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

HPLC 3. µm BEH Stephan Koza, Susan Serpa, Hua Yang, Edouard Bouvier, and Kenneth J. Fountain Waters Corporation, Milford, MA, USA HPLC 200 0 HPLC 2 2010 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 200 0 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 7.8 300 mm 17600396 XBridge Protein BEH SEC 0 3. µm 7.8 10 mm 17600398 7.8 300 mm 17600399 10 10 µl 0.8 ml/min 2 mm 10 mm ph7.2 Auto Blend Plus Technology BEH200 SEC Protein Standard Mix :18600618 BEH0 SEC Protein Standard Mix :18600682 Intact mab Mass Check Standard : 1860062 12 32 mm PTFE/ 1 ml : 1860038DV Silica-DIOL SEC 20 µm 7.8 300 m Silica-DIOL SEC 0 8 µm 7.8 300 m Alliance HPLC TUV ACQUITY UPLC TUV mm Waters Empower 2 3 HPLC 3. µmbeh 2

UPLC SE-UPLC BEH 2,3 UPLC HPLC HPLC BEH 200 0 3. µm BEH 7.8 mm 2 10 kda 2 MDa Rh SE-HPLC 8 µm HPLC 200 0 2 BEH UPLC HPLC UPLC HPLC SE-HPLC BEH 20 SEC µm 7.8 300 mm 200 BEH SEC 3. µm 7.8 300 mm Alliance HPLC 1 0.16 0.1 0.12 0.16 0.1 0.12 A B 1 2 3 6.00 7.00 8.00 9.00 1 11.00 12.00 13.00 1.00 1.00 UV Absorbance (21 nm) 0 0 0.030 0 0 0.070 0 0 0 0.030 0 0 - C D dimer 0.80 0.60 0.0 0.20.00 6.00 8.00 1 12.00 1.20 1.00 0.80 0.60 0.0 0.20.00 6.00 8.00 1 12.00.00 6.00 7.00 8.00 9.00 1 11.00 12.00 13.00 1. Waters BEH200 SEC Protein Standard Mix 18600618Intact mab Mass Check Standard 18600622 1 mg/ml 20 µm A C 200 BEH 3. µm B D SEC 7.8 300 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

Protein Standard Mix BEH 3. µm 10 KDa 300 KDa USP µm BEH 3. µm 0% 2 RS L 0 SEC 8 µm 7.8 300 mm 0 BEH SEC 3. µm 7.8 300 mm 2 7 20 µm BEH 200 3. µm 8 µm 3. µm BEH 0 SEC 0.18 0.16 A 0.1 0.12 1b 1a 0.18 0.16 B 0.1 0.12 6.00 8.00 1 12.00 1.00 16.00 UV Absorbance (21 nm) 0 0.018 0.016 0.01 0.012 8 6 2 0 0 0.01 0 C D dimer dimer multimer Buffer Buffer 9.00 1 11.00 12.00 13.00 1.00 2. Waters BEH0 SEC Protein Standard Mix 18600682 Intact mab Mass Check Standard 18600622 1mg/mL 0 8 µm A C 0 BEH 3. µm B D SEC 7.8 300 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

BEH BEH SEC SE-HPLC SEC SEC SE-HPLC 3. µm BEH SE-HPLC 20 µm SE-HPLC 7.8 300 mm 200 3. µm BEH SE- HPLC 7.8 300 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 200 3. µm BEH 2 1 ml/min 20 µm BSA2.0 3. µm BEH 2. 2.0 ml/min 3. µm BEH 2% 1.9 0 Flow Rate= 1.0 ml/minute 0 0.030 1 0 0 0 0 0.0 18.0 Flow Rate= 2.0 ml/minute 0 0.030 0 0 0.0 Time (minutes) 7.0 3. Waters BEH200 SEC Protein Standard Mix 18600618 20 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 200 3. µm BEH thyroglobulin XBridge Protein BEH SEC 200 3. µm XBridge Protein BEH SEC 0 3. µm HPLC 3. µmbeh

XBridge Protein BEH SEC 200 &0 3. µm SEC 7.8 300 mm 200 0 3. µm SEC Protein Standard Mix 3 6 SEC 0.8 ml/min 200 SEC 0.037 1 0 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 2 6.00 8.00 1 12.00 1.00 7.00 8.00 9.00 1 11.00 12.00 13.00 1.00 1.00. Waters BEH200 SEC Protein Standard Mix :18600618 BEH0 SEC Protein Standard Mix 18600682 200 0 BEH 3. µm SEC 3 2 7.8 300 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

200 0 3. µm SEC 7.8 300 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) 0.01 0 0.01 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 18600618Intact mab Mass Check Standard 18600622 1mg/mL 200 BEH 3. µm SEC 7.8 300 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 0 00 6.00 8.00 1 12.00 1.00 6.00 7.00 8.00 9.00 1 11.00 280 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 =1.9 6. Waters BEH0 SEC Protein Standard Mix 18600682 Intact mab Mass Check Standard 18600622 1 mg/ml 0 BEH 3. µm SEC 7.8 300 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 0 6.00 8.00 1 12.00 1.00 1 12.00 HPLC 3. µmbeh 7

XBridge Protein BEH SEC 0 & 200 3. µm 7 200 10K 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 1,000 100 (10 KDa) Amyloglucosidase (97 KDa) Conalbumin (7KDa) Ovalbumin ( KDa) Myoglobin (17 KDa) RNAseA (1 KDa) Aprotinin (6. KDa) Uracil (112 Da) 10 0.3 0. 0. 0.6 0.7 0.8 0.9 1 Normalized Retention Volume (Vr/VC) 7. uracil BEH200 0 3. µm SEC IgM Dipentamers IgM Multimers IgM Pentamer 6.00 8.00 1 8. 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

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; (7200012EN). 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; (72000713EN).. 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; (72000193EN). HPLC SEC 1.7 µm 2. µm.6 mm I.D. UPLC Protein BEH SEC UPLC UPLC Protein BEH SEC XBridge Protein BEH SEC 200 0 3. µm 10-1,00 KDa HPLC SEC SEC ACQUITY UPLC Protein BEH SEC www.waters.com 10-0001 1-3-12 TEL 03-371-7191 FAX 03-371-7118 32-0011 -1-1011F TEL 06-630-8888 FAX 06-6300-173 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. 201 2 72000202JA PDF