Agilent Technologies April 20,

Agilent Technologies April 20, 2015 1

Improving HPLC Characterization of Biomolecules Agilent Solutions to Separation Challenges Paul Dinsmoor Technical Specialist, Bio- Columns Agilent Technologies April 20, 2015 2

Agilent Solutions for Therapeutic Protein Characterization Titer determination and purification (Affinity) Protein A Bio-Monolith Protein identification and impurity profiling (RP) 120A Advancebio Peptide Mapping Column and 450A RP mab Column Glycan analysis (HILIC) Advancebio Glycan Mapping Column Charge variant analysis (IEX) Bio-IEX 1.7um Column Aggregation analysis (SEC) Bio-SEC 3 Column 3

Agilent Bio-LC Column Portfolio Agilent Bio-LC Columns Affinity Reversed Phase HILIC Size Exclusion Ion Exchange Bio-Monolith Protein A AdvanceBio Peptide Mapping AdvanceBio Glycan Mapping Bio SEC-3 Bio-Monolith (QA, DEAE, SO 3 ) Multiple Affinity Removal System ZORBAX RRHD 300A 1.8um ZORBAX RRHD 300-HILIC Bio SEC-5 Bio mab Poroshell 300 ProSEC 300S Bio IEX (SAX, SCX, WAX, WCX) AdvanceBio RP mab ZORBAX GF-250 PL SAX ZORBAX 300SB ZORBAX GF-450 PL SCX ZORBAX Amino Acid Analysis PLRP-S Products in red are new! Agilent Technologies April 20, 2015 4

REVERSED-PHASE BIOCHROMATOGRAPHY Agilent Technologies April 10, 2015 5

Agilent Technologies April 20, 2015 6

RP chromatography separates proteins through the interaction of the hydrophobic foot of the protein with a non-polar surface of the particle RP columns are nearly always based on silica particles Mechanical stability, easy to make, surface can be modified, excellent peak shape & efficiency Solvent Organic modifier: Acetonitrile, isopropanol, methanol Ion pair additive: Trifluoroacetic acid (TFA), formic acid Gradient separation Agilent Technologies April 20, 2015 7

Strategy for RP-HPLC Method Development of Proteins and Peptides Start at low ph (acidic mobile phase) and choose the initial column and conditions Initial selection parameters include: pore size (120A, 300A, 450A), mobile phase, bonded phase, particle size, column length, and internal diameter Obtain best resolution by optimizing: gradient steepness, bonded phase, temperature, column configuration Obtain best recovery by optimizing: Bonded phase, temperature, sample solubility Evaluate alternative columns/ technologies for improved selectivity and efficiency Agilent Technologies April 20, 2015 8

Common Challenges with RP Bioseparations Poor characterization in the separation results in poor identification - Need best stationary phase to perfect the separation - Need high resolution/ efficiency Reduced lifetime Lack of analytical consistency/ method robustness LC system pressure limitations Long run times for peptide maps Agilent Technologies April 19, 2015 9

Primary Structure Characterization Workflows - mab Improve accuracy and resolution Agilent Technologies April 20, 2015 10

Agilent Technologies April 20, 2015 11

Protein/Peptide Separations by Reversed-phase Larger Molecules = Slower Diffusion So, a need to decrease the diffusion time for macromolecules! To improve, we can increase the Diffusion Rate by: elevating operating temperature decreasing solvent viscosity and, or..

Agilent Technologies April 20, 2015 13

Agilent Technologies April 20, 2015 14

ZORBAX 300SB RRHD for Proteins Stablebond 300 silica/bonding C18, C8, C3, and Diphenyl bonded phase 1.8 µm particle size for high resolution 1200 Bar pressure limit for UHPLC 2.1 x 50 mm and 2.1 x 100 mm Agilent Technologies April 10, 2015 15

Agilent Technologies April 20, 2015 16

Four Rapid Resolution High Definition Phases Family of Four Reversed-Phase Ligands C18 C8 C3 Diphenyl Peptide Maps Increasing protein size/hydrophobicity Small Proteins ZORBAX 300Å, 1.8 µm Ligand C18 C8 C3 Diphenyl Application Small intact proteins/peptide maps Intact proteins Larger /hydophobic proteins, including MAbs Unique selectivity Agilent 1290 Infinity 17

C3 versus Diphenyl

The Separation Challenge - Does Size Matter? Effect of Pore Size and Molecular Size on Peak Width 0.2 0.18 0.16 300SB-C18 (300Å) SB-C18 (80Å) 0.14 PW 1/2 0.12 0.1 0.08 0.06 0.04 0.02 0 0 min 12 0 min 11 Proper pore size selection results in sharper peaks for large molecules 19

