HALO Bioclass catalog

Customer Service About Canadian Life Science Au suject de Canadian Life Science Founded in 1997, Canadian Life Science (CLS) has steadily grown Fondée en 1997, Canadian Life Science (CLS) n’a cessé de croître to Canada’s leading supplier of LC/GC columns, chromatography pour devenir un chef de file canadien dans la vente de colonnes and dissolution accessories as well as 2d long-term sample HPLC / GC, d’accessoires de chromatographie et dissolution, storage tubes and racks for bio-banking and compound ainsi que dans la vente de tubes et systèmes d’entreposage long management. terme pour échantillons. For over 22 years, CLS has provided customers with unparalleled Depuis plus de 22 ans, CLS offre à ses clients un service et service, expertise and a wide selection of quality and cost- une expertise inégalés, en plus d’un large éventail de produits effective products that offer maximum flexibility to solve your économiques de qualité offrant une flexibilité maximale afin de analytical challenges. CLS is your foundation for what matters. résoudre vos problèmes analytique. CLS est votre pilier pour ce qui compte. Everyone is accessible to you Nous sommes très accessible We believe that our customers like our personal touch. Nous avons une approche personnelle et nous répondons à tous We take your calls personally, not a computer, not voicemail . les appels lorsque vous téléphonez au bureau chef. Nous n’avons We have dedicated inside customer service staff who care, and pas de boîte vocale. take care for specific areas. We have knowledgeable outside staff Notre personnel est dévoué au service à la clientèle et ce à based in most of the larger cities accross Canada. travers le Canada. Si vous désirez parler directement à notre We supply world class products Directrice de comptes au Québec, vous pouvez contacter Canadian Life Science distributes for top-quality manufacturers. Genevieve Lemieux au 514.428.8034. Our product offering has been chosen in a way that will give you Nous choisissons les meilleurs produits au monde the maximum flexibility solving your analytical challenges. En tant que distributeur nous avons l’avantage de choisir les Technical Support meilleurs fournisseurs et notre gamme complète de produit vous Our technical staff is happy to respond to customer enquiries. donne l’embarras du choix. We can also supply extensive application information from one Support Technique of the world’s largest data bases. Notre équipe technique se fait toujours un plaisir de vous aider à Plan the best delivery faire le meilleur choix pour vos applications. When you decide to purchase a product from Canadian Life Service et logistique Science our customer service department will find the fastest Lorsque vous placez une commande chez nous, nous nous and most economical way to send your order to you. For assurons de choisir le moyen le plus efficace et économique your convenience we have two warehouse locations, one in d’acheminer la marchandise. Edmonton, AB and one in Peterborough, ON. Nous avons deux entrepôts dont un en Alberta pour servir nos We’re glad to help clients de l’Ouest et un en Ontario pour servir nos clients de l’Est. We are never too busy to take your call. C’est un plaisir de vous servir Try us. You’ll notice the difference. Nous ne sommes jamais trop occupés pour vous répondre. Canadian Life Science, friendly people to do business with! Essayez-nous et vous verrez la différence. Il est agréable de faire affaire avec nous! I-22 CANADIAN Proud member of the Chrom4 buying group Fier membre du groupe dʼacheteur Chrom4 LIFE SCIENCE 1 888-226-2775 :: [email protected] :: www.lifescience.ca

TABLE OF CONTENTS 3 HALO® PROTEIN, PEPTIDE AND GLYCAN OVERVIEW 4 PROTEIN SOLUTIONS 5 HALO 1000 Å PROTEIN BONDED PHASE PORTFOLIO 8 HALO 400 Å PROTEIN BONDED PHASE PORTFOLIO 10 PEPTIDE SOLUTIONS 11 HALO® PEPTIDE BONDED PHASE PORTFOLIO 14 GLYCAN SOLUTIONS 16 HALO® UHPLC AND HPLC GUARD COLUMNS 17 HALO® BIOCLASS SPECIFICATIONS TABLES 18 PART NUMBER LISTING BIOCLASS 1000 Å 2.7 micron particle 400 Å 3.4 micron particle 90 Å 2.7 micron particle PROTEIN GLYCAN 160 Å 2 micron particle 160 Å 2.7 micron particle 160 Å 5 micron particle 2   |  PEPTIDE HALO® and Fused-Core® are registered trademarks of Advanced Materials Technology, Inc.

HALO® PROTEIN, PEPTIDE AND GLYCAN Advanced Materials Technology led the revolution in biosimilars and other products of bioengineering in Fused-Core® particle technology with the and manufacturing. HALO® BioClass columns have development of the first commercially available been developed to simplify and promote a more sub-3 μm superficially porous particle (SPP) with the comprehensive understanding - faster. original HALO® 2.7 μm particle. Our manufacturing expertise has carried forward into being the wide • Intact proteins, monoclonal antibodies (mAbs), pore particle leader. This innovative culture has biosimilars, and other large biomolecules such been transferred to developing novel materials for as pegylated proteins, antibody drug conjugates the biotherapeutic market. HALO® particles are (ADCs), etc. ready to solve the characterization challenges of these complex separations. • P eptide mapping (analysis of enzyme digests) for characterization and monitoring of synthetic Today, researchers are keenly interested in both protein drugs fast and high resolution separations of numerous biomolecules to support the development of • A nalysis of therapeutic peptides and peptide novel therapeutic proteins and peptides in biomarkers (protein surrogates) pharmaceutical drug development, advance academic understanding in modern University • H igh resolution separations of complex mixtures laboratories, characterize protein post-translation of glycans released from N- and O-linked modifications, and to fully assess subtle differences glycoproteins The HALO® BioClass column family is comprised of the following product lines. PROTEIN PEPTIDE GLYCAN • W ide pore portfolio for • SPP technology for fast, high • Improved retention of acidic and unrestricted bonded phase resolution peptide separations zwitterionic analytes access capable of characterizing very large proteins with good • High peak capacities delivering • V ery low sensitivity to buffer peak shape and recovery rugged, reliable performance concentration for use with either UHPLC, • C ompatible chemistries for HPLC, or LC-MS • A ble to separate isobaric UHPLC, HPLC, and mass oligosaccharides with different spectrometry • E xtensive portfolio of particle linkages sizes and chemistries • Variety of bonded phase options for a tailored solution Guidance for Pore Size Selection Molecule Size Pore Size (Å) Application Particle Sizes (µm) 2.7 SMALL 90 Glycan (< 20 kDa*) 2, 2.7, 5 160 Peptide 3.4 MEDIUM 2.7 (100 Da < MW < 15 kDa) 400 Protein 1000 |www.fused-core.com    3 LARGE (2 kDa < MW < 500 kDa) LARGE (> 50 kDa) * for glycans, glycopeptides and glycoproteins

