Waters Sigma-Aldrich 1.0 mg/ml LC Waters Alliance HPLC ACQUITY UPLC H-Class Bio 30 cm Alliance HPLC UV ACQUITY UPLC TUV mm nm XBridge Protein B

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1 HPLC 3. µm BEH Stephan Koza, Susan Serpa, Hua Yang, Edouard Bouvier, and Kenneth J. Fountain Waters Corporation, Milford, MA, USA HPLC HPLC UPLC 200 SEC 1 UPLC SE-UPLC 2 µm BEH BEH 100% SE-UPLC BEH.6 mm SE-UPLC HPLC BEH HPLC 3. µm 7.8 mm HPLC SE- HPLC SEC HPLC HPLC -8 µm µm XBridge Protein BEH SEC 200 & 0 3. µm Alliance HPLC Auto Blend Plus Waters BEH200 & BEH0 SEC Protein Standard Mixture SEC HPLC SE-HPLC IgM 1

Waters Sigma-Aldrich 1.0 mg/ml LC Waters Alliance HPLC ACQUITY UPLC H-Class Bio 30 cm Alliance HPLC UV ACQUITY UPLC TUV mm 280 21 nm XBridge Protein BEH SEC 200 3. µm 7.8 10 mm 1760039 7.

2 Waters Sigma-Aldrich 1.0 mg/ml LC Waters Alliance HPLC ACQUITY UPLC H-Class Bio 30 cm Alliance HPLC UV ACQUITY UPLC TUV mm nm XBridge Protein BEH SEC µm mm mm XBridge Protein BEH SEC 0 3. µm mm mm µl 0.8 ml/min 2 mm 10 mm ph7.2 Auto Blend Plus Technology BEH200 SEC Protein Standard Mix : BEH0 SEC Protein Standard Mix : Intact mab Mass Check Standard : mm PTFE/ 1 ml : DV Silica-DIOL SEC 20 µm m Silica-DIOL SEC 0 8 µm m Alliance HPLC TUV ACQUITY UPLC TUV mm Waters Empower 2 3 HPLC 3. µmbeh 2

3 UPLC SE-UPLC BEH 2,3 UPLC HPLC HPLC BEH µm BEH 7.8 mm 2 10 kda 2 MDa Rh SE-HPLC 8 µm HPLC BEH UPLC HPLC UPLC HPLC SE-HPLC BEH 20 SEC µm mm 200 BEH SEC 3. µm mm Alliance HPLC A B UV Absorbance (21 nm) C D dimer Waters BEH200 SEC Protein Standard Mix Intact mab Mass Check Standard mg/ml 20 µm A C 200 BEH 3. µm B D SEC mm 0.8 ml/min A B 1 thyroglobulin 669 KDa 2 10 KDa 3 BSA 67 KDa myoglobin 1 KDa uracil 112 Da C D 10 KDa 300K Da HPLC 3. µmbeh 3

4 Protein Standard Mix BEH 3. µm 10 KDa 300 KDa USP µm BEH 3. µm 0% 2 RS L 0 SEC 8 µm mm 0 BEH SEC 3. µm mm µm BEH µm 8 µm 3. µm BEH 0 SEC A b 1a B UV Absorbance (21 nm) C D dimer dimer multimer Buffer Buffer Waters BEH0 SEC Protein Standard Mix Intact mab Mass Check Standard mg/mL 0 8 µm A C 0 BEH 3. µm B D SEC mm 0.8 ml/min A B 1 thyroglobulin 669 KDa 2 10 KDa 3 BSA 67 KDa myoglobin 1 KDa uracil 112 Da C D 10 KDa 300 KDa 0K Da HPLC 3. µmbeh

5 BEH BEH SEC SE-HPLC SEC SEC SE-HPLC 3. µm BEH SE-HPLC 20 µm SE-HPLC mm µm BEH SE- HPLC mm 3 µm SE-HPLC 1.0 ml/min 1.2 ml/min 3. µm BEH SE-HPLC 2.0 ml/min 2.7 ml/minprotein Standard Mix thyroglobulin 1.3M Da20 µm µm BEH 2 1 ml/min 20 µm BSA µm BEH ml/min 3. µm BEH 2% Flow Rate= 1.0 ml/minute Flow Rate= 2.0 ml/minute Time (minutes) Waters BEH200 SEC Protein Standard Mix µm SEC 1.0 ml/min A 200 BEH 3. µm SEC 2.0 ml/min B A B 1 thyroglobulin 669 KDa 2 10 KDa 3 BSA 67 KDa myoglobin 1 KDa uracil 112 Da Protein Standard Mix thyroglobulin 1.3 MDa 20 µm µm BEH thyroglobulin XBridge Protein BEH SEC µm XBridge Protein BEH SEC 0 3. µm HPLC 3. µmbeh

6 XBridge Protein BEH SEC 200 &0 3. µm SEC mm µm SEC Protein Standard Mix 3 6 SEC 0.8 ml/min 200 SEC SEC 8 0 XBridge 200Å XBridge 0Å Batch 1, Column 1 1b 2 1a 3 Batch 1, Column 1 1b 1a Batch 1, Column 2 Batch 1, Column 2 Batch 2, Column 1 Batch 2, Column 2 Batch 3, Column 1 Batch 2, Column 1 Batch 2, Column 2 Batch 3, Column 1 Batch 3, Column 2 Batch 3, Column Waters BEH200 SEC Protein Standard Mix : BEH0 SEC Protein Standard Mix BEH 3. µm SEC mm 1a thyroglobulin dimer 1.3 MDa 1b thyroglobulin 669 KDa 2 10 KDa 3 BSA 67 KDa myoglobin 1 KDa uracil 112 Da ACQUITY UPLC H-Class Bio HPLC 3. µmbeh 6