NEW! AdvanceBio Peptide Mapping Column for HPLC and UHPLC: 2.7 µm Superficially Porous 120Å pore size 600 bar pressure limit 2 µm frit to reduce clogging 0.5 um 1.7 um 2.7 um Greater analytical confidence: Each batch is tested with a rigorous peptide mix to ensure suitability and reproducibility Save Time: 2 to 3 times faster than fully porous particles Increased Flexibility: Highly compatible with TFA and formic acid mobile phases for efficient LC UV and LC/MS analysis Agilent Technologies April 10, 2015 20

Agilent Technologies April 20, 2015 21

Quality Assurance Testing with Agilent Peptide Mix Each batch of AdvanceBio Peptide Mapping media is tested with the Agilent Peptide Standard (PN 5190-0583) to ensure batch to batch reproducibility Peptide Mapping Standard P/N# 5190-0583

AdvanceBio Peptide Mapping Column Highlights Peptide Mapping 2.1 x 150mm AdvanceBio Peptide Mapping Column Mobile phase: A-water (0.1%TFA), B- ACN (0.08%TFA), 40 C, flow: 0.52mL/min mau 70 BSA tryptic digest 60 50 40 30 20 10 0 0 2.5 5 7.5 10 12.5 15 17.5 20 min Hydrophilic peptide retention Narrow Peaks w baseline resolution Hydrophobic peptide retention Reduced and fast analysis time Critical and desired peptide mapping components to achieve fast, selective and highly efficient peptide separations across a wide dynamic range.

Peptide Mapping by LC/MS Critical Post Translational Modifications (PTM) Identified in Fast and Slow Analyses x10 8 3.75 2.75 +ESI TIC Scan Frag=200.0V igg023.d 40 min. Run, 2.1 x 100 mm 140 bar 0.2 ml/min 10-40% B Native peptide 1.75 0.75 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 Counts vs. Acquisition Time (min) Deamidated form 1 Deamidated form 2 Heavy Chain Peptide 357-366 and its two deamidated forms conserved x10 8 4.6 +ESI TIC Scan Frag=200.0V igg011.d 14 min. Run, 2.1 x 100 mm 3.6 2.6 433 bar 0.6 ml/min 10-40% B Native peptide 1.6 0.6 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5 10 10.5 11 11.5 12 12.5 13 Counts vs. Acquisition Time (min) Deamidated form 1 Deamidated form 2 Agilent Technologies April 10, 2015 24

Poroshell 300 300Å pore size StableBond and Extend chemistry Available in SB-C3, SB-C8, SB-C18, and Extend-C18 5 µm particle size Agilent Technologies April 1, 2015 25

Column Internal Diameter Porous Particle Flow Rate Range Poroshell Flow Rate Range 2.1 mm 0.1 0.3 ml/min 0.3 3 ml/min 1.0 mm 30 60 µl/min 0.08-0.75 ml/min Very high flow rates can be used effectively with Poroshell columns Agilent Technologies April 20, 2015 26

High Flow Rates with 2.1 mm id Poroshell for High Resolution and Fast Separations Columns: Poroshell 300SB-C18 2.1 x 75 mm, 5 µm MP: A: 0.1% TFA B: 0.07% TFA in ACN Gradient: 5 100% B in 1.0 min. Flow Rate: 3.0 ml/min. Temperature: 70 C Pressure: 250 bar Detection: UV 215 nm 1 2 3 4 5 6 7 8 Pub No# 5989-9899EN for complete app note Sample: 1. Angiotensin II 2. Neurotensin 3. Rnase 4. Insulin 5. Lysozyme 6. Myoglobin 7.Carbonic Anhydrase 8.Ovalbumin 0 0.5 1.0 Time (min) Agilent Technologies April 1, 2015 27

High Flow Rate, High Sensitivity LCMS Column: Poroshell 300SB-C18, 1.0 x 75 mm 1E8 80000000 60000000 40000000 20000000 pmoles of protein 0.5 0.75 1 2.5 5 Mobile Phase Gradient: 20-100% B in 5.5 min. A: water + 0.1% formic acid B: ACN + 0.1% formic acid Flow Rate: 0.6 ml/min Temperature: 80 C Injection volume: 1 ul LC/MS: Pos. Ion ESI Peakwidth: 0.06 min Sample: Mixture of insulin, lysozyme, cytochrome C, myoglobin, BSA, carbonic anhydrase 0 0 0.5 1 1.5 2 2.5 3 3.5 min

NEW! AdvanceBio RP mab Column for HPLC and UHPLC Particle 3.5um SP 450 Å pore diameter 600bar pressure limit 2um frit to reduce clogging The optimum high speed, large molecule resolution for use with both HPLC and UHPLC systems 0.25 um 3.0 um 3.5 um Phases C4 SB-C8 Diphenyl The most popular phases for proteins, plus a unique selectivity