HALO® PROTEIN 2.7 µm PROTEIN SOLUTIONS 2.7 µm 1.7 µm • T he advantages of using wide pore silica based superficially porous particles (SPP) for high resolution analysis of large 0.5 µm proteins has been well established and as the innovator Shell with 1000 Å pores of the 1000 Å Fused-Core® particle, AMT recognizes the benefit of unrestricted pore access which combines the HALO® PROTEIN 3.4 µm power of ultrafast and high resolution separations to the biologics workflow 3.0 µm 3.4 µm • F used-Core® particles provide narrower peak widths and 0.2 µm improved resolution for characterization of biomolecules in Shell with 400 Å pores comparison to fully porous particles (FPPs) • A s complex biotherapeutics development continues to grow, understanding structural modifications requires separation options. Often these minor variants consist of subtle differences in protein chains, glycosylation sites and free sulfhydryl groups • H ALO® delivers a comprehensive portfolio of both 400 Å and 1000 Å silica phase selectivities to choose from APPLICATIONS FEATURES • mAbs •Outstanding temperature stability up to 90 °C • ADCs •Compatible with UHPLC, HPLC and MS • Biosimilars • E lution of very large proteins with excellent peak • H/D exchange • Fragments shape and recovery • Very low LC-MS bleed ANTIBODY-DRUG CONJUGATES BIOSIMILARS H/D EXCHANGE FRAGMENTS mAb PROTEIN 4   | 

HALO 1000 Å PROTEIN BONDED PHASE PORTFOLIO HALO 1000 Å C4 Ligand: DIMETHYLBUTYLSILANE CH3 USP Designation: L26 CH3 Available Particle Sizes: 2.7 µm O Si CH3 Pore Size: 1000 Å HALO 1000 Å ES-C18 H3C Ligand: DIISOBUTYLOCTADECYLSILANE O Si CH3 CH3 USP Designation: L1 H3C (CH2)17 Available Particle Sizes: 2.7 µm Pore Size: 1000 Å CH3 HALO 1000 Å DIPHENYL O Si CH3 Ligand: DIPHENYLMETHYL USP Designation: L11 Available Particle Sizes: 2.7 µm Pore Size: 1000 Å |www.fused-core.com    5

INADAQUATE PORE SIZE RESULTS IN BROADER PEAKS AND LOWER RESOLUTION The larg pores of Absorbance, mAU 40 1 (B) HALO 1000 Å C4, 2.7 µm TEST CONDITIONS: the HALO 1000 Å 35 P = 120 bar Columns: HALO 1000 Å C4, 2.7 µm, 2.1 x 150 mm C4 column allow 30 2 (B) Part Number: 92712-714 improved access 25 (A/B) 7 peaks Mobile Phase A: 88/10/2 water/ACN/n-propanol/0.1% DFA 20 4 Mobile Phase B: 70/20/10 n-propanol/ACN/water/0.1% DFA 15 3 Gradient: 14-24% B in 20 min 10 Flow Rate: 0.2 mL/min 2b 5 (A) 5 Twice the 0 6 (A*) peaks Temperature: 80 °C to the stationary Almost half -5 9 10 11 12 13 Injection: 2 µL of 2 mg/mL denosumab in water/0.1% DFA 8 Time, min phase and increased the back 40 Detection: 280 nm, PDA resolution for IgG2 pressure 35 LC System: Shimadzu Nexera 30 Absorbance, mAU FPP 300 Å C4, 1.7 µm isoforms compared 25 P = 205 bar PEAK IDENTITIES to the smaller 300 Å 20 1. IgG2-B 4. IgG2-A/B 15 2. IgG2-B 5. IgG2-A 10 pores of the FPP C4 5 2b. IgG2-B 6. IgG2-A* column. 3. IgG2-A/B Comparative results presented 0 here may not be representative -5 9 10 11 12 13 for all applications 8 Time, min Comparative results presented here may not be representative for all applications. WIDE PORE BONDED PHASE OPTIONS AMT recognizes Absorbance, mAU HALO Diphenyl TEST CONDITIONS: mAbs are unique HALO ES-C18 Columns: as indicated, 2.1 x 150 mm and; therefore, Abs (280 nm) HALO C4 Mobile phase A: water/0.1% TFA developed three Mobile phase B: ACN/0.1% TFA 1000 Å bonded Gradient: 32–40% B in 16 min phase options Flow rate: 0.4 mL/min for tailored Temperature: 80 °C charaterization Injection volume: 2 μL screening. Instrument: Shimadzu Nexera Detection: 280 nm, PDA FPP 300Å C4, 1.7µm Comparative results presented here may not be representative for all applications. 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 min Time, min HALO® OUTPERFORMS THE COMPETITION While many protein Absorbance, mAU TEST CONDITIONS: separations occur at Columns: 2.1 x 150 mm higher temperatures, Mobile Phase A: water/0.1% TFA the desire to carry Mobile Phase B: ACN/0.1% TFA them out at lower Gradient: 30-45% B in 15 min temperature exists. Flow rate: 0.4 mL/min In this example of Temperature: 40 °C​ the HALO® Diphenyl Injection volume: 2 µL of 2 mg/mL trastuzumab versus a competitor in water/0.1% TFA phenyl phase note Detection: 280 nm, PDA the exemplary performance of the Comparative results presented here may not be HALO® Diphenyl. representative for all applications. Time, min 6   | 

