution... Expanded Resolution Windows... Batch-to-Batch Reproducibility... Highly Inert Material for Better Recovery and Quantitation... 7 Applications and Technical Support Proteins...9- Peptides and Small Proteins... Aggregates... NEW PEGylated Proteins...- Method Development Services... Column Care and Use... 7 Ordering Information Easy Column Selection... 8 Cross Reference Chart... 9

4 Higher Efficiency for Greater Resolution Achieve greater baseline separation between your analytes due to tight particle size distribution and packing specifications Protein Separation of - kda MW on BioSep-SEC-s vs. TSKgel GSW XL mau BioSep-SEC-s All four proteins are resolved Co-migration of peaks &, BioSep-SEC-s has a wider molecular weight window than TSKgel SW XL, which enables increased resolution of proteins on the higher end of the molecular weight range. As illustrated in the chromatogram, peaks and co-migrate with the TSKgel column, but are resolved with the BioSep-SEC-s column. TSKgel GSW XL APP ID 889 Conditions for both columns: Columns: BioSep-SEC-s TSKgel GSW XL Mobile Phase: mm Tris ph 7., mm Sodium Chloride Flow Rate:. ml/ Detection: nm Sample:. Hu IgA kda. b-amylase kda. BSA kda. Ovalbu kda Efficiency (imum number theoretical plates on x 7.8 mm column) SEC-S, Plates SEC-S, Plates SEC-S, Plates Terms and Conditions Phenomenex reserves the right to refuse sale at its discretion. Offer applies to endusers only. Trademarks TSKgel is a registered trademark of Tosoh Corporation. BioSep is a trademark of Phenomenex, Inc. Disclaimer Px Advanced Technology Solutions Pvt. Ltd. is a part of Phenomenex. Comparative separations may not be representative of all applications. Columns used for comparison were manufactured by Tosoh Corporation. Phenomenex is in no way affiliated with Tosoh Corporation. Phenomenex India Tel: - Fax: - indiainfo@phenomenex.com Web:

5 Expanded Resolution Windows Expect equal or better resolution than your current GFC column, guaranteed! Higher optimal molecular weight selectivity window and greater resolution of the analytes Human IgGk Aggregates on BioSep -SEC-s and TSKgel GSW XL mau mau TSKgel GSW XL BioSep- SEC-s, 8 8 High MW Proteins on BioSep -SEC-s and TSKgel GSW XL TSKgel GSW XL BioSep- SEC-s APP ID 89 APP ID 8898 Conditions for both columns: Columns: BioSep-SEC-s TSKgel GSW XL Mobile Phase: mm Sodium Phosphate ph.8 Detection: nm Sample:. HMW aggregates. IgG Trimer. IgG Dimer. IgG kappa dimer. Hu IgG kappa monomer. Low MW impurity Conditions for both columns: Columns: BioSep-SEC-s TSKgel GSW XL Mobile Phase: mm Sodium Phosphate ph.8 Detection: nm Sample:. HMW impurity. IgM 9 kda. Thyroglobulin 7 kda. IgA kda. b-amylase kda. BSA kda 7. Ribonuclease A.7 kda 8. Uridine Da If BioSep analytical columns do not provide you with at least an equivalent separation as any other GFC column of similar porosity, type, and dimensions, return the column with comparative data within days for a Full REFUND. Disclaimer Comparative separations may not be representative of all applications. Columns used for comparison were manufactured by Tosoh Corporation. Phenomenex is in no way affiliated with Tosoh Corporation. Trademarks TSKgel is a registered trademark of Tosoh Corporation. BioSep is a trademark of Phenomenex, Inc. Phenomenex India Tel: - Fax: - indiainfo@phenomenex.com Web:

6 Batch-to-Batch Reproducibility Reproducibility is of the utmost importance when validating methods Each batch of material is carefully monitored to ensure that particles have the proper size, shape, and pore characteristics batch-to-batch Every column is performance and QC tested to ensure the same high quality separation column-to-column Batch-to-Batch Variations on BioSep-SEC-s mau 7 8 Separate Batches Overlaid APP ID Three different silica batches were overlaid to show the batch-to-batch reproducibility of the media. Conditions same for all batches: Column: BioSep-SEC-s Part No.: H-7-K Mobile Phase: mm Sodium Phosphate ph.8 Detection: nm Sample:. HMW Impurity. lgm 9 kda. Thyroglobulin 7 kda. lga kda. b-amylose kda. BSA kda 7. Ribonuclease Å.7 kda 8. Uridine Da Phenomenex India Tel: - Fax: - indiainfo@phenomenex.com Web:

7 Highly Inert Material for Better Recovery and Quantitation BioSep experiences a noal amount of non-specific interactions which provides an extremely inert media demonstrating clear advantages for accurate quantitation of proteins and aggregates. Human Serum under Different Mobile Phases. Mobile phase containing NaCI. Mobile phase containing Arginine. Mobile phase without Arginine 8 APP ID 89 Equal recovery of proteins and aggregates under different mobile phase conditions is indicative of a highly inert column. Conditions same for all separations except for mobile phase: Column: BioSep-SEC-s Part No.: H--K Mobile Phase:. mm Sodium Phosphate ph 7., mm Sodium Chloride. mm Sodium Phosphate ph.8, mm Arginine. mm Sodium Phosphate ph.8 Detection: 8 nm Sample: Human Serum Phenomenex India Tel: - Fax: - indiainfo@phenomenex.com Web: 7

8 Recommended Applications for Each Phase SEC-S Biogeneric aggregate applications Peptide therapeutics Small proteins PEGylated peptides and small proteins SEC-S Immunoglobulin aggregate applications Medium to large size proteins Serum proteins SEC-S Large proteins PEGylated IgG Depending on the size and type of sample you have, there is a BioSep phase to fit your needs. BioSep SEC-s, SEC-s and SEC-s are all available in narrow bore, analytical, and preparative dimensions.

9 Applications Proteins MW Calibration on Proteins 7 8 APP ID 89 Column: BioSep-SEC-s Part No.: H-7-K Mobile Phase: mm Sodium Phosphate ph 7., mm Sodium Chloride Detection: nm Sample:. HMW impurity. lgm 9 kda. Thyroglobulin 9 kda. lga kda. b-amylase kda. BSA kda 7. Ribonuclease A.7 kda 8. Uridine Da High MW Protein Mixture 7 APP ID 899 Column: BioSep-SEC-s Part No.: H--K Mobile Phase: mm Sodium Phosphate ph.8, mm Sodium Chloride Detection: nm Sample:. IgM 9 kda. Thyroglobulin 7 kda. IgA kda. b-amylase kda. BSA kda. Ribonuclease A.7 kda 7. Uridine Da 8 Phenomenex India Tel: - Fax: - indiainfo@phenomenex.com Web: 9

10 Applications Proteins (cont d) Recombinant Human Erythropoietin (HuEPO) mau Column: BioSep-SEC-s Dimensions: x. mm Part No.: H--E Mobile Phase: mm Sodium Phosphate ph.8, mm Sodium Chloride Flow Rate:. ml/ Detection: nm Sample: Recombinant Human Erythropoietin. HMW impurity. EPO dimer. EPO monomer. LMW impurity APP ID 97 Human Serum and HSA Column: BioSep-SEC-s Part No.: H--K Mobile Phase: mm Sodium Phosphate ph 7., mm Sodium Chloride Detection: nm Sample: Human Serum Human Serum Albu (HSA) Human Serum Proteins Human Serum Albu APP ID 89 8 Phenomenex India Tel: - Fax: - indiainfo@phenomenex.com Web:

11 Applications Proteins (cont d) Protein Mixture () Column: BioSep-SEC-s Part No.: H--K Mobile Phase: mm Sodium Phosphate ph 7., mm Sodium Chloride Detection: 8 nm Sample:. Thyroglobulin 9 kda. lgg kda. BSA kda. Ovalbu kda. Myoglobin.9 kda. Uridine Da APP ID 898 Protein Mixture () 8 Column: BioSep-SEC-s Part No.: H--K Mobile Phase: mm Sodium Phosphate ph 7., mm Sodium Chloride Detection: 8 nm Sample:. lgm 9 kda. lga kda. Transferrin 8 kda. ß-Lactoglobin kda. Ribonuclease A.7 kda. Uridine Da APP ID Phenomenex India Tel: - Fax: - indiainfo@phenomenex.com Web:

12 Applications Peptides and Small Proteins Unlike protein separations that resemble physiological conditions, peptide separations require different conditions to get good, low molecular weight resolution. Low MW Protein and Peptide Mixture. L-Glutathione 7 Da. Leu-Leu-Leu 8 Da. Bradykinin - 7 Da. Bradykinin -7 7 Da. Angiotensin Da 9. Angiotensin II Da 8. Bradykinin Da 7. Substance P -9 Da. Angiotensin I 97 Da. Glucagon 8 Da. Insulin 7 Da. Apotinin Da. Cytochrome c Da. Myoglobin 78 Da 8 The use of acetonitrile and the weak ion pairing buffer TFA (. %) imizes secondary interactions between peptides and the stationary phase, leading to sharper peaks and better resolution in the low molecular weight ranges. BioSep SEC-s is the recommended media as it has the smallest pore size of the BioSep GFC media. APP ID 89 Column: BioSep-SEC-s Part No.: H--K Mobile Phase: % Acetonitrile,. % TFA Detection: nm Sample:. Myoglobin 78 Da. Cytochrome c Da. Apotinin Da. Insulin 7 Da. Glucagon 8 Da. Angiotensin I 97 Da 7. Substance P -9 Da 8. Bradykinin Da 9. Angiotensin II Da. Angiotensin Da. Bradykinin -7 7 Da. Bradykinin - 7 Da. Leu-Leu-Leu 8 Da. L-Glutathione 7 Da Medium MW Protein Mixture APP ID 89 Column: BioSep-SEC-s Part No.: H--K Mobile Phase: mm Sodium Phosphate ph.8, mm Sodium Chloride Detection: nm Sample:. HMW impurity. Thyroglobulin 7 kda. IgA kda. b-amylase kda. IgG kda. Transferrin 8 kda 7. Ovalbu kda 8. b-lactoglobulin A kda 9. Uridine Da 8 Phenomenex India Tel: - Fax: - indiainfo@phenomenex.com Web:

13 Applications Aggregates Protein aggregation is a common application in biotherapeutics. Optimal resolution is necessary in order to separate the monomer peak from associated dimers and possible trimers in the sample. Using BioSep-SEC-S columns allows accurate quantitation of monomer and aggregate. Human IgGk Aggregates 8 Column: BioSep-SEC-s Part No.: H--K Mobile Phase: mm Sodium Phosphate ph.8 Detection: nm Sample:. IgG aggregate peak. IgG trimer peak. IgG dimer peak #. IgG dimer peak #. IgG Monomer. IgG low MW fragment 8 Results show that the dimer peak of IgG is well resolved from the monomer peak. There appears to be two different dimer forms that are partially resolved, aggregate at the total excluded void of the column, and the appearance of a possible trimer peak ahead of the dimer peak. Finally, there is a fragment peak that elutes after the IgG monomer peak, most likely attributed to an IgG that is missing one of its Fab fragments. These results show the utility of using BioSep-SEC-s for antibody aggregate analysis. APP ID 89 mau Murine IgGk Aggregates Column: BioSep-SEC-s Part No.: H--K Mobile Phase: mm Sodium Phosphate ph.8, mm Sodium Chloride Detection: nm Sample:. HMW aggregates. IgG kappa dimer. IgG kappa dimer. IgG Monomer. Low MW impurity. Void Volume Peak APP ID Phenomenex India Tel: - Fax: - indiainfo@phenomenex.com Web:

14 Applications PEGylated Proteins Therapeutic proteins are often PEGylated to increase their serum lifetime; however, such reactions typically generate a heterogeneous product that can be difficult to characterize and purify. It is common that proteins can be PEGylated at multiple sites even with N-teral specific chemistries; thus the need for time course monitoring. BioSep-SEC-s is typically used as it provides optimal resolution of molecular weights below kda, the range of most PEGs, proteins, and their conjugates. Resolution of each component on a BioSep can be used for monitoring or purification capacity to get high recovery and purity of the desired PEGylated protein. PEGylated Ribonuclease A (ae PEG kda) mau 7 Overnight Intact 8 APP ID 89 Column: BioSep-SEC-s Part No.: H--K Mobile Phase: mm Sodium Phosphate ph.8 Detection: nm Sample:. Ribo A + PEG Complex. Ribo A + PEG Complex. PEGylated Ribonuclease A. Unmodified Ribonuclease A. PEG Reagents PEGylated L-Chymotrypsinogen A (N-teral PEG kda) mau 8 Overnight Column: BioSep-SEC-s Part No.: H--K Mobile Phase: mm Sodium Phosphate ph.8 Detection: nm Sample:. PEG + Chymo A Complex. PEG + Chymo A Complex. PEG + Chymo A Complex. PEGylated Chymotrypsinogen A. Chymotrypsinogen A Intact APP ID 89 8 Phenomenex India Tel: - Fax: - indiainfo@phenomenex.com Web:

15 Applications PEGylated Proteins (cont d) PEGylated b-lactoglobulin A (N-Teral PEG kda) mau 8 Column: BioSep-SEC-s Part No.: H--K Mobile Phase: mm Sodium Phosphate ph.8 Detection: nm Sample:. PEG Modified Complex. PEGylated b-lactoglobulin. b-lactoglobulin. PEG Reagent Overnight Intact 8 APP ID 89 PEGylated IgG (N-Teral PEG kda) Overnight APP ID 899 Column: BioSep-SEC-s Part No.: H--K Mobile Phase: mm Sodium Phosphate ph.8 Detection: nm Sample:. High MW PEG/IgG Complex. IgG Dimer + IgG/ PEG Complex. IgG monomer unmodified. Low MW impurity. Low MW impurity. PEG reagent impurity Intact 8 Phenomenex India Tel: - Fax: - indiainfo@phenomenex.com Web:

16 Now Available! Method Development, Re-Validation and Optimization Services Too busy to re-validate your current methods onto BioSep columns? Need help optimizing your current gel filtration method? Looking for assistance designing the best method for your separation? Give us a call. We can help! Phenomenex is pleased to offer method development, re-validation and optimization services to our customers. We approach our service efforts with over years of industry experience, technical expertise and an unsurpassed dedication to our customer s needs. We are committed to supporting you and your work, every step of the way. The process is simple, and it s FREE*:. Contact us and fill out a project request form. Mail your sample to our services team. You will receive a comprehensive report with detailed results and an optimal method within business days* For more information on any of the Phenomenex service offerings, or to begin a project today, please call your local Phenomenex office or contact us via at phenologix@phenomenex.com Additional Services Available for: HPLC UHPLC LC/MS GC GC/MS Chiral Separations Solid Phase Extraction (SPE) Preparative Bulk Synthetic Oligonucleotides On-Site Training * Depending on the complexity of a project, extended timelines and certain fees may be involved. These are detered at the start of a project. Phenomenex India Tel: - Fax: - indiainfo@phenomenex.com Web:

17 Easy Column Care and Use Completely regenerate by flushing with water overnight Restore to non-denaturing conditions quickly and easily Adsorbed materials are easily removed by washing with sodium phosphate buffer at ph. Strongly retained proteins may be removed by washing with acetonitrile or methanol without compromising performance Technical Data and Specifications BioSep SEC-s BioSep SEC-s BioSep SEC-s Resin Type Silica Silica Silica Particle Size (µm) Pore Size (Å) 9 ph Range Maximum Backpressure (psi),,, Typical Backpressure (psi) Efficiency (imum number theoretical plates x 7.8 mm),,, Maximum Flow Rate This is a function of pressure. Columns can withstand up to, psi, but avoid sudden pressure changes. Column Hardware Standard: stainless steel column with stainless steel frits. Titanium frits available. Maximum Temp. C Maximum Salt Conc. M Denaturants. % SDS, M Guanidine HCI, or 8 M urea Regeneration After exposure to denaturants, wash with water overnight. Max. Organic Modifier Up to % CH CN. Start with % H O, linear gradient to % CH CN over. Up to 9 % CH CN, % DMSO or mm b-mercaptoethanol. Cleaning Procedure General protein removal: wash with ml of. M NaH P, ph.. Hydrophobic protein removal: use acetonitrile gradient. Strongly adsorbed proteins: wash with ml of. % SDS or M Guanidine thiocyanate or % DMSO. Storage Overnight storage: run mobile phase at. ml/ute. Prolonged storage: use. % NaN in H O or % methanol in H. Column Protection Use of a SecurityGuard is recommended to prolong column lifetime. Phenomenex India Tel: - Fax: - indiainfo@phenomenex.com Web: 7