Fast Intact mab Analysis AdvanceBio RP-mAb provides superior peak shape at a lower pressure and resolves more fine detail than a UHPLC protein column from competitor AdvanceBio RP-mAb C4, 450Å, 3.5 µm 490 bar Competitive C4, 300Å, 1.7 µm 910 bar Method Parameters Column dimensions: 2.1 x 100 mm Mobile phase A: 0.1% TFA in water:ipa (98:2) Mobile phase B: IPA:acetonitrile:MPA* (70:20:10) Flow rate: 1.0 ml/min Gradient: 10-58% B in 4 min, 1 min wash at 95% B, 1 min re-equilibration at 10% B Sample: 5 µl injection of Humanized Recombinant Herceptin Variant IgG1 Intact from Creative Biolabs (1 mg/ml) Temperature: 80 C * MPA = Mobile Phase A Detection: UV @ 254 nm Agilent Technologies April 10, 2015 31

Fast, High Resolution mab Fragment Analysis AdvanceBio RP-mAb provides superior peak shape and resolution than other columns designed for protein separations mau 200 mau DAD1 A, Sig=220,8 Ref=off (AEM_PS450_...C_MD\AEM_PS450_FAB-FC_MD_4 2014-09-10 09-02-53\1443508-69-0005.D) 0 2 2.25 2.5 2.75 3 3.25 3.5 3.75 4 4.25 DAD1 A, Sig=220,8 Ref=off (AEM_PS450_...B-FC\AEM_PS450_IGG1_FAB-FC 2014-09-11 13-54-40\USRIT001297-003.D) 200 mau 0 2 2.25 2.5 2.75 3 3.25 3.5 3.75 4 4.25 DAD1 A, Sig=220,8 Ref=off (AEM_PS450_...B-FC\AEM_PS450_IGG1_FAB-FC 2014-09-11 14-37-19\706785-1-000003.D) 200 mau 0 2 2.25 2.5 2.75 3 3.25 3.5 3.75 4 4.25 DAD1 A, Sig=220,8 Ref=off (AEM_PS450_...B-FC\AEM_PS450_IGG1_FAB-FC 2014-09-10 11-45-32\CD4F123-0000003.D) 200 AdvanceBio RP-mAb C4, 450Å, 3.5 µm Competitor A Protein C4, 400Å, 3.4 µm Competitor B WIDEPORE C4, 200Å, 3.6 µm Competitor C C4-30, 300Å, 2.6 µm min min min 0 2 2.25 2.5 2.75 3 3.25 3.5 3.75 4 4.25 min Method Parameters Column dimensions: 2.1 x 100 mm Mobile phase A: 0.1% TFA in water Mobile phase B: n-propanol/acetonitrile/mpa (80/10/10) Flow rate: 0.8 ml/min Gradient: 5-40% B in 5 min, 1 min wash at 95% B, 1 min re-equilibration at 10% B Sample: 1 µl injection of Fc/Fab, Papain Digested Humanized Recombinant Herceptin Variant IgG1 from Creative Biolabs (2 mg/ml) Temperature: 60 C Detection: UV @ 220nm Agilent Technologies April 10, 2015 32

AdvanceBio RP-mAb C4 Separtes Proteins with small differences: Biosimilars in development Remicade Remicade clone Remicade Remicade clone Shift of Fc Shift of Fc Using 2.1 % B/mL gradient Using 1.0 % B/mL gradient Annotated shift of the Fc part implicates differences in hydrophobicity of the Fc part due to a 2-point mutation in the AA sequence of the biosimilar compared to the originator. The shift is observed with either a fast or slow gradient. 33

Fast Intact mab Analysis AdvanceBio RP-mAb Diphenyl resolves additional fine detail - the Diphenyl phase is unique to Agilent mau 140 120 DAD1 H, Sig=254,8 Ref=off (AEM_PS450_...\AEM_PS450_IGG-INTACT_MD_4 2014-08-21 08-02-26\1443508-52-0006.D) DAD1 E, Sig=254,8 Ref=off (AEM_PS450_...\AEM_PS450_IGG-INTACT_MD_4 2014-08-20 09-06-44\1435601-25-0037.D) DAD1 E, Sig=254,8 Ref=off (AEM_PS450_...\AEM_PS450_IGG-INTACT_MD_4 2014-08-19 15-36-09\DIP143501-3-047.D) AdvanceBio RP-mAb C4 AdvanceBio RP-mAb SB-C8 AdvanceBio RP-mAb Diphenyl mau 14 12 DAD1 H, Sig=254,8 Ref=off (AEM_PS450_...\AEM_PS450_IGG-INTACT_MD_4 2014-08-21 08-02-26\1443508-52-0006.D) DAD1 E, Sig=254,8 Ref=off (AEM_PS450_...\AEM_PS450_IGG-INTACT_MD_4 2014-08-20 09-06-44\1435601-25-0037.D) DAD1 E, Sig=254,8 Ref=off (AEM_PS450_...\AEM_PS450_IGG-INTACT_MD_4 2014-08-19 15-36-09\DIP143501-3-047.D) 10 100 8 6 4 80 2 0 60-2 -4 1.7 1.8 1.9 2 2.1 2.2 2.3 2.4 2.5 min 40 20 0 1.7 1.8 1.9 2 2.1 2.2 2.3 2.4 2.5 min Method Parameters Column dimensions: 2.1 x 100 mm Mobile phase A: 0.1% TFA in water/ipa (98/2) Mobile phase B: IPA/acetonitrile/MPA* (70/20/10) Flow rate: 1.0 ml/min Gradient: 10-58% B in 4 min, 1 min wash at 95% B, 1 min re-equilibration at 10% B Sample: 5 µl injection of Humanized Recombinant Herceptin IgG1 Intact from Creative Biolabs (1 mg/ml) Temperature: 80 C Detection: UV @ 254nm * MPA = Mobile Phase A April 10, 2015 34