OPTIMIZED TRASTUZUMAB SEPARATION USING A HALO 1000 Å DIPHENYL COLUMN The separation using this highly efficient HALO 1000 Å Protein Abs (280 nm) TEST CONDITIONS: Diphenyl column is Mobile phase A: water (0.1% DFA) completed in less Mobile phase B: 50/50 ACN/n-propanol/0.1% DFA than 30 minutes, Gradient: 29–33% B in 29 min while being Flow rate: 0.25 mL/min compatible with Temperature: 60 °C both UV detection, Injection volume: 2 μL of 2mg/mL trastuzumab in as well as online water/0.1% TFA Instrument: Shimadzu Nexera Detection: 280 nm, PDA high resolution MS detection. 0 5 10 15 20 25 30min Time, min IMPROVED PEAK WIDTH ACROSS VARIOUS MABS The improved PW1/2 (min) TEST CONDITIONS: performance of Columns: 2.1 x 150 mm ultra wide pores Mobile Phase A: water/0.1% DFA is not just realized Mobile Phase B: ACN/0.1% DFA with one mAb. Gradient: 27-37% B in 20 min Note the advantage Flow rate: 0.4 mL/min to five various large Temperature: 80 °C proteins. Injection Volume: 2 µL (1 µg) Instrument: Shimadzu Nexera 75% narrower peak widths! Detection: 280 nm, PDA RUGGEDNESS AND RELIABILITY TEST CONDITIONS: Columns: HALO 1000 Å ES-C18, The HALO 1000 Å Absorbance, mAU 2.7 µm, 2.1 x 50 mm stationary phases Mobile Phase A: water/ 0.1% TFA offer rugged and Time, min Mobile Phase B: ACN/ 0.1% TFA reliable performance Gradient: 32-60% B in 6 min every time. In the Flow Rate: 0.4 mL/min example of HALO Injection Volume: 1.0 μL 1000 Å ES-C18, the Temperature: 80 °C retention times for Detection: 280 nm, PDA trastuzumab show Sample: trastuzumab extreme phase stability for over |www.fused-core.com    7 500 injections.

HALO 400 Å PROTEIN BONDED PHASE PORTFOLIO HALO 400 Å C4 CH3 Ligand: DIMETHYLBUTYLSILANE O Si USP Designation: L26 CH3 Available Particle Sizes: 3.4 µm CH3 Pore Size: 400 Å HALO 400 Å ES-C18 Ligand: DIISOBUTYLOCTADECYLSILANE H3C USP Designation: L1 Available Particle Sizes: 3.4 µm CH3 Pore Size: 400 Å O Si (CH2)17 CH3 H3C CH3 ANTIBODY-DRUG CONJ ANTIBODY FRAGMENTS POLYPEPTIDE 8   | 

HIGH RESOLUTION OF LIGHT AND HEAVY CHAIN VARIANTS OF IgG1 Masses deconvoluted TEST CONDITIONS: 3.5 using MagTran software* Columns: HALO 400 Å C4, 3.4 µm, 2.1 x 100 mm 1366 .0 3 1290 .0 Very high 2.5 1221 .9 Part Number: 93412-614 resolution is 1161 .4 Mobile Phase A: 0.5% formic acid with 20 mM obtained between variants of light 2 1105 .9 1451 .6 Ammonium Formate and heavy chains of a reduced Mobile Phase B: 45% ACN/45% IPA/10% A solvent and alkylated monoclonal 1.5 Gradient: 29–32% B in 20 min antibody (IgG1) sample using a 1 1546 .7 1655 .6 Temperature: 80 ˚C HALO 400 Å Protein C4 column. 1010 .1 1783 .1 Injection Volume: 2 μL of 2 μg/μL reduced and 7 LC1: 23,204 0.5 1931 .5 alkylated lgG1 1859 .1 Detection: 280 nm, UV and MS using 2pps scan rate 930 .2 1604 .6 773 .6859 .4 from 500 to 2000 m/z 6 0 Sample Solvent: 0.25% (v/v) formic acid in water 500 750 1000 1250 1500 1750 m/z Absorbance, mAU 5 MS Parameters: Positive ion mode, ESI at +4.5 kV, HC2: 50,424 HC3: 50,668 400 ˚C heatblock, 225 ˚C capillary 4 HC1: 50,539 LC= light chain LC-MS System: Shimadzu Nexera and LCMS-2020 HC5: 28,862 (single quadrupole MS) 3 HC= heavy chain LC3: 23,203 2 LC2: 23,192 1 HC4: 50,680 0 -5 0 2.5 5 7.5 10 12.5 15 17.5 20 Time, min PROTEIN SEPARATIONS: 3.4 µm FUSED-CORE VS. 3 µm FULLY POROUS PARTICLE 85 HALO 400 Å ES-C18, 3.4 µm 0.0065 TEST CONDITIONS: 75 Pressure: 234 bar 0.0011 Columns: 4.6 x 100 mm 65 Mobile Phase A: water/0.1% TFA 55 0.1 0.2 0.3 0.0065 0.0069 0.0082 Mobile Phase B: ACN/0.1% TFA 45 Gradient: 23-85% B in 1 min Improved peak 35 0.4 0.5 0.6 0.7 0.8 0.9 1.0 widths are observed 25 3 + 6 µL heat exchangers with the HALO 15 Flow rate: 3 mL/min 400 Å versus the Temperature: 60 °C competitor 300 Å 5 Injection Volume: 5 µL fully porous column. -5 Instrument: Agilent 1200 SL Detection: 215 nm, PDA 0.0 85 Fully Porous 300 Å C18, 3 µm 0.0114 0.0102 75 Pressure: 244 bar 65 0.0104 0.0107 0.0150 PEAK IDENTITIES 13.7 kDa 55 0.1 0.2 0.3 12.4 kDa 45 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1. Ribonuclease A 14.3 kDa 35 Time, min 2. Cytochrome c 14.2 kDa 25 3. Lysozyme 250 kDa total; tetramer of 15 4. α-Lactalbumin ~60 kDa each 5. Catalase 5 -5 0.0 Comparative results presented here may not be representative for all applications. JUGATES BIOSIMILARS PROTEIN FRAGMENTS mAb |www.fused-core.com    9