18 Easy Column Selection Molecular Weight Separation Ranges Three BioSep phase options to separate samples of varying molecular weight (MW) ranges:,,, as described below: Under M GnHCI S- S-, - 7, S-, -, -, Under. % SDS S- - 7, S-, -, S-, -, Native S-, -,, S-, - 7, S-, -, 7 Molecular Weight (Daltons) Log MW 7 MW Calibration Curves for Protein Separation Utilize calibration curves to help guide your column selection Low to High MW 7 Protein Calibration Curves 8 9 Elution Volume (Ve) APP ID 897 Conditions for all columns: Columns: BioSep-SEC-s BioSep-SEC-s BioSep-SEC-s Mobile Phase: mm Sodium Phosphate ph 7., mm Sodium Chloride Detection: 8 nm Samples:. IgM 9 kda. Thyroglobulin 9 kda. IgA kda. IgG kda. Transferrin 8 kda. BSA kda 7. Ovalbu kda 8. b-lactoglobin kda 9. Myoglobin.9 kda. Ribonuclease A.7 kda. Uridine Da BioSep-SEC-s BioSep-SEC-s BioSep-SEC-s Calibration curves are used to identify the MW of an unknown analyte and/or to select the appropriate column phase based on the ideal linear MW range for analytes of interest. If you need assistance using these curves, please contact your Phenomenex Technical Consultant. 8 Phenomenex India Tel: - Fax: - indiainfo@phenomenex.com Web:

19 Ordering Information Global support and availability in over countries batches available for validation Large inventory for immediate shipment Stainless Steel Columns (mm): Narrow Bore Analytical Preparative SecurityGuard Cartridges (mm) Phases x. x 7.8 x 7.8 x. x.* x.** /pk ea BioSep-SEC-s H--E H--K K--K H--P AJ-87 AJ-888 BioSep-SEC-s H--E H--K K--K H--P AJ-88 AJ-889 BioSep-SEC-s H-7-E H-7-K K-7-K H-7-P AJ-89 AJ-89 for ID:.-7.8 mm for ID:. mm Stainless Steel Guard Columns (mm) Narrow Bore Express Analytical Phases x. x x 7.8 BioSep-SEC-s A--E Q--K C--K BioSep-SEC-s A--E Q--K C--K BioSep-SEC-s A-7-E Q-7-K C-7-K *SecurityGuard Analytical cartridges require holder, Part No.: KJ-8 ** PREP SecurityGuard Cartridges require holder, Part No.: AJ-8 Aqueous SEC Column Check Standard (for BioSep-SEC-S and other protein SEC columns) Part No.: AL- Unit quantity: Dry; reconstituted to ml Contains: Bovine thyroglobulin; Human gamma globulin; Ovalbu; Myoglobin; Uridine (reconstitute with ml of mm Sodium phosphate ph.8) Diluent: mm Sodium phosphate ph.8 Storage: Add. % NaN to the solution and refrigerate Test Conditions Mobile phase: mm Sodium phosphate, ph.8 Flow rate:. ml/ for a x 7.8 mm column Injection volume: μl Detection: 8 nm If BioSep analytical columns do not provide you with at least an equivalent separation as any other GFC column of similar porosity, type, and dimensions, return the column with comparative data within days for a Full REFUND. Cross Reference Chart Phenomenex BioSep Phases TSK-Gel Shodex Sepax Bio-Rad Waters BioSuite Zorbax GSW PROTEIN KW-8. SRT SEC-s -* Bio-Sil SEC BioSuite GF- GSW XL SRT - GSW PROTEIN KW-8 SRT SEC-s - Bio-Sil SEC BioSuite GF- GSW XL SEC-s GSW PROTEIN KW-8 SRT -** Bio-Sil SEC BioSuite ** GSW XL Terms and Conditions Phenomenex reserves the right to refuse sale at its discretion. Offer applies to endusers only. Subject to Phenomenex Standard Terms & Conditions, which may be viewed at Trademarks BioSep is a trademark of Phenomenex, Inc. TSK-Gel, Shodex, Bio-Rad, Waters, SRT, Bio-Sil, BioSuite, and Zorbax are trademarks of their respective owners. These owners are in no way affiliated with Phenomenex. Disclaimer Px Advanced Technology Solutions Pvt. Ltd. is a part of Phenomenex. ** Only up to,, MW * Only above, MW Phenomenex India Tel: - Fax: - indiainfo@phenomenex.com Web: 9

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