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Large fibrous proteins

Exploiting chemical stability

PLRP-S: Features ph 1-14 Extreme buffer concentrations High temperature stability Durable and Resilient Inherently hydrophobic so does not require a bonded alkyl chain to confer hydrophobicity 1000A & 4000A pores Benefits Acid and base cleanup Typically < 8M 200 C Long Lifetimes Avoids typical silica problems of silanol group Analysis of very large biomolecules or high speed separations

RP Summary AdvanceBio RP-mAb is an addition to the market leading Agilent reversedphase bio-column portfolio and complements existing columns Agilent Column Positioning ZORBAX RRHD 300SB AdvanceBio Peptide Mapping High resolution UHPLC analysis of proteins, including intact mabs, and protein fragments Fast, high resolution HPLC and UHPLC analysis of peptides Poroshell 300 Fast, HPLC analysis of large intact proteins, including intact mabs AdvanceBio RP mab Designed for mab separations Fast, high resolution HPLC and UHPLC analysis of intact mabs and mab fragments PLRP-S Polymeric for high ph stability, alternate selectivity, and large pores April 10, 2015 39

How the New Columns Fit: Agilent Positioning Match The Column To System Pressure Capabilities Agilent Column Particle Pressure Rating Phases AdvanceBio RP-mAb SPP, 3.5 µm, 450Å 600 bar SB-C8, C4, Diphenyl ZORBAX RRHD 300SB TPP, 1.8 µm, 300Å 1200 bar SB-C18, SB-C8, SB-C3, Diphenyl Poroshell 300 SPP, 5 µm, 300Å 400 bar SB-C18, SB-C8, SB-C3, Extend-C18 ZORBAX 300SB TPP, 3.5 & 5 µm, 300Å 400 bar SB-C18, SB-C8, SB-C3, SB-CN PLRP-S TPP, 3, 5, 8 um 100, 300, 1000, and 4000A 400 bar NA April 15, 2015 40

Benefits Pain Features and Advantages Benefit Insufficient resolution Long analysis times Short column lifetime Superficially porous particles and sub2um fully porous particles increase diffusion rate, mass transfer, and efficiency for biomolecules Analysis time can be decreased using faster flow rates and shorter superficially porous & sub2um fully porous particle columns without loss in efficiency Column with robust Poroshell packed bed and with 2 um frit decreases chances of bedcollapse or inlet blockage Improved confidence in analysis results (accuracy) Improved throughput - reduced costs Improved resource use - reduced costs HPLC pressure limitations Superficially porous particles maintain compatibility with all LC instruments System flexibility Agilent Technologies April 20, 2015 41

HYDROPHILIC INTERACTION LIQUID CHROMATOGRAPHY(HILIC) Agilent Technologies April 10, 2015 42

Why are glycans important to profile? Bio-Therapeutics Are Glycosylated Changes in glycosylation may affect drug safety and efficacy. Monoclonal Antibody Therapeutic Protein Reducing Risk, Variation through controlling glycosylation Fab The structural characterization and quantitative estimation of glycans is highly essential in biopharmaceutical projects. Glycans FDA Classification & Regulations Fc It is tremendously challenging to comprehensively characterize glycan profiles and determine the structures of glycans. Glycans Early stages

Principles of HILIC (glycans) Glycans are very hydrophilic They may be neutral or posses charges that will interact with silica and bonded phase Column bonded phases will greatly impact the separation performance of glycans Agilent Technologies April 20, 2015 44

Agilent Technologies April 20, 2015 45

Glycan Analysis Common Challenges Very long analysis times Instrument limitations Difficulty achieving reproducible results Agilent Technologies April 19, 2015 46

N-Glycan Mapping Glycoprotein N-Glycan HILIC column LC/MS 2-AB labelled glycan HILIC column LC/FLD (-MS) Majority process Agilent Technologies April 10, 2015 47

Glycan Analysis Workflow Deglycosylation Kit Description (24 or 96 samples) Reaction buffer 5X Glycoprotein Deglycosylation Denaturant Detergent PNGase F Work-up Deglycosylation Work-up Description (24 or 96 samples) SPE cartridges N-Glycans 2-AB Labeling Kit Work-up 2-AB Labelling Description (24 or 96 samples) 2-AB solution Reductant solution 2-AB Labeling Work-up 2-AB Labelled N-glycans Description (24 or 96 samples) SPE cartridges HILIC FLD / MS April 10, 2015 48

