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1260 Infinity Bio-inert

1260 Infinity Bio-inert
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Bio-inert UHPLC The 1260 Infinity Bio-inert LC System Sebastian Krahe Produktspezialist Agilent Technologies 1

Good Reasons for Having a Dedicated Bio-inert HPLC Protein Analysis: • Unspecific surface binding • Low throughput, low resolution • Peak tailing for critical proteins • Corrosion and pH issues with standard LC • Decreased column lifetime and chromatographic performance Other Applications: • • high pH applications • 2 metal-free chromatography Phosphorylated compounds, oligonucleotides

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1260 Infinity Bio-inert HPLC What is similar to standard 1260 Infinity? Based on proven technology System performance specification System Robustness 600 bar (UHPLC) for fast or high resolution bio-separations without any compromises! 3

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1260 Infinity Bio-inert HPLC What´s new ? No corrosion due to high salt Sample management completely metal-free No usage of MP35N capillaries with cobalt, nickel, chromium… pH-range 1-13, shortterm pH 14 low and high pressure possible large volume injections • Widest pH range • High salt tolerance • Lowest surface activity no passivation needed !! 4

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New Capillary Design Ensures Bio-inertness Capillaries Metal cladded PEEK capillary Newest fitting design: Hybrid technique New capillary technolgy enables 600 bar AND is completely metal free !! 5

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Proof of concept: surface activity 300 200 100 90 % MeOH 400 70 % MeOH 500 Solvents Buffer A: 10 mM ammonium acetate Buffer B: 10 mM ammonium acetate +10% methanol Buffer C: 10 mM ammonium acetate +50% methanol Buffer D: 10 mM ammonium acetate +70% methanol Buffer E: 10 mM ammonium acetate +90% methanol 50 % MeOH 600 Chromatographic conditions Flow rate: 0.5 mL/min Isocratic run with buffer A, B, C, D or E Stop time: 5 minutes Injection volume: 0.2 μL Temperature TCC: 40 °C Diode array detector: 254 nm No column, PEEK restriction 10 % MeOH mAU 0 % MeOH ATP Analysis – 1260 Infinity standard...

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Proof of concept: low surface activity 90 % MeOH 70 % MeOH 700 50 % MeOH mAU 10 % MeOH 0 % MeOH ATP Analysis – 1260 Infinity Bio-inert LC Chromatographic conditions Flow rate: 0.5 mL/min Isocratic run with buffer A, B, C, D or E Stop time: 5 minutes Injection volume: 0.2 μL Temperature TCC: 40 °C Diode array detector: 254 nm No column, PEEK restriction 600 500 400 Solvents Buffer A: 10 mM ammonium acetate Buffer B: 10 mM ammonium acetate +10% methanol Buffer C: 10 mM ammonium acetate +50% methanol Buffer D: 10 mM ammonium acetate +70% methanol Buffer E: 10 mM ammonium acetate +90% methanol...

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Quaternary Buffer Mixing with Buffer Advisor and 1260 Infinity Bio-inert LC System 8

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1. Select Buffer S Gradient Mode (• Cation Exchange C Pnion Exchange 3. Compose Stock Solutions Ionic Strength Dilute online or add salt for salt Agilent Technologies

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…Saving Time and Resources…. Instead of setting up different bottles with different pH… 10 …you use a quaternary Bioinert LC with Buffer Advisor software to instantly run salt- and pH gradients

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Simplifies Ion Exchange Workflows Buffer Advisor SW Customer benefits: Ease of use • Automates preparation of separation buffers for IEX from stock solutions • Eliminates the need to prepare multiple buffer solutions • Provides recipes for buffer preparation Increase of productivity • Simplifies pH scouting studies • Simplifies pH gradient setup Increase of Robustness and run to run repeatibility • Compensates for pH shifts in salt gradients • Calculates optimized gradient steps 11

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Process Help 1. Select Buffer & Gradient Mode Single Buffer *- Composite Buffer {pH / Salt Gradient} [Wide Range pH Gradient} f* Cation Exchange C Anion Exchange 2. Define Gradient Table j. Compose Stock Solutions Ionic Strength Export Gradient Time Table... Minimum Buffering Capacity (BC) =3.15 j Agilent Technologies

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Single Buffer [pH / Salt Gradient) Cation Exchange Sodium Citrate [Citric + Tri-Sodium Citrate) Sodium Citrate [Citric + NaOH) ; /Formic/Na [acid + Na salt) | Formic/Na [acid + NaOH) ; /Lactic/Na [acid + Na salt) I Lactic/Na [acid + NaOH) ; /AcetidNa (Acetic+Acetate/Na) ^J/SuccinidNa [acid + Na salt) Succinic'Na [acid + NaOH) .^MalonidNa [acid + Na salt) | . MalonidNa [acid + NaOH) ^MES/Na (MES+MES/Na) MESiNa [MES+NaOH) 1. Select Buffer & Gradient Mode [Wide Range pH Gradient) (* Cation Exchange Agilent Technologies

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2. Define Gradient Table 3. Compose Stock Solutions Stock Solution Recipes Bottle C |NaH2P04: Monosodium phosphate Bottle D |Na2HP04: Sodium phosphate dibasic Print Preview. Agilent Technologies

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Step 3: Press Process and obtain your new optimized gradient table and the corresponding display Export the Gradient Timetable as xml file 15

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Step 4: Import the Gradient timetable into OpenLAB CDS ChemStation 1. Go to the OpenLAB CDS ChemStation and set up and save a new method, 2 . Go to the Quat Pump tab and click Import Solvent Blending File 3.Select the file that you have generated in the Buffer Advisor software 16

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Determination of Low Metal Release in the 1260 Infinity Bio-inert LC The Agilent 1260 Infinity Bio-inert Quaternary LC system releases less metals using acidic, basic, and salt containing buffers compared to • • 17 the Agilent 1260 Infinity LC stainless steel system and to the bio-inert LC system from another vendor

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Agilent Technologies

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Applications Agilent has a complete solution for bioanalytical workflows including LC-Instrument, column and applications Typical applications for the 1260 bio-inert LC in Biopharma: 1. Aggregate analysis of mABs with SEC 2. Charge variant analysis with WCX 3. Peptide Mapping with RP 4. Glycan analysis with HILIC, RP or AEX 5. Structural analysis with HIC 19

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Characterization of therapeutic proteins and monoclonal Antibodies drugs 1. Physical and chemical characterization (product characterization) 2. Potency/activity 3. Product related impurities 4. Process related impurities Heavy Chain Antigen binding Light Chain Pyroglutamate Disulfide shuffling Fab Deamidation/oxidation Hinge Fc 20 Truncation (lysine) Glycosylation site

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