PEPTIDE SOLUTIONS HALO® 2 µm Peptide • H ALO® BioClass Peptide solutions offer an extensive 2 µm portfolio of particle sizes and phases to tailor a solution ideal 1.2 µm for both ultrafast and ultrahigh resolution separations of 0.4 µm peptides and polypeptides up to 20 kDa Shell with 160 Å pores • F ast, high resolution, high peak capacity peptide separations HALO® 2.7 µm Peptide at 40-50% the back pressure compared to sub-2 µm particle columns 2.7 µm 1.7 µm • D ue to the lower back pressure of Fused-Core® design, 0.5 µm columns can be used in series to maximize peak capacity for Shell with 160 Å pores UHPLC and HPLC analyses of complex tryptic digest samples HALO® 5 µm Peptide • C ompatible for UHPLC and LC-MS applications offering high efficiency and stability • ~ 20% higher peak capacity than sub-2 µm non-core columns (2 µm) APPLICATIONS 3.3 µm 4.6 µm • T ryptic digests • P ost-Translational Modifications (PTMs) 0.6 µm • Variants Shell with 160 Å pores • Polypeptides POLYPEPTIDES 2 µm FEATURES ES-C18 • F ast separations VARIANTS 160 Å• H igh peak capacity • Rugged, reliable performance • A lternative selectivity with ES-C18, ES-CN, and Phenyl-Hexyl PTM |10    

HALO® PEPTIDE BONDED PHASE PORTFOLIO HALO 160 Å ES-C18 H3C CH3 Ligand: DIISOBUTYLOCTADECYLSILANE (CH2)17 L1 O Si CH3 USP Designation: Sizes: 2, 2.7, 5 µm Available Particle Pore Size: 160 Å H3C CH3 HALO 160 Å ES-CN Ligand: DIISOPROPYLCYANOPROPYLSILANE H3C CH3 CN USP Designation: L10 O Si Available Particle Sizes: 2.7, 5 µm Pore Size: 160 Å H3C CH3 S HALO 160 Å Phenyl-Hexyl CH3 Ligand: DIMETHYLPHENYL-HEXYLSILANE O Si USP Designation: L11 Available Particle Sizes: 2.7 µm CH3 Pore Size: 160 Å |www.fused-core.com    11

FAST TRYPTIC DIGEST SEPARATIONS WHILE MAINTAINING RESOLUTION HALO® Peptide separations can be increased 2-fold while maintaining high resolution due to the Fused Core® particle design. 140 90 005..-776550%mmLLB//mminiin3n0 min 40 5-60% B in 30 min -10 0 5 10 15 20 25 30 5 140 TEST CONDITIONS: 90 1.0 mL/min Columns: 2.1 x 100 mm HALO 160 Å ES-C18, 2.7 µm 40 51-.600m%LB/minin20 min 5-60% B in 20 min Mobile Phase A: water/0.1% TFA -10 0 Mobile Phase B: 80% ACN/0.1% TFA 150 Gradient: as indicated 130 110 Temperature: 60 °C 10 15 20 Injection volume: 15 µL 90 70 Detection: 215 nm, PDA 50 30 1551-.-.56560m0m%%LL/B/Bmmiininnin1155mminin Sample: apotransferrin tryptic digest 10 -10 0 5 Time, min 10 15 Time, min FAST, HIGH RESOLUTION SEPARATION WITH HALO® Due to the Fused-Core® design and excellent mass transfer ability, ultra fast peptide separations are achievable with the HALO® Peptide TEST CONDITIONS: Columns: HALO 160 Å ES-C18, 2 µm, 3.0 x 50 mm Part Number: 91123-402 240 Mobile Phase A: water/0.1% TFA 11 Mobile Phase B: 80/20 ACN/water/0.1% TFA PEAK IDENITITIES Gradient: Hold at 12.5% B until 0.1 min; 1. Gly-Tyr 190 12.5-63% B from 0.1 – 1.0 min Absorbance , mAU 140 Flow rate: 2.2 mL/min 2. Val-Tyr-Val 3. Angiotensin 1/2 (107) amide 8 Pressure: 556 bar 4. Met-enk 90 Temperature: 60 °C 5. Angiotensin 1/2 (1-8) amide Injection Volume: 0.5 µL 6. Angiotensin II 40 1 23 4 5 67 9 10 Detection: 215 nm, PDA 7. Leu-enk Sample Solvent: water/0.1% TFA Response Time: 0.025 8. Ribonuclease A 9. Angiotensin (1-12) (mouse) -10 Data Rate: 200 Hz 10. Porcine Insulin 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 LC System: Shimadzu Nexera X2 11. Angiotensin (1-12) (human) Time, min |12    