Glycan Analysis Workflow Glycoprotein Deglycosylation Work-up Unlabelled Standards N-Glycans Work-up 2-AB Labelling Labelled Standards 2-AB Labelled N-glycans HILIC FLD / MS April 10, 2015 49

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NEW! AdvanceBio Glycan Mapping Column for HPLC and UHPLC:

Glycan Analysis by LC-FLD + AdvanceBio Glycan Mapping, 2.7 um 1260 Infinity Bio-inert HPLC + AdvanceBio Glycan Mapping, 1.7 um 1290 Infinity UHPLC Agilent Technologies April 10, 2015 53

Superficially Porous Glycan Mapping Column High Speed and Resolution with Less Backpressure

AdvanceBio glycan column delivers faster separations with 1.8 µm particles FLD1 A, Ex=260, Em=430 (GLYCANAMIDE_RRHD\GLYCANS_RRHD040.D) 0.50mL/min, LU 55 C Inj = 2uL 1.2 in 70/30 ACN/water Mobile phase A: 100mM NH4 Formate ph 4.5, 1 Mobile phase B: ACN FLD: Ex=260 0.8 nm, Em=430 nm Sample: Agilent human IgG glycan library 0.6 0.4 0.2 0 0 27 2.5 80 0.505 7.5 10 12.5 15 17.5 20 22.5 FLD1 A, Ex=260, Em=430 (GLYCANAMIDE_RRHD\GLYCANS_RRHD050.D) LU 1.2 1 0.8 0.6 0.4 0.2 Time %B ml/min 0 80 0.50 25 60 0.50 26 0 0.50 Agilent AdvanceBio Glycan Mapping, 1.8 um 9.612 Waters Acquity UPLC BEH Glycan 1.7um 10.535 11.169 1 11.668 2 12.017 3 12.348 4 12.984 13.390 5 13.966 6 14.373 16.000 16.006 17.005 17.076 1 18.240 18.247 18.567 2 3 18.926 4 19.550 5 20.587 6 Same condition: 40% faster Better Rs Narrower peaks Same elution order 23.661 min 0 0 2.5 5 7.5 10 12.5 15 17.5 20 22.5 min Column RT (min) Rs 2,1 Rs 3,2 Rs 4,3 Rs 6,5 avg. PW (min) Peak Capacity Waters BEH Glycan 20.2 1.77 1.94 3.39 2.10 0.1085 214 AdvanceBio Glycan 12.7 2.60 2.90 5.43 2.81 0.0741 221 April 20, 2015

Monoclonal Antibody Glycan Mapping Rapid Separation of 2-AB labeled N-linked Human IgG glycan Library (Agilent): 2.1 x 150mm 1.8um AdvanceBio Glycan Mapping Column mobile phase: A: 100mM NH4 formate LUpH 4.5: B: ACN Inj.: 2ul 70:30 ACN: water Temp: 55 C FLD: 1 Excitation 260nm Emission 430nm 2 2.476 4 3.304 Isomer separation Time %A %B Flow rate (ml/min) 0 25 75 1.0 12 40 60 1.0 12.15 60 40 0.5 12.5 60 40 0.5 0.8 0.6 0.4 0.2 2-AB labelling reagent 0.345 0.363 1.965 2.093 12.9 25 75 0.5 13.05 25 75 1.0 15 25 75 1.0 Column Rs 2,1 Rs 3,2 Rs 4,3 Rs 6,5 avg. PW (min) Peak Capacity AdvanceBio Glycan Mapping Column, 1.8um 1 3 2.840 2.992 5 3.460 3.624 3.770 3.923 1 2 3 4 5 6 0 0 1 2 3 4 5 6 7 8 4.411 10 4.636 5.214 1.63 1.70 3.05 2.09 0.059 135 + 5.680 6.167 6.480 7.382 7.819 7.991 9 Separation < 10 min min April 20, 2015 56

HILIC Summary Characterization Product Features Advantage Benefit Pain Point Glycan Mapping AdvanceBio Glycan Mapping 1.8um FPP, bonded phase 2.7um SPP, bonded phase 1.8um FPP, bonded phase 2.7um SPP, bonded phase High efficiency, right selectivity Efficiency at higher flow rate, fast gradients Improved accuracy and reproducibility of data reliable results, cost saving Improved analysis efficiency, cost saving Resolution Throughput FPP: Fully Porous Particle, SPP: Superficially Porous Particle Agilent Technologies April 19, 2015 57

SIZE EXCLUSION CHROMATOGRAPHY Agilent Technologies April 10, 2015 58

Agilent Technologies April 20, 2015 59

Aggregation Studies Proteins aggregate! Protein aggregates can greatly enhance the immune responses to the protein of interest Protein aggregates can also enhance immune responses and may cause adverse event These responses may impact safety and efficacy Protein aggregates can be induced under forced conditions and can occur simultaneously during biopharmaceutical manufacturing Manufacturing Processes Steps and Products Fermentation Purification Formulation Storage Shipping Administration Stress Conditions Heat Freeze-thaw Cross-linking Protein concentration Formulation change ph, salt Chemical modification Mechanical stress / surface April 20, 2015 60

