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Keep Your CQA Analysis Biocompatible from Start to Finish...Peptide Plus Bio IEX (SAX, WAX, SCX,...
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Keep Your CQA Analysis Biocompatible from Start to Finish PEEK-lined columns and bio-inert supplies to complement the Agilent 1290 Infinity II Bio LC system
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Biotherapeutic proteins are highly complex molecules, which typically are produced by fermentation using recombinant methodologies. This production process, however, results in the generation of many different variants of these proteins. Ensuring the quality of such materials is paramount. This means confirming the product is correctly manufactured, any impurities are identified and quantified, and the potency of the protein is determined.
As a result, it is necessary to perform tests on the intact, nondenatured molecule. Something as large as a monoclonal antibody may contain more than 1,300 individual amino acids and have a mass of more than 145,000 Daltons. However, identifying a single minor impurity, such as deamidation of asparagine resulting in a mass difference of just one Dalton, is challenging. Only by breaking down the molecule into fragments, and then into smaller polypeptide chains through enzymatic treatment, is it possible to begin to pinpoint some of these subtle differences.
Many types of variants can be created and these are often referred to as post-translational modifications (PTMs), which arise after the protein has been expressed, and can be a consequence of the manufacturing conditions or exposure to conditions that cause changes to occur. Fluctuations in temperature, pH, concentration, or exposure to enzymes can all lead to the development of variants.
The Agilent 1290 Infinity II Bio LC system—together with PEEK-lined, bio-inert Agilent AdvanceBio columns and standards—can help you characterize PTMs and other critical quality attributes (CQAs). For molecules that are susceptible to metal-induced artifacts, we have also designed a biocompatible flow path. Importantly, this includes PEEK-lined columns, because columns are the single largest contributor of metal to the flow path.
Confidently Measure the Efficacy and Stability of Your Therapeutic Proteins with Agilent Consumables and InfinityLab Instruments
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Agilent AdvanceBio LC columns
Be Agilent Sure in Your CQA Monitoring
Learn more at www.agilent.com/chem/advancebio
Agilent AdvanceBio LC columns are designed and produced to provide results you can be sure of when analyzing highly complex biotherapeutic molecules and monitoring their purity, potency, and other critical quality attributes.
To learn more, click the blue, green, and gray boxes.
Titer Determination
Aggregate Analysis Intact Purity & PTM Analysis
Peptide Mapping & PTM Analysis
Charge Variant Analysis
Glycan Analysis
Amino Acid / Cell Culture Media Analysis
Affinity Size Exclusion Reversed Phase > 150 Å
Hydrophobic Interaction
Reversed Phase < 150 Å
Ion Exchange Hydrophilic Interaction
Reversed Phase < 150 Å
Hydrophilic Interaction
AdvanceBio SEC 1.9 µm PEEK
PLRP-S 1000 Å 5 µm PEEK
AdvanceBio EC-C18 PEEK
Bio mAb / Bio IEX NP5 PEEK
AdvanceBio MS Spent Media PEEK
Bio-Monolith Protein A
AdvanceBio SEC 1.9 µm
PLRP-S AdvanceBio HIC
AdvanceBio Peptide Mapping
Bio mAb AdvanceBio Glycan Mapping
AdvanceBio Amino Acid Analysis (AAA)
Bio-Monolith Protein G
AdvanceBio SEC 2.7 µm
AdvanceBio RP mAb 450 Å
AdvanceBio Peptide Plus
Bio IEX (SAX, WAX, SCX, WCX)
ZORBAX AAA
Bio SEC-3 ZORBAX RRHD 300 Å, 1.8 µm
ZORBAX 300 Extend-C18
PL SCX, SAX
Bio SEC-5 ZORBAX 300SB 3.5, 5, & 7 µm
Bio-Monolith (QA, DEAE, SO3)
ProSEC 300S Poroshell 300 5 µm
Key
ZORBAX GF250 & GF450
Stainless steel (SS) column hardware
Solid PEEK or PEEK-lined bio-inert column hardware
Built-in sampling versatilityThe 1290 Infinity II Bio multisampler allows for adaptable injection volumes, thermostatting of temperature-sensitive bio-analytes, and ultralow carryover.
