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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893.1828

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1696.1161 1884.4532 2119.8912 2422.5596

Counts vs. Mass- to - Charge (m/z)

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16951.22

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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2196.84651952.8671

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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

See ordering information

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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

See ordering information

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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

Ordering Information One-click ordering

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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 agilent_inquiries@agilent.com

Europe info_agilent@agilent.com

Asia Pacific inquiry_lsca@agilent.com

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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