Making the Leap Small Molecule – Biologics Rita Steed LC Columns Application Engineer May 8, 2019 1 May 8, 2019 Small molecule – Biologics Agilent Restricted Outline • Small and/or biomolecules; similarities/differences • Column choice • Things to consider • Method conditions • Troubleshooting 2 May 8, 2019 Small molecule – Biologics Agilent Restricted Small and/or Biomolecule; Similarities/Differences Define Types of chromatography • Similar • Different Sample types • Simple • Complex Functional groups • Polarity 3 May 8, 2019 Small molecule – Biologics Agilent Restricted Small v. Biomolecules Small molecules Biologics • Size – MW<1000 (although some may be • Size – Mostly >1000 with some exceptions somewhat larger) • Structure – Diverse; need to consider chemistry, • Structure – Simple drugs, pesticides, chemicals biology, and biochemistry • Manufacturing – Chemical synthesis, MAbs, aggregates, charge variants, conjugates, predictable process peptides, amino acids, nucleic acids • Characterization – Straightforward • Manufacturing – Living cells; “dirty” (early) process with similar components; difficult to control • Stability – Stable • Characterization – Complex molecular makeup; heterogeneity • Stability – Sensitive to external conditions; temp, pH, shearing, folding, glycoforms 4 May 8, 2019 Small molecule – Biologics Agilent Restricted HPLC Common Separation Mechanisms Small molecules Biomolecules • Reversed phase* • Reversed phase* • Ion exchange • Ion exchange • HILIC • Size exclusion/Gel filtration • Normal phase • Hydrophobic interaction (HIC) • Chiral • HILIC • Affinity *Of the many different HPLC separation mechanisms and column types, reversed phase is commonly used for small molecules and biomolecules. 5 May 8, 2019 Small molecule – Biologics Agilent Restricted Nonpolar and Polar Functional Groups* Nonpolar (Hydrophobic) Polar (Hydrophilic) -CH3 -COOH • Methyl • Carboxyl groups -(CH2)nCH3 -NH2 • Alkyl groups • Amino groups -C6H5 -OH • Phenyl groups • Hydroxyl Groups -CONH2 • Amide groups *Affect solubility and elution order 6 May 8, 2019 Agilent Restricted Do You Know What This Is? C22H30N6O4S MW=476.6 g/mol O CH3 N N O O N S N N H N H3C O CH3 CH3 Sildenafil 7 May 8, 2019 Small molecule – Biologics Agilent Restricted Do You Know What This Is? C6470H10012N1726O2013S42 MW = 145421.50 g/mol DIQMTQSPSS LSASVGDRVT ITCRASQDVN TAVAWYQQKP GKAPKLLIYS ASFLYSGVPS RFSGSRSGTD FTLTISSLQP EDFATYYCQQ HYTTPPTFGQ GTKVEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC EVQLVESGGG LVQPGGSLRL SCAASGFNIK DTYIHWVRQA PGKGLEWVAR IYPTNGYTRY ADSVKGRFTI SADTSKNTAY LQMNSLRAED TAVYYCSRWG GDGFYAMDYW GQGTLVTVSS ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKKVEP KSCDKTHTCP PCPAPELLGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSREE MTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPG EVQLVESGGG LVQPGGSLRL SCAASGFNIK DTYIHWVRQA PGKGLEWVAR IYPTNGYTRY ADSVKGRFTI SADTSKNTAY LQMNSLRAED TAVYYCSRWG GDGFYAMDYW GQGTLVTVSS ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKKVEP KSCDKTHTCP PCPAPELLGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSREE MTKNQVSLTC LVKGFYPSDI AVEWESNGQPHerceptin ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPG DIQMTQSPSS LSASVGDRVT ITCRASQDVN TAVAWYQQKP GKAPKLLIYS ASFLYSGVPS RFSGSRSGTD FTLTISSLQP EDFATYYCQQ HYTTPPTFGQ GTKVEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC 8 May 8, 2019 Small molecule – Biologics Agilent Restricted Choosing the Right Column Sorbent characteristics • Particle size • Plate count • Back pressure • Pore size • Bonding chemistry – Small Molecule – C18, C8 – Proteins – C4, C8 Column characteristics • Internal diameter • Length • Material Instrument compatibility 9 March 6, 2019 Small molecule – Biologics Agilent Restricted Particle Size ➢ Diameter ➢ Plate count ➢ Back pressure Column Resolving Resolving Resolving Typical Length Power Power Power Pressure* (mm) N(5 µm) N(3.5 µm) N(1.8 µm) Bar (1.8 µm) 10 µm 150 12,500 21,000 32,500 724 100 8,500 14,000 24,000 420 75 6000 10,500 17,000 320 50 4,200 7,000 12,000 210 30 N.A. 4,200 6,500 126 3 µm 1.8 µm 15 N.A. 2,100 2,500 55 5 µm • pressure determined with 60:40 MeOH/water, 1ml/min, 4.6mm ID 10 March 6, 2019 Small molecule – Biologics Agilent Restricted Increase Resolution No Run Time Increase! mAU mAU 40 μm μm 40 3.5 3.5 30 5 20 20 10 4.6 x 150 mm,x 4.6 150 4.6 x 150 mm, x 150 4.6 0 0 0 2 4 6 8 min 0 2 4 6 8 min mAU • Increased resolution due to reduced peak μm width 60 1.8 1.8 • Increased peak height due to reduced peak width gives increased sensitivity 40 20 4.6 x 150 mm, x 150 4.6 0 0 2 4 6 8 min Pore Size and Molecular Size Effect on Peak Width 0.2 0.18 300SB-C18 (300Å) 0.16 SB-C18 (80Å) 0.14 0.12 0.1 PW 1/2 PW 0.08 0.06 0.04 0.02 0 12 March 6, 2019 Small molecule – Biologics Agilent Restricted Pore Size: Effect on the efficiency of a large molecule 13 May 8, 2019 Small molecule – Biologics Agilent Restricted Columns for intact protein analysis No “one size fits all” column … Particle Diameter Diffusion Distance Pore Size Matrix Porosity ZORBAX RRHD 300SB 1.8µm 0.9µm 300Å Silica 100% (fully porous) 0.25µm AdvanceBio RP-mAb 3.5µm 450Å Silica 37% (ultra thin shell) (3.0µm solid core) 0.25µm Poroshell 300 5µm 300Å Silica 27% (ultra thin shell) (4.5µm solid core) ZORBAX 300SB 5µm 2.5µm 300Å Silica 100% (fully porous) PLRP-S 1000Å 5µm 2.5µm 1000Å Polymer 100% (fully porous) • Smaller particle diameter for sharper peaks and increased resolution, but higher operating pressures • Superficially porous particles for sharper peaks and lower operating pressures • Polymeric particles for increased pH stability and reduced non-specific interactions • Pore size should be large enough to accommodate the molecule of interest 14 August, 2018 Agilent Technologies Bonding Chemistry Sorbent characteristics • Particle size • Plate count • Back pressure • Pore size • Bonding chemistry – Small Molecule – C18, C8 – Proteins – C3, C4, C8 Column characteristics • Internal diameter • Length • Material Instrument compatibility Page 15 May 8, 2019 Agilent Restricted Selectivity Differences Across InfinityLab Poroshell Bonded Phases mAU 3 4 7 150 InfinityLab Poroshell 120 EC-C18 8 1 100 2,5 6 50 0 0 2 4 6 8 10 12 mAU 3 4 7 InfinityLab Poroshell 120 SB-C18 8 150 1 100 2,5,6 50 0 0 2 4 6 8 10 12 mAU InfinityLab Poroshell 120 Phenyl Hexyl 3 4 7 150 8 1 100 2 5 6 50 0 0 2 4 6 8 10 12 40-80 % Methanol in 14 min, DAD 260, 80 nm 0.4 ml/min, 1. Hydrocortisone 2. B Estradiole, 3. Andostadiene 3. 