mabs and ADCs analysis by RP

Transcription

1 mabs and ADCs analysis by RP Shanhua Lin, Ph.D. The world leader in serving science

2 Protein and mab Separation by HPLC Size difference? YES Size Exclusion Chromatography (SEC) MAbPac SEC-1 NO NO Charge difference? YES Ion Exchange Chromatography (IEX) MAbPac SCX-1 ProPac WCX-1 CX-1 ph gradient Buffer NO NO YES Reverse Phase Chromatography (RPC) MAbPac RP Hydrophobicity difference? YES Hydrophobic Interaction Chromatography (HIC) MAbPac HIC-1 MAbPac HIC-2 MAbPac HIC-Butyl ProPac HIC-1 2

3 Executive Summary Thermo Scientific MAbPac RP Column Designed for high-resolution separation of monoclonal antibodies (mabs) and mab fragments (including LC, HC, Fc, Fab, scfc, and F(ab ) 2 ) by reverse phase chromatography Developed for analytical chemists who need High Resolution Accurate Mass determination of monoclonal antibody variants, antibody drug conjugates (ADC), and proteins 3

4 RP Columns Are Important for mab Analysis mabs are highly heterogeneous and are susceptible to degradation Glycosylation Modifications on the termini Oxidation Deamidation Isomerization Reduction of disulfide bond Aggregation LC/MS analysis of mab and mab fragments can reveal these modifications without complete digestion and peptide mapping. 4

5 Fast Separation of Proteins Column: MAbPac RP, 4 µm Format: mm Mobile phase A: H 2 O/TFA (99.9 :.1 v/v) Mobile phase B: MeCN/ H 2 O/TFA (9: 9.9 :.1 v/v/v/) Gradient: Time (min) %A %B Temperature: 8 ºC Flow rate: 6mL/min.6 Inj. volume: 1 µl Detection: UV (28 nm) Sample: 1. Ribonuclease A (.5 mg/ml) 2. Cytochrome C (.5 mg/ml) 3. Lysozyme (.5 mg/ml) 4. mab (1 mg/ml) Retention Time (min) 5

6 Reproducibility of MAbPac RP #111 #91 #81-2 #71 #651 #51-4 #41 #31-6 #21 # Column: MAbPac RP, 4 µm Format: mm Mobile phase A: H 2 O/TFA (99.9 :.1 v/v) Mobile phase B: MeCN/ H 2 O/TFA (9: 9.9 :.1 v/v/v/) Gradient: Time (min) %A %B Temperature: 8 ºC Flow rate:.6 ml/min Inj. volume: 1 µl Detection: UV (28 nm) Sample: 1. Ribonuclease A (.5 mg/ml) 2. Cytochrome Ct C(5 (.5 mg/ml) 3. Lysozyme (.5 mg/ml) 4. mab (1 mg/ml) Retention Time (min) 6

7 Loadability: Three Orders of Magnitude (a).2 µg 18. (b).2 µg (c) 2 µg Column: MAbPac RP, 4 µm Format: mm Mobile phase A: H 2 O/TFA (99.9 :.1 v/v) Mobile phase B: MeCN/ H 2 O/TFA (9: 9.9 :.1 v/v/v/) Gradient: Time (min) %A %B Temperature: 8 ºC Flow rate:.6 ml/min Inj. volume: 1 µl Detection: UV (28 nm) Sample: mab (d) 2 µg Retention Time (min) Area y = 2.311x R² = mab (µg) 7

8 Carryover : mab injection (peak area = 37.97) Column: MAbPac RP, 4 µm Format: 3 5 mm Mobile phase A: H 2 O/TFA (99.9 :.1 v/v) Mobile phase B: MeCN/ H 2 O/TFA (9: 9.9 :.1 v/v/v) Gradient: Time (min) %A %B Carryover <.62% 2: blank (Peak area =.233) Temperature: 8 ºC Flow rate:.5 ml/min Inj. volume: 5 µl Detection: UV (28 nm) Sample: mab (5 mg/ml) Retention Time (min) 8

9 Stability at High ph Condition After 6 hrs of.8 M NaOH wash at 8 C 1 Before NaOH wash Column: MAbPac RP, 4 µm Format: mm Mobile phase A: H 2 O/TFA (99.9 :.1 v/v) Mobile phase B: MeCN/ H 2 O/TFA (9: 9.9 :.1 v/v/v/) Gradient: Time (min) %A %B Temperature: 8 ºC Flow rate:.6 ml/min Inj. volume: 1µL Detection: UV (28 nm) Sample: 1. Ribonuclease A (.5 mg/ml) 2. Cytochrome C (.5 mg/ml) 3. Lysozyme (.5 mg/ml) 4. mab (1 mg/ml) Retention Time (min) 9

10 1 Separation of mab Fragments

11 Structure of IgG and Typical Forms of Heterogeneity 11

12 mab Fragments (a) (b) (c) 12

13 mab and mab Fragments Analysis 14 mab (a) (b) (c) LC Fc HC Fab 1 F(ab ) (d) 2 5 scfc Column: MAbPac RP, 4 µm Format: 3 5 mm Mobile phase A: H 2 O/FA/TFA (99.88 :.1:.2 v/v/v) Mobile phase B: MeCN/ H 2 O/FA/TFA (9: 9.88 :.1:.2 v/v/v/v) Gradient: Time (min) %A %B Temperature: 8 ºC Flow rate:.5 ml/min Inj. volume: 5 µl Detection: UV (28 nm) Sample: (a) trastuzumab (5 mg/ml) (b) trastuzumab + DTT (4 mg/ml) (c) trastuzumab + Papain (2 mg/ml) (d) trastuzumab + IdeS (2 mg/ml) Retention Time (min) 13