Versatile column handling The 1290 Infinity II MCT, featuring the Thermal
Equilibration Device, gives high-temperature stability for the most challenging applications.
Various bio-inert heat exchangers, Quick Change and Quick Connect capillaries, fittings,
and kits are also available.
Reliable solvent delivery Built for robustness under high-salt or high/
low-pH conditions, the 1290 Infinity II Bio high-speed pump maintains precise flow rates at
pressures up to 1300 bar. It is ideal for applications, such as long, shallow
gradients or high throughput.
Multiple detection options Choose from several optical detection
capabilities with varying flow cells, including variable wavelength, diode array and
fluorescence detectors, and the bio-MDS system.
Biocompatible valve automationInfinityLab Quick Change valves enable sample enrichment and cleanup, automated column regeneration, and enhanced system flushing.
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Agilent offers robust, reliable solutions for analyzing purity, potency, and other critical quality attributes (CQAs). For molecules that are susceptible to metal-induced artifacts, we also have designed a biocompatible flow path from start to finish.
Meet Your Most Complex Biopharma Challenges
Agilent 1290 Infinity II Bio LC system
AdvanceBio SEC 1.9 µm columnsThe best choice for SEC/MS applications
These PEEK-lined columns alleviate interactions with stainless steel surfaces, which can lead to additional aggregation. They feature a narrow 2.1 mm internal diameter for operating flow rates of just 0.05 to 0.10 mL/min, and are ideal for sensitive, native-mode protein analysis. What’s more, their inert hydrophilic coating allows you to use volatile mobile phases in lower concentrations while minimizing nonspecific interactions.
Because most stainless steel columns have exposed active metal sites, they require priming with biologic samples in order to obtain an acceptable level of reproducibility. PEEK-lined stainless steel columns have no active metal sites, which reduces the need for column priming—and saves you time and sample. You can also use AdvanceBio SEC standards to check these columns for quality, just as you would with stainless steel SEC columns.
Eliminate Metal-Induced Artifacts PEEK-lined stainless steel columns
Size exclusion chromatography of myoglobin under denaturing conditions (using formic acid as ion pair reagent) in aqueous acetonitrile on an Agilent 6545XT AdvanceBio LC/Q-TOF system. Under denaturing conditions, the deconvoluted mass corresponds to apo-myoglobin, where the heme group is no longer present. But under native mode conditions, the deconvoluted mass corresponds with the holo-myoglobin (heme group still intact), with only a small proportion of apo-myoglobin present.
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17064.91 17178.79 17293.3816893.95 17407.0416780.94 17235.52 17520.74 17634.7717579.81 17748.88 17863.0517349.59 17976.8617689.53
Counts vs. deconvoluted mass (amu)16550 16600 16650 16700 16750 16800 16850 16900 16950 17000 17050 17100 17150 17200 17250 17300 17350 17400 17450 17500 17550 17600 17650 17700 17750 17800 17850 17900 17950 18000
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2510.46721131.0743 1352.2619 2928.73601597.9507
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Apo-myoglobin (heme missing)
Holo-myoglobin (heme intact)
Denaturing mode 0.1% FA in 30% acetonitrile
Native mode 50 mM ammonium acetate
PEEK-lined SEC/MS analysis of myoglobin—deconvoluted
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Agilent PLRP-S 1000 Å 5 µm columnsExcellent inertness for intact protein analysis
PLRP-S media is an industry leader for intact protein analysis. Available in a wide pore size for excellent mass transfer, the inert PLRP-S 1000 Å polystyrene-divinylbenzene stationary phase ensures excellent protein recovery with minimal carryover. Its polymer base confers inherent hydrophobicity for reverse-phase separations using MS-compatible ion pair reagents like formic acid. Plus, the absence of residual silanol groups allows for excellent peak shape. In addition, the column’s PEEK-lined hardware minimizes the risk of artifacts—such as oxidation and peak tailing—due to secondary metal interactions.