Size Exclusion Process Smaller molecules spend longer in the pores and elute later. Larger molecules spend less time in the pores and elute sooner. 61

Mechanism of SEC Separation Pore Size Determines Linear Separation Range

Some General Guidelines for SEC 1. When methods are to be validated, test for ruggedness with several different column lots, mobile phase preparations, and operators. 2. As a rule of thumb, SEC will only provide baseline separation of molecules with more than a 2 fold difference in MW. 3. Sample volume should be limited to below 5% of the total column volume. Max resolution.5 2% CV.

Common SEC challenges Insufficient/incorrect pore sizes can reduce resolution Non-specific interactions contribute to loss of sample, lead to inconsistent results, rework SEC is typically slow Consistent and robust results High salt conditions puts excessive wear on instrument, parts Agilent Technologies April 19, 2015 64

Pore Choice for Antibody Analysis 2 4 Eluent: 50mM NaH 2 PO 4 + 0.15M NaCl, ph6.8 Columns: Agilent Bio SEC-3,4.6x300mm Flow: 0.35ml/min Detector: UV@220nm System: Agilent 1260 Infinity Bio-Inert LC Sample: Mouse IgG 150Å 300Å 1 3 5 1. Dimer 2. Monomer 3. Monomer Fragment 4. Azide 5. Retained Molecule 100Å 65

SEC Column Choice: Resolving Ranges Agilent Technologies April 10, 2015 66

Size Exclusion Columns 5µm Particle 100Å, 150Å, 300Å, 500Å, 1000Å, 2000Å pore sizes High stability and long lifetime Great reproducibility Unique, 3µm particle 100Å, 150Å, 300Å pore sizes Highest resolution Highest efficiency Faster SEC separations

Fast, High resolution SEC characterization Step 1: Improve Resolution Technology: Small Particle SEC Columns Results: Higher Resolution SEC Separations

Use Higher Resolution to Achieve Faster Separations Step 2 Go Faster Increase flow rate and use a shorter column for faster separations. 2.0ml/min 1.5ml/min 1.0 ml/min monomer dimer Column: Agilent Bio SEC-3, 7.8 x150mm Sample: mab (2mg/ml) Injection: 5ul Flow rate: 1.0, 1.5 and 2ml/min (56 bar, 75 bar, 105 bar) Eluent: 150mM sodium phosphate + 100mM Na-sulfate Detection: 220nm Flow Rate Resolution Monomer/Dimer Monomer Efficiency Percentage Dimer 1.0ml/min 1.53 3,510 0.64 1.5ml/min 1.43 2,502 0. 47 2.0ml/min 1.13 1,917 0.64 4 Minutes Technology: Small Particle SEC Columns Results: Faster SEC separations Fast SEC TB5990-8613EN 69

Agilent Technologies April 20, 2015 70

Examples of Additives to reduce non specific interactions 100-150mM NaCl 100-150mM NaSO4 5-10% AcN 5-10% MeOH 50-100mM urea or Guanidine.1% SDS 71

Size Exclusion Summary Characterization Product Features Advantage Benefit Key Pain Point Aggregation Bio SEC 3um particles, porosities High efficiency, right selectivity High efficiency, right MW range Improved accuracy and reproducibility of data reliable results, cost saving Resolution 3um particles High efficiency Improved analysis efficiency, cost saving Throughput Agilent Technologies April 19, 2015 72

AFFINITY CHROMATOGRAPHY Agilent Technologies April 20, 2015 73

AFFINITY CHROMATOGRAPHY Agilent Technologies April 19, 2015 74

What is a Monolith General Monolith Characteristics Continuous material with porous channels that connect to other channels Mass transfer is convective (not diffusive) therefore faster - important for biomolecules Low back pressure High flow rates Agilent Technologies April 20, 2015 75

Agilent Technologies April 20, 2015 76

Analytical Bio-Monolith Protein A Columns Used for: Fast screening of harvest cell culture samples for IgG process optimization Accurate analysis of mab quantities to determine protein harvest Capture and purification of protein for further characterization Features and benefits: Bio-Monolith Protein A (immunoaffinity) Monolith type material for fast, flow rate independent separations Monolith material does not clog easily with cell debris Attaches easily to all LCs with standard fittings Agilent Technologies April 20, 2015 77

2 Minute Analysis Antibody Titer from Cell Culture Supernatant Column Agilent Bio-Monolith Protein A Sample: Cell lysate spiked with IgG1 Equilibration buffer: 50 mm NaPO 4, ph 7.4 Elution buffer: 0.1 M citric acid, ph 2.8 Flow Rate: 1.0 ml/min Detector: UV 280nm System: 1200 Infinity Absorbance ( (mau at 280 mm) mau 250 200 150 100 50 0 IgG1 wash elute re-equilibrate Flow through (8 µg) IgG1 (2.5 µg) Lysate proteins containing mab 0 0.25 0.5 0.75 1 1.25 1.5 1.75 2 2.25 Time (min) min Agilent Technologies April 20, 2015 78