7 µm SEC mm 600 SEC ph ph7.2 6 Protein Standard Mix 2 BSA 3. µm XBridge Protein BEH SEC UV Absorbance (280 nm) Protein Standard Mix Injection #2 R s =2. A 1 Protein Standard Mix Injection #610 R s =2.2 B UV Absorbance (21 nm) mab Standard Injection #6 R s =1.87 C dimer mab Standard Injection #611 D R s =1.83. Waters BEH200 SEC Protein Standard Mix Intact mab Mass Check Standard mg/mL 200 BEH 3. µm SEC mm A B 1 thyroglobulin 669 KDa 2 10 KDa 3 BSA 67 KDa myoglobin 1 KDa uracil 112 Da C D 10 KDa 300K Da ACQUITY UPLC H-Class Bio nm) UV Absorbance (2 Protein Standard Mix Injection #2 1a Protein Standard Mix Injection #600 1b R s =1. R s =1. 21 nm) UV Absorbance (2 0 mab Standard Injection #6 R s =1.6 dimer mab Standard Injection #601 R s = Waters BEH0 SEC Protein Standard Mix Intact mab Mass Check Standard mg/ml 0 BEH 3. µm SEC mm A B 1 thyroglobulin 669 KDa 2 10 KDa 3 BSA 67 KDa myoglobin 1 KDa uracil 112 Da C D 10 KDa 300K Da ACQUITY UPLC H-Class Bio HPLC 3. µmbeh 7

8 XBridge Protein BEH SEC 0 & µm K Da 0K Da 0 0K Da 1.3M Da 0 thyroglobulin 669 KDa1.3 MDa 2 IgM pentamer 900 KDa IgM dipentamer 1.8 MDa 0 8 dipentamer 1.8M Da SEC XBridge Protein BEH SEC 0Å, 3. µm, 100K 1,00K Daltons 10,000,000 XBridge Protein BEH SEC 200Å, 3. µm, 10K 0K Daltons 1,000,000 IgM(900 KDa) Thyroglobulin (669 KDa) Protein Molecular Weight (KDa) 100,000 10,000 1, (10 KDa) Amyloglucosidase (97 KDa) Conalbumin (7KDa) Ovalbumin ( KDa) Myoglobin (17 KDa) RNAseA (1 KDa) Aprotinin (6. KDa) Uracil (112 Da) Normalized Retention Volume (Vr/VC) 7. uracil BEH µm SEC IgM Dipentamers IgM Multimers IgM Pentamer IgM pentamer IgM dipentamer IgM pentamer BEH 0 3. µm SEC IgM pentamer 900 KDa IgM dipentamer 1.8 MDa IgM 2.7 MDa ACQUITY UPLC H-Class Bio HPLC 3. µmbeh 8

9 HPLC 3. µm XBridge Protein BEH SEC 200Å 0Å µm SEC LC SEC BEH SEC 1. Fountain KJ, Hong, P, Serpa, S, Bouvier, ESP and Morrison, D. Analysis of Biomolecules by Size-Exclusion UltraPerformance Liquid Chromatography. Waters Corporation, Application Note [serial on the Internet]. 2010; (WA6226 ). 2. Paula Hong, Stephan Koza, and Kenneth J. Fountain. Advances in Size- Exclusion Chromatography for the Analysis of Small Proteins and Peptides: Evaluation of Calibration Curves for Molecular Weight Estimation. Waters Corporation, Application Note [serial on the Internet]. 2012; ( EN). 3. Stephan Koza, Matthew Lauber, and Kenneth J. Fountain. The Analysis of Multimeric Monoclonal Antibody Aggregates. Waters Corporation, Application Note, [serial on the Internet]. 2013; ( EN).. Stephan Koza, and Kenneth J. Fountain. Successful Transfer of Size-Exclusion Separations between HPLC and UPLC. Waters Corporation, Application Note, [serial on the Internet]. 201; ( EN). HPLC SEC 1.7 µm 2. µm.6 mm I.D. UPLC Protein BEH SEC UPLC UPLC Protein BEH SEC XBridge Protein BEH SEC µm 10-1,00 KDa HPLC SEC SEC ACQUITY UPLC Protein BEH SEC TEL FAX F TEL FAX Waters ACQUITY UPLC UPLC Allience XBridge Empower The Science of W hat s Possible Waters Corporation Auto Blend Plus BEH Technology Waters Corporation 201 Waters Corporation. Produced in Japan JA PDF

LC ACQUITY UPLC H-Class Bio TUV Sigma, mg/ml 0.01 N 276 nm μl 0.4 ml/min L g/l / 99% / 65/15/20 v/v/v 10% 10% 0.01 N ACQUITY

LC ACQUITY UPLC H-Class Bio TUV Sigma, mg/ml 0.01 N 276 nm μl 0.4 ml/min L g/l / 99% / 65/15/20 v/v/v 10% 10% 0.01 N ACQUITY Ultra Performance Liquid Chromatography Stephan Koza, Paula Hong, and Kenneth J. Fountain Waters Corporation, 34 Maple Street, Milford, MA, USA & SEC 2010 2 8 5 2030 4 3 8 1982 2009 131 USP EPHMW USP34