Ramucirumab (1 mg/mL; 1 µL injection) 0.21 mL/min, 70 °C, 20–80% ACN (0.1% TFA) over 20 minutes, UV 280 nm
For intact analysis, PEEK-lined stainless steel columns can provide significant improvements in symmetry by reducing peak tailing due to secondary metal interactions while retaining similar elution profiles and retention times.
PLRP-S 2.1 x 50 mm (PEEK-lined)
166.1 bar
0.112 peak width at half height
2.329 tailing (at 5% peak height)
1.844 symmetry (at 10% peak height)
PLRP-S 2.1 x 50 mm (SS)
121.7 bar
0.139 peak width at half height
3.285 tailing (at 5% peak height)
2.511 symmetry (at 10% peak height)
Master your chromatographyControlled through Agilent OpenLab software, InfinityLab LC Series instruments optimize your bio-analysis LC workflows by reducing the time you spend on data processing, review, and reporting.
Learn more about OpenLab software suite: www.agilent.com/chem/openlab
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Analytical success depends upon the quality of your reference materialAgilent standards are rigorously tested and manufactured using ISO certifications—including ISO 17025 and 17034. So you can calibrate with confidence and maximize accuracy. Learn more about Agilent NISTmAb
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AdvanceBio 2.7 µm EC-C18 columnsDesigned for biocompatible (U)HPLC instrumentation
Our fully end-capped C18 silica stationary phase features excellent retention characteristics for a wide range of peptides typically found in a digested protein. What’s more, their superficially porous Poroshell particles offer high performance without exceptional operating pressures. We recommend EC-C18 columns for applications where stainless steel columns might interact with components like phosphorylated peptides—or increase on-column oxidation of susceptible amino acids like methionine.
Ten peptide standard (5190-0583)3–47% ACN (0.1% FA), 2–46 min, 0.4 mL/min; 55 °C; UV, 220 nm
For intact analysis, PEEK-lined stainless steel columns can provide significant improvements in symmetry by reducing peak tailing due to secondary metal interactions while retaining similar elution profiles and retention times.
AdvanceBio EC-C18 2.1 x 150 mm PEEK-lined (Peak capacity 207)
AdvanceBio Peptide Mapping 2.1 x 150 mm (SS) (Peak capacity 214)
To improve peak width and capacity, use an ultralow dispersion kit Ultralow dispersion kits allow for the least possible on-column dispersion for UHPLC methods by use of optimized tubing components. Here, the 0.07 mm ID tubing produces a much sharper peak than the 0.17 mm ID tubing. This is especially important for SEC, where low flow rates contribute to increased dispersion.
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Expand your scope with LC/MSWhether you’re analyzing large biomolecules, peptides, or glycans, the Agilent 6545XT AdvanceBio LC/Q-TOF is designed for biopharma. Together with the 1290 Infinity II bio LC and Agilent MassHunter BioConfirm software, the system automates intact protein, peptide mapping, and glycan analysis workflows.
Learn more by downloading our brochure here
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Is your flow path biocompatible?Removing stainless steel from your flow path allows for increased recovery, improved peak shape, and higher sensitivity
As illustrated here, stainless steel column hardware (bottom) is the largest barrier to overcoming poor recoveries. PEEK-lined columns (top) maximize recovery and sensitivity while dramatically improving peak shape.
HILIC-DAD chromatograms of adenosine (A), AMP, ADP, and ATP on a stainless steel and PEEK-lined InfinityLab Poroshell 120 HILIC-Z column installed on a 1260 Infinity II bio-inert LC and 1260 Infinity II LC.