17 dione, 4. Testosterone 2.1 x 100 mm column, 40 C, 0.1% Formic Acid in Water and 5. Ethyestradione 6. Estrone 7. Norethindone acetate 8. Progestreone Methanol, Agilent 1260 Method Development Solution 16 May 8, 2019 Agilent Technologies Bonding Chemistry • AdvanceBio RP-mAb C4 provides a sharp peak and resolves fine detail in less than 2-minutes • AdvanceBio RP-mAb Diphenyl resolves additional fine detail • AdvanceBio RP-mAb C4 • AdvanceBio RP-mAb SB-C8 mAU 14 mAU • AdvanceBio RP-mAb Diphenyl 12 140 10 8 120 6 Method Parameters 4 Column: 2.1 x 100 mm 100 Mobile phase A: 2 0.1% TFA in water:IPA (98:2) 80 0 Mobile phase B: -2 IPA:acetonitrile:MPA* (70:20:10) Flow rate: 1.0 mL/min, 60 -4 Temperature: 80 °C 1.7 1.8 1.9 2 2.1 2.2 2.3 2.4 2.5 min Gradient: 10-58% B in 4 min, 40 Injection: 5 μL Sample: Herceptin IgG1 20 All phases provides good separation of Temperature: 80 °C intact mAb however AdvanceBio RP-mAb Detection: UV @ 254 nm 0 Diphenyl resolves additional fine detail. 1.7 1.8 1.9 2 2.1 2.2 2.3 2.4 2.5 min 17 August, 2018 May 8, 2019 Agilent Technologies Agilent Restricted Agilent InfinityLab Poroshell 120 Portfolio Small Molecules and Peptides Best for Low Best for High Best for Best for Polar Best All Round pH Mobile pH Mobile Alternative Best for Chiral Analytes Phases Phases Selectivity InfinityLab Poroshell InfinityLab Poroshell InfinityLab Poroshell InfinityLab Poroshell InfinityLab Poroshell InfinityLab Poroshell EC-C18 SB-C18 HPH-C18 Bonus-RP HILIC Chiral-V 1.9 µm, 2.7 µm, 4 µm 2.7 µm 1.9 µm, 2.7 µm, 4 µm 2.7 µm 1.9 µm, 2.7 µm, 4 µm 2.7 µm InfinityLab Poroshell InfinityLab Poroshell InfinityLab Poroshell InfinityLab Poroshell InfinityLab Poroshell InfinityLab Poroshell EC-C8 SB-C8 HPH-C8 PFP HILIC-Z Chiral-T 1.9 µm, 2.7 µm, 4 µm 2.7 µm 2.7 µm, 4 µm 1.9 µm, 2.7 µm, 4 µm 2.7 µm 2.7 µm InfinityLab Poroshell InfinityLab Poroshell InfinityLab Poroshell Phenyl-Hexyl HILIC-OH5 Chiral-CD 1.9 µm, 2.7 µm, 4 µm 2.7 µm 2.7 µm InfinityLab Poroshell InfinityLab Poroshell SB-Aq Chiral-CF 2.7 µm 2.7 µm 4 µm 2.7 µm 1.9 µm InfinityLab Poroshell EC-CN Reversed phase chemistries 2.7 µm 18 May 8, 2019 Small molecule – Biologics Agilent Restricted Agilent Biomolecule Columns Agilent Biomolecule HPLC Columns Sequence Charge Titer Aggregate Glycan Amino Acid / Cell Culture Media Intact Purity & PTM Analysis Variant & PTM Variant Determination Analysis Analysis Analysis Analysis Analysis Reverse Phase Hydrophobic Reverse Phase Hydrophilic Reverse Phase Hydrophilic Affinity Size Exclusion Ion Exchange >150 Å Interaction < 150 Å Interaction < 150 Å Interaction Bio-Monolith AdvanceBio AdvanceBio AdvanceBio AdvanceBio AdvanceBio PLRP-S