Agilent Technologies April 20, 2015 79

ION-EXCHANGE CHROMATOGRAPHY Agilent Technologies April 10, 2015 80

Charge variants What we know? Surface charge changed mainly by post-translational modifications - Directly, as a change in the number of charged residues - Indirectly as a chemical or physical alteration that changes surfacecharge distribution May result during manufacturing or under storage conditions Originates from - Deamidation - Oxidation - Glycosylation or glycation - Proteolytic degradation - Amino acid substitutions - Amino acid deletions 81

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Ion Exchange Chromatography Charge Isoform Analysis of Monoclonal Antibodies 0.60 0.55 0.50 0.45 0.40 0.35 AU 0.30 0.25 0.20 0.15 0.10 0.05 0.00 (Acidic Isoforms) weakly bound (Basic Isoforms) Strongly bound 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 Minutes Column: Agilent Bio MAb, NP5, 4.6mm x 250mm Buffer A: 10 mm Sodium Phosphate, ph 7.50 Buffer B: A + 100 mm NaCl, ph 7.50 Gradient: 15-95% B in 60 min Flow rate: 0.8 ml/min. Sample: 5 µl, 5 mg/ml, mab

Ion Exchange Chromatography Separation based on charge

Some Guidelines for IEX 1.The General Rule for choosing a Bio IEX column - Acidic proteins: SAX or WAX - Basic proteins: SCX or WCX 2. Consider the isoelectric point (pi) of your protein when choosing the ph of your mobile phase: - If ph>pi, your protein will have a net negative charge - If ph<pi, your protein will have a net positive charge 3. The ph of your starting buffer should be 0.5 to 1 ph unit from your pi - Above pi for anion-exchange - Below pi for cation-exchange 4. If your pi is unknown - Start with ph 6 for cation-exchange - Start with ph 8.0 for anion-exchange Agilent Technologies April 19, 2015 85

Ion-exchange sorbents Weak ion-exchange sorbents have a narrower operating ph range than strong ion-exchange sorbents

Charge Variant Analysis Common challenges Resolution can be limited and inconsistent can require troubleshooting and rework - Need capability to handle complex analyses consistently - mabs present special challenges, due to their complexity Column contamination can lead to early column failure and produce incomplete sample recovery Method development is time-consuming - Need capability for faster, systematic method development with different buffer strengths Agilent Technologies April 19, 2015 87

Ion Exchange Product Families Particle Porosity Functionalities Particle Sizes Pore Size Application Agilent Bio-IEX Polymer Non-porous SAX, WAX, SCX, WCX 1.7um, 3um, 5um 10um Agilent Bio MAb Polymer Non-Porous WCX 1.7um, 3um, 5um 10um PL-SAX PS/DVB Fully Porous SAX 5um, 8m, 10um, 30um PL-SCX PS/DVB Fully Porous SCX 5um, 8m, 10um,30um N/A N/A Peptides, proteins IgG 1000A, 4000A Peptides, oligos, proteins. Larger column sizes 1000A, 4000A Peptides, proteins. Larger column sizes Bio-Monolith IEX Polymer Monolith QA, DEAE, SO 3 N/A N/A BioMacromoleucles 1. Non-porous particles for high efficiency analytical separations 2. Porous particles for scale up to purification 3. Monoliths for high speed separations Page 46 88

Ion Exchange Columns Non-porous PS/DVB particles Uniform polymeric coating and WCX layer, specifically designed for antibody separations Available in 10 µm, 5 µm, 3 µm, 1.7 µm particle sizes Non-porous PS/DVB particles Uniform polymeric coating with SCX, WCX, SAX, WAX layers, designed for protein and peptide separations Available in 10 µm, 5 µm, 3 µm, 1.7 µm particle sizes High surface area High capacity 89

Specifications Parameter Particle Size Binding Capacity Agilent Bio IEX 1.7, 3, 5, and 10um > 100 ug protein on column ph Range 2-12 Temp Limit 80 C Hardware/Pressure Pressure Limit Recommended Flow Rate: Mobile phase Compatibility 600 bar for SS 400 bar for PEEK 10um 275 bar (4000 psi) 5um 413 bar (6000 psi) 3um 551 bar (8000 psi) 1.7um - 689 bar (10000 psi) 0.1 1 ml/min Acetonitrile/acetone/methanol mixtures 90

Optimum Flow Rate for NP3 and NP1.7 Proteins are not small molecules! Proteins are sluggish! Proteins are complex three dimensional structures! NP1.7 NP3 NP5 NP10 Higher efficiency, higher pressure