Application Note
Pharma & Biopharma
AuthorsKoen Sandra, Jonathan Vandenbussche, Gerd Vanhoenacker, Ruben t’Kindt, and Pat Sandra Research Institute for Chromatography (RIC), President Kennedypark 26, Kortrijk, Belgium
Sonja Krieger, Sonja Schneider, and Udo Huber Agilent Technologies, Inc.Waldbronn, Germany
AbstractPhosphorylated compounds such as nucleotides are known to interact with stainless steel components leading to recovery issues in HPLC and LC/MS. This Application Note describes the analysis of nucleotides using the Agilent 1260 Infinity II Bio-Inert LC System and a PEEK-lined Agilent InfinityLab Poroshell 120 HILIC-Z column. The fully inert flowpath, governed by the setup, leads to substantially improved recoveries of these challenging phosphorylated solutes.
HPLC-DAD Analysis of Nucleotides Using a Fully Inert Flowpath
Agilent 1260 Infinity II Bio-Inert LC System and a PEEK-lined Agilent InfinityLab Poroshell 120 HILIC-Z Column
Improved recoveries of challenging phosphorylated analytesThis application was performed using InfinityLab Poroshell 120 HILIC-Z columns—both SS and PEEK-lined SS. Superficially porous particle technology is a great way to save time and solvent while increasing throughput and recovery.
Want to know more? Read the complete application note: HPLC-DAD Analysis of Nucleotides Using a Fully Inert Flow Path
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You can always count on Agilent to support your entire workflow—including sample preparation, columns, supplies, standards, and instruments. To add items to your shopping cart at the Agilent online store, simply click the part number links. Then, enter the quantities for the products you need.
Download our glycan standards chart, which includes structures.
Agilent AdvanceBio SEC Protein Standards – uniquely designed for AdvanceBio SEC Columns
Highlights• Wide range of molecular weight proteins
• Optimize for use with Agilent’s AdvanceBio SEC columns for maximum peak to peak resolution
• Manufactured and shipped in moisture and light resistant packaging to ensure stability of standard
Agilent AdvanceBio SEC Protein Standards
END UNCERTAINTY
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Only Agilent AdvanceBio SEC Protein standards are manufactured from a select group of proteins and peptides optimized to represent the widest range of molecular weights for optimal calibration and performance verification of your AdvanceBio SEC columns.
AdvanceBio SEC 300Å Protein Standard separation on AdvanceBio SEC 300Å column
AdvanceBio SEC 130Å Protein Standard separation on AdvanceBio SEC 130Å column
AdvanceBio SEC 130Å Protein Standard p/n 5190-9416, 1.5 mL vial)Analyte MW1. Ovalbumin 45,0002. Myoglobin 17,0003. Aprotinin 6,7004. Neurotensin 1,7005. Angiotensin II 1,000
AdvanceBio SEC 300Å Protein Standard(p/n 5190-9417, 1.5 mL vial)Analyte MW1. Thyroglobulin 670,0002. g-globulin 150,0003. Ovalbumin 45,0004. Myoglobin 17,0005. Angiotensin II 1,000
Agilent-NISTmAb and SEC standards
Description Part Number
Agilent NISTmAb 25 µL (QTY 1) 5191-5744
Agilent NISTmAb 25 µL (QTY 4) 5191-5745
AdvanceBio SEC 130 Å (1.5 mL) 5190-9416
AdvanceBio SEC 300 Å (1.5 mL) 5190-9417
AdvanceBio Gly-X N-glycan prep with InstantPC kits (formerly ProZyme)
Description Part Number
24 count 96 count
Gly-X with InstantPC kit GX24-IPC GX96-IPC
Gly-X with 2AB kit GX24-2AB GX96-2AB