ENHANCE SELECTIVITY WITH HALO 160 Å PHENYL-HEXYL FOR A TRYPTIC DIGEST The HALO 160 Å Phenyl-Hexyl column provides improved resolution between tryptic digest fragments 2 and 3 compared to the 160 Å ES-CN column and the 160 Å ES-C18 c14o0lumn. Peptide identification accomplished by using MS-MS fragmentation spectra. PEAK IDENTITIES: 4. LLIYSASFLYSGVPSR (592 m/z) TEST CONDITIONS: 1. FTISADTSKNTAYLQMNSLR (754 m/z) 5. SGTASVVcLLNNFYPR (899 m/z) Columns: H ALO 160 Å ES-CN, 2.7 µm, 2.1 x 100 mm 2. LScAASGFNIKDTYIHWVR (747 m/z) 6. ScDKTHTcPPcPAPELLGGPSVFLFPPKPK (834 m/z) Part Number: 92122-604 HALO 160 Å Phenyl-Hexyl, 2.7 µm, 3. GFYPSDIAVEWESNGQPENNYK (849 m/z) 7.VVSVLTVLHQDWLNGKEYK (1115 m/z) 2.1 x 100 mm Part Number: 92112-606 HALO 160 Å ES-C18, 2.7 µm, 2.1 x 100 mm Part Number: 92122-602 Mobile Phase A: water + 10 mM difluoroacetic acid (DFA) Mobile Phase B: ACN + 10 mM difluoroacetic acid Gradient: 2–50% B in 60 min Flow Rate: 0.3 mL/min Temperature: 60 °C Injection Volume: 5 μL of 0.2 mg/mL digest Detection: 220 nm, PDA Sample Solvent: 50 mM Tris-HCl/1.5 M Guanidine-HCl with 0.25% formic acid LC System: Shimadzu Nexera Flow Cell: 2.5 μL semi-micro NANOSCALE HIGH RESOLUTION LC-MS ANALYSIS OF TRASTUZUMAB TRYPTIC DIGEST In this example, two 0.2 x 250 mm HALO 160 Å ES-C18, 5 µm nanoscale columns were connected in series to provide additional resolving power for LC analysis of the tryptic digest of reduced and alkylated trastuzumab. TEST CONDITIONS: Columns: HALO 160 Å ES-C18, 5 µm two 0.2 mm x 250 mm columns in series 100 Base Peak Mass Chromatogram Mobile Phase A: 0.1% difluoroacetic acid in water 90 Mobile Phase B: 0.1% difluoroacetic acid in ACN 80 Flow Rate: 3 µL/min Relative Abundance 70 Gradient: 60 Time (min) % B 50 0 5 40 2 5 30 137 42.5 20 10 Temperature: 60 °C 0 Injection Volume: 5 µL 10 20 30 40 50 60 70 80 90 100 110 120 130 Sample:10 µg/µL trastuzumab tryptic peptides TiTmimee(,mimn) in (Sample loaded at 2% B at 10 µL/min) HPLC: Dionex Ultimate 3000 HALO 160 Å columns are also available in capillary and nano dimensions for proteomics experiments and other sample-limited situations. Mass Spectrometer:Thermo Orbitrap Velos Pro™ Hybrid Ion Trap-Orbitrap BENEFITS OF DFA L = leucine enkephalin, A = angiotensin I, S = substance P, β = β-endorphin, M = melittin Switching to Difluoroacetic Acid (DFA), a |www.fused-core.com    13 less fluorinated ion pairing acid mobile phase modifier provides MS sensitivity improvement relative to TFA, particularly with small to mid size molecules. DFA has the practical advantage of similar chromatographic benefits of TFA, (including excellent peak shape and recovery), along with easier removal from instrument components. DFA can be easily removed in 10-15 minutes with 50:50 ACN/water.

GLYCAN SOLUTIONS HALO® Glycan The HALO® Glycan incorporates a highly polar ligand 1.7 µm 2.7 µm that contains 5 hydroxyl groups tethered to 2.7 µm Fused-Core® silica particles via novel, proprietary 0.5 µm linkage chemistry resulting in a high-resolution separation of complex glycan mixtures. Shell with 90 Å pores • Improved retention of acidic and zwitterionic analytes • Ideal for hydrophilic interaction liquid chromatography (HILIC) separations of oligosaccharides, and particularly, of released and labeled glycans from glycoproteins and proteoglycans • E ach lot of HALO® Glycan material is tested for HALO 90 Å GLYCAN quality assurance by separation of a procainamide- reducing-end-labeled glycan ladder of Ligand: PROPRIETARY POLY-HYDROXY oligosaccharides having 2–25 glucose units (GU) USP Designation: L95 Available Particle Sizes: 2.7 µm -Peaks for oligosaccharides composed of 5 Pore Size: 90 Å and 10 GU must meet tight specifications for retention and peak width before lot is approved for glycan analysis QA ANALYSIS OF HALO® GLYCAN Example QA Chromatogram for HALO® Glycan column. Each HALO® Glycan packing lot is tested using this glycan ladder mixture to assess and ensure lot-to-lot reproducibility. TEST CONDITIONS: Columns: HALO 90 Å Glycan, 2.7 µm, 2.1 x 150 mm 40 G# = DP of maltooligosaccharide Part Number: 92922-705 35 For example, G3 = maltotriose G5 Mobile Phase A: 50 mM Ammonium Formate, pH 4.45 Mobile Phase B: water G6 Gradient: 80-55% B in 25 min 30 G4 Flow Rate: 0.6 mL/min Absorbance, mAU Pressure: 190 bar 25 G7 Temperature: 60 ˚C 20 Injection Volume: 3 μL Detection: 300 nm, UV 15 G8 Sample Solvent: 70/30 ACN/water G3 Response Time: 0.5 sec 10 G9 Data Rate: 3.3 Hz Flow Cell: 2.5 μL semi-micro 5 G10 LC System: Shimadzu Nexera G11 G12 0 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 Time, min |14    