Bio-WCX Working Flow Rates/ Pressures Column Dimension Flow Rate Agilent Bio IEX Bio-WCX NP10 4.6x250mm.8 ml/min 50-60bar Bio-WCX NP5 4.6x250mm.8 ml/min 150-250bar Bio-WCX NP 3 4.6x50mm.5 ml/min 80-120 bar Bio-WCX NP 1.7 4.6x50mm.5 ml/min 110-320 bar 92

Reasons for High Column Pressure? CAUTION: Risk of blockage with NP1.7 is significantly higher. 93

Agilent Bio IEX Columns Comparing Separations on Each Particle Size 1.7 µm 3 µm 5 µm Peak N 10 µm Peak N Column: Bio WCX, 4.6 x 50 mm Buffer A: 20 mm PBS Buffer B: A+1.0 M NaCl Gradient: 0-100%B (20 min) Flow rate: 1.0 ml/min for 10 µm, 5 µm, 3 µm 0.75 ml/min for 1.7 µm Sample: 1) Ribonuclease A 2) Cytochrome C 3) Lysozyme Concentration: 1.0 mg/ml Detector: 280 nm Average N ~80,000 for WCX 1.7 µm 0 2 4 6 8 10 12 14 16 18 20 22 Min Agilent Technologies April 10, 2015 94

Fast, High Resolution IEX Separations Bio WCX NP3, 4.6 x 50 mm Bio WCX NP1.7, 4.6 x 50 mm 1.0 ml/min 1.0 ml/min Technology Used: small particle columns (3um, non porous) Result: 5 minute, high resolution IEX 95

Standard Protein Separation: Bio WCX Conventional HPLC (400 bar) mau 500 400 Column: Bio WCX NP3 (4.6x50mm SS) Change a 5um standard to 3um short column: saves time maintains resolution 300 200 100 Gradient: 20mM sodium phosphate buffer, ph 6.5 0-800mM NaCl (0-20 mins) Flow rate: 1.0mL/min Sample: 10µl inj. Detection: UV, 220nm 0 mau 350 300 250 0 1 2.5 5 7.5 10 12.5 15 17.5 20 22.5 Column: Bio WCX NP5 (4.6x250mm SS) 2 4 1. Ovalbumin pi 4.5 2. RNase A pi 9.4 3. Cytochrome C pi 9.8 4. Lysozyme pi 11 min 200 3 150 100 50 0 0 2.5 5 7.5 10 12.5 15 17.5 20 22.5 min 96

Selectivity Comparison 3 Sample: 1. Ribonuclease 2. Cytochrome C 3. Lysozyme - SCX 1 2 3 3 Sample concentration: 2 mg/ml Injection volume: 2 ul Columns: Agilent Bio SCX, NP 3, 4.6x50 mm, SS Agilent Bio WCX, NP 3, 4.6x50 mm, SS Agilent Bio MAb, NP 3, 4.6x50 mm, SS - WCX - MAb 2 1 1 Eluent A:10 mm NaH 2 PO 4.2H 2 O, ph 5.75 Eluent B: A + 1 M NaCl Gradient: 0 to 100% b in 25 mins Flow rate: 0.5 ml/min Temperature : Ambient Detector: UV at 220 nm April 20, 2015 97

Analytical Bio-Monolith Ion Exchange Columns Polymer based monolithic discs Fast, high resolution ion-exchange Key applications are for large proteins and biomolecules (virus particles, pdna, antibodies [IgG and IgM]) Agilent Bio-Monolith QA (strong anion-exchanger) Agilent Bio-Monolith DEAE (weak anion-exchanger) Agilent Bio-Monolith SO3 (strong cation-exchanger) Agilent Technologies April 10, 2015 98

Resources for More Information www.agilent.com/chem/getbioguides Selection Guide Application-focused Brochures Reversed-Phase Ion-Exchange Size Exclusion How To Guides Your Reference Guide to the Analysis of Biopharmaceuticals and Biomolecules 5990-9384EN Protein Identification and Impurity Profiling using Reversed- Phase HPLC/UHPLC 5991-0625EN Characterize Charged Variants of Proteins with Speed and Confidence 5991-2449EN Affinity Resolve Protein Aggregates and Degradants with Speed and Confidence 5991-2898EN Characterize Charged Variants of Proteins with Speed and Confidence 5991-2449EN Keys for Enabling Optimum Peptide Characterizations: A Peptide Mapping How To Guide 5991-2348EN Size Exclusion Chromatography for Biomolecule Analysis: A How To Guide 5991-3651EN Ion-Exchange Chromatography for Biomolecule Analysis: a How to Guide 5991-3775EN Agilent Technologies April 20, 2015 99

BioHPLC Columns on the Agilent Website To learn more and order online visit www.agilent.com/chem/biocolumns Agilent Technologies April 20, 2015 100

NEED ASSISTANCE? LC Column help desk 1-800-227-9770 orders customer service (option 1,1) technical support applications assistance (option 3,3,2) lc-column-support@agilent.com 101

Thank you! Questions?