AdvanceBio InstantPC Human IgG N-glycan library GKPC-005
AdvanceBio 2-AB Human IgG N-glycan library GKSB-005
Agilent NISTmAb standards p/n 5191-5745
AdvanceBio SEC standards p/n 5190-9416 / 5190-9417
Agilent columns
Description Part Number
AdvanceBio SEC 1.9 µm 200 Å, 2.1 x 150 mm PEEK-lined PL1980-3201PK
AdvanceBio SEC 1.9 µm 200 Å, 2.1 x 50 mm PEEK-lined PL1980-1201PK
AdvanceBio SEC 1.9 µm 120 Å, 2.1 x 150 mm PEEK-lined PL1980-3250PK
AdvanceBio SEC 1.9 µm 120 Å, 2.1 x 50 mm PEEK-lined PL1980-1250PK
PLRP-S 5 µm 1000 Å, 2.1 x 100 mm PEEK-lined PL1912-2502PK
PLRP-S 5 µm 1000 Å, 2.1 x 50 mm PEEK-lined PL1912-1502PK
AdvanceBio EC-C18 2.7 µm, 2.1 x 100 mm PEEK-lined 675775-902
AdvanceBio EC-C18 2.7 µm, 2.1 x 50 mm PEEK-lined 679775-902
AdvanceBio MS Spent Media 100 Å, 2.1 x 100 mm 675775-901
AdvanceBio MS Spent Media 100 Å, 2.1 x 50 mm 679775-901
AdvanceBio MS Spent Media, 100 Å, 2.1 x 150 mm, 2.7 µm 673775-901
AdvanceBio PEEK-lined column p/n PL1980-3201PK
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InfinityLab Quick Connect bio-inert capillaries and supplies
Description Part Number
LC mobile phase filtration
InfinityLab solvent filtration assembly (includes 250 mL funnel, membrane holder base, 1 L flask, and aluminum clamp)
5191-6776
InfinityLab solvent filtration 2 L glass flask (optional) 5191-6781
Regenerated cellulose filter membrane, 47 mm diameter, 0.20 μm pore size, 100/pk 5191-4340
Regenerated cellulose filter membrane, 47 mm diameter, 0.45 μm pore size, 100/pk 5191-4337
Column connection
InfinityLab Quick Connect LC fitting 5067-5965
Quick Connect Capillary MP35N 0.12 x 105 mm 5500-1578
Quick Connect Capillary MP35N 0.12 x 150 mm 5500-1579
Quick Connect Capillary MP35N 0.12 x 220 mm 5500-1580
Quick Connect Capillary MP35N 0.12 x 280 mm 5500-1581
Quick Connect Capillary MP35N 0.12 x 400 mm 5500-1582
Quick Connect Capillary MP35N 0.12 x 500 mm 5500-1583
Quick Connect Capillary MP35N 0.17 x 105 mm 5500-1584
Quick Connect Capillary MP35N 0.17 x 150 mm 5500-1585
Quick Connect Capillary MP35N 0.17 x 220 mm 5500-1586
Quick Connect Capillary MP35N 0.17 x 280 mm 5500-1587
Quick Connect Capillary MP35N 0.17 x 500 mm 5500-1588
Well plates
Well plate 96/0.5 mL, round, U shape, 14 mm, PP, 120/pk 5043-9311
Well plate 96/1.0 mL, round, U shape, 32 mm, PP, 50/pk 5043-9305
Mat 96, round, preslitted, silicone, 50/pk 5042-1389
Detector bio flow cells
Max-light cartridge cell LSS 10 mm G7117-60020
Bio micro flow cell VWD, 3 mm, 2 µL, RFID G1314-60189
Bio standard flow cell VWD, 10 mm, 14 µL, RFID G1314-60188
The Gly-X N-glycan rapid release and labeling with InstantPC kit uses an in-solution enzymatic protein deglycosylation, followed by rapid labeling of released N-glycans with InstantPC dye. After a simple clean-up step, the glycan samples are ready for analysis by UHPLC, LC/MS, MS/MS, and other methods. Learn more about AdvanceBio Gly-X technology
InfinityLab Quick Connect LC fitting p/n 5067-5965
Learn more: www.agilent.com/chem/advancebio
Buy online: www.agilent.com/chem/store
Find a local Agilent customer center in your country: www.agilent.com/chem/contactus
U.S. and Canada 1-800-227-9770 [email protected]
Europe [email protected]
Asia Pacific [email protected]
DE44200.1288657407
This information is subject to change without notice.
© Agilent Technologies, Inc. 2021 Published in the USA, July 14, 2021 5994-2983EN
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