SEPARATION OF N-LINKED GLYCANS FROM RIBONUCLEASE B Gradient HILIC-MS separation of N-linked glycans, which had been released using PNGase from ribonuclease B, using the HALO® Glycan column. 70 60 Man8 Ribonuclease B TEST CONDITIONS: N-Linked Glycans Columns: HALO 90 Å Glycan, 2.7 µm, 2.1 x 150 mm Part Number: 92922-705 Absorbance, mAU 50 Man5 Man6 Mobile Phase A: 50 mM Ammonium Formate, pH 4.45 Mobile Phase B: ACN 40 Gradient: 77.5–56.5% B in 52.5 min Flow Rate: 0.6 mL/min 30 Man9 Temperature: 60 °C Injection Volume: 2 μL 20 Man7 Detection: 300 nm, UV 10 0 2.5 5 7.5 10 12.5 15 17.5 20 22.5 25 27.5 30 32.5 35 37.5 40 42.5 45 47.5 50 52.5 55 0.0 Time, min SEPARATION OF N-LINKED GLYCANS FROM HUMAN IgG Released- and procainamide-labeled glycans from human IgG were separated using a 2.1 x 150 mm HALO® Glycan column and detected using UV and selected-ion-monitoring MS detection. 3.0 0.843 17.595 21.817 21.516 2.5 1.510 25.586 2.0 4.871 G0F G1F G2F 1.5 1.0 Absorbance, mAU 10.961 13.924 15.001 18.357 22.420 UV 0.5 11.875 11.432 18.669 23.25423.529 0.418 0.247 14.490 16.318 30.008 33.788 TEST CONDITIONS: 0 12.518 15.481 19.527 24.289 30.870 30.366 -0.5 20.190 26.365 Columns: HALO 90 Å Glycan, 2.7 µm, 31.273 0 27.342 31.937 34.740 35.834 39.537 2.1 x 150 mm 40.024 300,000 32.616 Part Number: 92922-705 41.617 250,000 42.916 Mobile Phase A: 50 mM Ammonium 200,000 Formate, pH 4.4 150,000 Mobile Phase B: ACN 5 10 15 20 25 30 35 40 45 50 Gradient: 77.5–60% B in 60 min 100,000 Flow Rate: 0.5 mL/min 50,000 Temperature: 60 °C 0 0 2:1536.50(+) Injection Volume: 4 μL 2:1479.50(+) 2:1682.60(+) G1F G2F Detection: 300 nm, UV 2:1454.50(+) LC System: Shimadzu Nexera 2:922.80(+) G0F 2:1024.20(+) MS: Shimadzu LCMS 2020 (single 2:1003.80(+) G0F-N quadrupole) Intensity 2:1105.20(+) G0 MS ESI: +4.7 kV 2:1076.30(+) 2:1149.30(+) Scan range: 500-2000 m/z 2:1250.70(+) 2:1396.80(+) Scan rate: 2 pps 2:1295.40(+) 2:1084.30(+) G1FB G2F+NeuAc 2:1157.30(+) 2:1258.90(+) G2 G2F+NeuAc2 2:930.80(+) G2FB G2+NeuAc G2FB+NeuAc G2FB+NeuAc2 5 10 15 20 25 30 35 40 45 50 |www.fused-core.com    15

HALO® UHPLC AND HPLC GUARD COLUMNS • Collect strongly retained • F inger-tight, direct-connect units • A vailable for all HALO® analytical compounds from the sample that auto-adjust to any column geometries (2.1, 3.0 and 4.6 mm and minimizes column fouling with a 10–32 inlet port ID) and phases • E asily replace guard cartridge • U ltra-low dispersion, easy to use, without removing guard holder operate at pressures up to 1000 from the flow path bar See below for an exploded view of the HALO® guard cartridge and guard holder. Please see pages 18-19 for ordering information. Finger-tight guard Finger-tight guard Titanium hybrid cartridge holder, Inlet cartridge holder, Outlet ferrule Allows for replacement Provides easy of guard cartridge without Adds durability replacement of guard car- removing the guard holder for multiple connections tridge from the flow path HALO® guard cartridge Auto-adjusting zero dead All HALO® phases available volume (ZDV) end fitting Ensures optimum, ZDV connection to the analytical column HALO® GUARD COLUMNS: PROTECTION + PERFORMANCE 30 26 with guard column 22 18 14 HALO® guard columns 10 provide optimum protection for your TEST CONDITIONS: HALO® HPLC and 6 Columns: HALO 90 Å C18, 2.7 µm, 4.6 x 50 mm UHPLC column without sacrificing column Absorbance, mAU 2 Mobile Phase: 60/40 ACN/water efficiency. -2 Flow Rate: 1.8 mL/min 0.0 0.2 0.4 0.6 0.8 1.0 1.2 Temperature: 30 ˚C Time, min Injection Volume: 1 µL 34 Detection: 254 nm, UV no guard column 30 Pressure: 158 bar with guard column 26 146 bar without guard column Instrument: Optimized Agilent 1100 22 bypassed semi-micro flow cell 18 0.05” ID tubing 14 14 Hz data rate 10 6 2 -2 0.0 0.2 0.4 0.6 0.8 1.0 1.2 Time, min The Optimize Technologies EXP® Direct Connect Holder: U.S. Patent No. 8,201,854 & 8,696,902 and Foreign Patents Pending. |16    

HALO® BIOCLASS SPECIFICATIONS TABLES PROTEIN SPECIFICATIONS Bonded Phase Pore Size Particle USP Carbon Surface Low pH/T High pH/T Endcapped (Å) Sizes (s) Designa- Load (%) Area (m2/g) Limit Limit (µm) tion 0.6 22 2/90 °C 0.4 15 1/90 °C C4 1000 2.7 L26 1.4 22 9/40 °C Yes 400 3.4 1.0 15 ES-C18 1000 2.7 L1 8/40 °C Yes 400 3.4 Diphenyl 1000 2.7 L11 1.0 22 2/90 °C 9/40 °C Yes PEPTIDE SPECIFICATIONS Bonded Phase Pore Size Particle USP Carbon Surface Low pH/T High pH/T Endcapped (Å) Sizes (s) Designa- Load (%) Area (m2/g) Limit Limit (µm) tion 4.0 65 1/90 °C 4.6 90 ES-C18 2 4.0 60 8/40 °C No 160 2.7 L1 5 ES-CN 160 2.7 L10 2.2 90 1/90 °C 8/40 °C Yes 5 1.5 60 Phenyl-Hexyl 160 2.7 L11 4.7 90 2/90 °C 9/40 °C Yes GLYCAN SPECIFICATIONS Bonded Phase Pore Size Particle USP Carbon Surface Low pH/T High pH/T Endcapped (Å) Sizes (s) Designa- Load (%) Area (m2/g) Limit Limit (µm) tion Proprietary 90 2.7 L95 3.2 135 2/65 °C 9/40 °C No Poly-Hydroxy |www.fused-core.com    17

HALO 1000 Å AND 400 Å PROTEIN HALO 90 Å GLYCAN COLUMNS COLUMNS HALO® Glycan columns are available in 2.1 and 4.6 mm Part numbers for nano, capillary, analytical and semi-preparative HALO diameters in the following lengths as a 2.7 µm particle 1000 Å and 400 Å in 2.7 and 3.4 µm phases are provided below. Guard size. Guard columns are available for UHPLC and HPLC columns are available in 2.1, 3.0 and 4.6 mm IDs for UHPLC and HPLC applications if additional protection is desired. applications to provide additional column protection when desired. Dimensions 400 Å, 3.4 µm 1000 Å, 2.7 µm ID x Length (in mm) HALO Glycan C4 ES-C18 Dimensions C4 ES-C18 Diphenyl 2.1 x 50 92922-405 ID x Length (in mm) 2.1 x 100 92922-605 2.1 x 150 92922-705 0.075 x 50 94319-414 94319-402 97219-414 97219-402 97219-426 92924-405 97219-614 97219-602 97219-626 4.6 x 50 92924-605 0.075 x 100 94319-614 94319-602 97219-714 97219-702 97219-726 4.6 x 100 92924-705 97218-414 97218-402 97218-426 4.6 x 150 0.075 x 150 94319-714 94319-702 97218-614 97218-602 97218-626 97218-714 97218-702 97218-726 0.1 x 50 94318-414 94318-402 97217-414 97217-402 97217-426 97217-614 97217-602 97217-626 0.1 x 100 94318-614 94318-602 97217-714 97217-702 97217-726 97216-414 97216-402 97216-426 0.1 x 150 94318-714 94318-702 97216-614 97216-602 97216-626 Guard Columns, 3-Pack 97216-714 97216-702 97216-726 Dimensions 0.2 x 50 94317-414 94317-402 97215-414 97215-402 97215-426 97215-614 97215-602 97215-626 ID x Length (in mm) 0.2 x 100 94317-614 94317-602 97215-714 97215-702 97215-726 HALO Glycan 92711-314 92711-302 92711-326 2.1 x 5 0.2 x 150 94317-714 94317-702 92711-414 92711-402 92711-426 92922-105 92711-514 92711-502 92711-526 4.6 x 5 92924-105 0.3 x 50 94316-414 94316-402 92711-614 92711-602 92711-626 92711-714 92711-702 92711-726 0.3 x 100 94316-614 94316-602 92712-214 92712-202 92712-226 92712-314 92712-302 92712-326 0.3 x 150 94316-714 94316-702 92712-414 92712-402 92712-426 Guard Column Holder 94900-001 92712-514 92712-502 92712-526 0.5 x 50 94315-414 94315-402 92712-614 92712-602 92712-626 92712-714 92712-702 92712-726 0.5 x 100 94315-614 94315-602 92712-914 92712-902 92712-926 92713-214 92713-202 92713-226 0.5 x 150 94315-714 94315-702 92713-314 92713-302 92713-326 92713-414 92713-402 92713-426 1.0 x 30 93411-314 93411-302 92713-514 92713-502 92713-526 92713-614 92713-602 92713-626 1.0 x 50 93411-414 93411-402 92713-714 92713-702 92713-726 92713-914 92713-902 92713-926 1.0 x 75 93411-514 93411-502 92714-214 92714-202 92714-226 92714-314 92714-302 92714-326 1.0 x 100 93411-614 93411-602 92714-414 92714-402 92714-426 92714-514 92714-502 92714-526 1.0 x 150 93411-714 93411-702 92714-614 92714-602 92714-626 92714-714 92714-702 92714-726 2.1 x 20 93412-214 93412-202 92714-914 92714-902 92714-926 92710-414 92710-402 92710-426 2.1 x 30 93412-314 93412-302 92710-514 92710-502 92710-526 92710-614 92710-602 92710-626 2.1 x 50 93412-414 93412-402 92710-714 92710-702 92710-726 2.1 x 75 93412-514 93412-502 2.1 x 100 93412-614 93412-602 2.1 x 150 93412-714 93412-702 2.1 x 250 93412-914 93412-902 3.0 x 20 93413-214 93413-202 3.0 x 30 93413-314 93413-302 3.0 x 50 93413-414 93413-402 3.0 x 75 93413-514 93413-502 3.0 x 100 93413-614 93413-602 3.0 x 150 93413-714 93413-702 3.0 x 250 93413-914 93413-902 4.6 x 20 93414-214 93414-202 4.6 x 30 93414-314 93414-302 4.6 x 50 93414-414 93414-402 4.6 x 75 93414-514 93414-502 4.6 x 100 93414-614 93414-602 4.6 x 150 93414-714 93414-702 4.6 x 250 93414-914 93414-902 10.0 x 50 93410-414 93410-402 10.0 x 75 93410-514 93410-502 10.0 x 100 93410-614 93410-602 10.0 x 150 93410-714 93410-702 Guard Columns, 3-Pack C4 ES-C18 C4 ES-C18 Diphenyl Dimensions ID x Length (in mm) 2.1 x 5 93412-114 93412-102 92712-114 92712-102 92712-126 92713-114 92713-102 92713-126 3.0 x 5 93413-114 93413-102 92714-114 92714-102 92714-126 4.6 x 5 93414-114 93414-102 Guard Column Holder 94900-001 |18    

HALO 160 Å PEPTIDE COLUMNS The part numbers are provided below for the nano, capillary, analytical and semi-preparative HALO 160 Å 2, 2.7 and 5 µm phases. Guard columns are available for 2.1, 3.0 and 4.6 mm internal diameters for UHPLC and HPLC applications, if additional protection is desired. 160 Å, 2 µm 160 Å, 2.7 µm 160 Å, 5 µm Dimensions ES-C18 ES-C18 ES-CN Phenyl-Hexyl ES-C18 ES-CN ID x Length (in mm) - 91229-402 91229-404 91229-406 91529-402 91529-404 0.075 x 50 - 91229-602 91229-604 91229-606 91529-602 91529-604 0.075 x 100 - 91229-702 91229-704 91229-706 91529-702 91529-704 0.075 x 150 - 91228-402 91228-404 91228-406 91528-402 91528-404 - 91228-602 91228-604 91228-606 91528-602 91528-604 0.1 x 50 - 91228-702 91228-704 91228-706 91528-702 91528-704 0.1 x 100 - 91227-402 91227-404 91227-406 91527-402 91527-404 0.1 x 150 - 91227-602 91227-604 91227-606 91527-602 91527-604 - 91227-702 91227-704 91227-706 91527-702 91527-704 0.2 x 50 - 91226-402 91226-404 91226-406 91526-402 91526-404 0.2 x 100 - 91226-602 91226-604 91226-606 91526-602 91526-604 0.2 x 150 - 91226-702 91226-704 91226-706 91526-702 91526-704 - 91225-402 91225-404 91225-406 91525-402 91525-404 0.3 x 50 - 91225-602 91225-604 91225-606 91525-602 91525-604 0.3 x 100 - 91225-702 91225-704 91225-706 91525-702 91525-704 0.3 x 150 - 92121-302 92121-304 92121-306 95121-302 95121-304 - 92121-402 92121-404 92121-406 95121-402 95121-404 0.5 x 50 - 92121-502 92121-504 92121-506 95121-502 95121-504 0.5 x 100 - 92121-602 92121-604 92121-606 95121-602 95121-604 0.5 x 150 - 92121-702 92121-704 92121-706 95121-702 95121-704 91122-202 92122-202 92122-204 92122-206 95122-202 95122-204 1.0 x 30 91122-302 92122-302 92122-304 92122-306 95122-302 95122-304 1.0 x 50 91122-402 92122-402 92122-404 92122-406 95122-402 95122-404 1.0 x 75 91122-502 92122-502 92122-504 92122-506 95122-502 95122-504 1.0 x 100 91122-602 92122-602 92122-604 92122-606 95122-602 95122-604 1.0 x 150 91122-702 92122-702 92122-704 92122-706 95122-702 95122-704 2.1 x 20 91122-902 92122-902 92122-904 92122-906 95122-902 95122-904 2.1 x 30 91123-202 92123-202 92123-204 92123-206 95123-202 95123-204 2.1 x 50 91123-302 92123-302 92123-304 92123-306 95123-302 95123-304 2.1 x 75 91123-402 92123-402 92123-404 92123-406 95123-402 95123-404 2.1 x 100 91123-502 92123-502 92123-504 92123-506 95123-502 95123-504 2.1 x 150 91123-602 92123-602 92123-604 92123-606 95123-602 95123-604 2.1 x 250 91123-702 92123-702 92123-704 92123-706 95123-702 95123-704 3.0 x 20 91123-902 92123-902 92123-904 92123-906 95123-902 95123-904 3.0 x 30 - 92124-202 92124-204 92124-206 95124-202 95124-204 3.0 x 50 - 92124-302 92124-304 92124-306 95124-302 95124-304 3.0 x 75 - 92124-402 92124-404 92124-406 95124-402 95124-404 3.0 x 100 - 92124-502 92124-504 92124-506 95124-502 95124-504 3.0 x 150 - 92124-602 92124-604 92124-606 95124-602 95124-604 3.0 x 250 - 92124-702 92124-704 92124-706 95124-702 95124-704 4.6 x 20 - 92124-902 92124-904 92124-906 95124-902 95124-904 4.6 x 30 - 92120-402 92120-404 92120-406 95120-402 95120-404 4.6 x 50 - 92120-502 92120-504 92120-506 95120-502 95120-504 4.6 x 75 - 92120-602 92120-604 92120-606 95120-602 95120-604 4.6 x 100 - 92120-702 92120-704 92120-706 95120-702 95120-704 4.6 x 150 - 95120-902 95120-904 4.6 x 250 - - - 10.0 x 50 10.0 x 75 10.0 x 100 10.0 x 150 10.0 x 250 Guard Columns, 3-pack ES-C18 ES-C18 ES-CN Phenyl-Hexyl ES-C18 ES-CN Dimensions 91122-102 92122-102 92122-104 92122-106 95122-102 95122-104 ID x Length (in mm) 91123-102 92123-102 92123-104 92123-106 95123-102 95123-104 92124-102 92124-104 92124-106 95124-102 95124-104 2.1 x 5 - 3.0 x 5 4.6 x 5 Guard Column Holder 94900-001 |www.fused-core.com    19

AMT19_05_BIOCLASS [email protected] www.advanced-materials-tech.com fused-core.com HALO and Fused-Core are registered trademarks of Advanced Materials Technology, Inc.