14 LC/MS Analysis of Intact mab and Glycosylation Profile RT: RelativeAbundance TIC 8.26 NL: 559E8 5.59E8 TIC MS Column: herceptin_5 mgperml Time (min) herceptin_5mgperml # RT: AV: 7 NL: 8.14E6 T: FTMS + p ESI sid=4. Full ms [1.-4.] MS spectrum Relative Abundance MS m/z herceptin_5mgperml # RT: AV: 7 NL: 6.26E6 T: FTMS + p ESI sid=4. Full ms [1.-4.] m/z at Column: MAbPac RP, 4 µm Format: 3 5 mm Mobile phase A: H 2 O/FA/TFA (99.88 :.1:.2 v/v/v) Mobile phase B: MeCN/ H 2 O/FA/TFA (9: 9.88 :.1:.2 v/v/v/v) Gradient: Time (min) %A %B Temperature: 8 ºC Flow rate:.5 ml/min Inj. volume: 1 µl Detection: positive-ion mode Mass Spec: Q Exactive Plus Sample: trastuzumab (5 mg/ml) 7 65 Relative Abundance m/z 14

15 LC/MS Analysis of Reduced mab RT: TIC Relative Abundance uau LC HC 8.28 NL: E8 TIC MS Herceptin_ HC+LC_1ul NL: 6 6.6E5 UV UV_VIS_1 55 UV Herceptin_ 5 HC+LC_1ul Time (min) Column: MAbPac RP, 4 µm Format: 3 5 mm Mobile phase A: H 2 O/FA/TFA (99.88 :.1:.2 v/v/v) Mobile phase B: MeCN/ H 2 O/FA/TFA (9: 9.88 :.1:.2 v/v/v/v) Gradient: Time (min) %A %B Relative Abundance LC HC NL: 2.52E7 Herceptin_HC+ LC_1ul#152 RT: 7.42 AV: 1 T: FTMS + p ESI sid=4. Full ms [1.-4.] NL: 6.19E6 Herceptin_HC+ LC_1ul#178 RT: 8.28 AV: 1 T: FTMS + p ESI sid=4. Full ms [1.-4.] Temperature: 8 ºC Flow rate:.5 ml/min Inj. volume: 1 µl UV Detection: 28 nm MS Detection: positive-ion mode Mass Spec: Q Exactive Plus Sample: reduced trastuzumab (4 mg/ml) Herceptin_HC+LC_1ul #178 RT: 8.28 AV: 1 NL: 6.19E6 T: FTMS + p ESI sid=4. Full ms [1.-4.] Relative Abundance m/z m/z 15

16 16 Separation of mab Fragments with Charge Variant

17 DTT Reduction and IdeS Digest: scfc, LC, and Fd 17

18 Separation of mab Fragments Containing Lys Variant scfc, 1. 1 Lys 8. scfc, Lys LC Fd Column: MAbPac RP, 4 µm Format: 3. 5 mm Mobile phase A: H 2 O/TFA (99.9 :.1 v/v) Mobile phase B: MeCN/ H 2 O/TFA (9: 9.9 :.1 v/v/v/) Gradient: Time (min) %A %B Temperature: 8 ºC Flow rate:.5 ml/min Inj. volume: 2 µl Detection: UV (28 nm) Sample: Infliximab+IdeS+DTT (2 mg/ml) Retention Time (min) 18

19 LC/MS Analysis: scfc, LC, and Fd remicade+ides+dtt_2mgperml 11/12/214 1:27:41 PM RT: ndance Relative Abu Abundance Relative scfc F(ab ) 2 Lysine variants LC Fd 8.68 NL: 7.27E8 TIC MS Remicade+ IDES IDES_2mgper ml IDES +DTT Time (min) scfc, 1Lys NL: 7.93E scfc, Lys F(ab ) LC Fd m/z NL: 1.65E8 TIC MS remicade+ ides+ dtt_2mgperml Remicade+IDES_2mgperml#144 RT: 6.92 AV: 1 T: FTMS + p ESI sid=4. Full ms [1.-4.] NL: 9.43E6 Remicade+IDES_2mgperml#149 RT: 7.1 AV: 1 T: FTMS + p ESI sid=4. Full ms [1.-4.] NL: 5.76E7 Remicade+IDES_2mgperml#19 RT: 8.68 AV: 1 T: FTMS + p ESI sid=4. Full ms [1.-4.] NL: 2.6E7 remicade+ides+dtt_2mgperml#173 RT: 7.89 AV: 1 T: FTMS + p ESI sid=4. Full ms [1.-4.] NL: 1.49E7 remicade+ides+dtt_2mgperml#187 RT: 8.32 AV: 1 T: FTMS + p ESI sid=4. Full ms [1.-4.] 19

20 LC/MS Analysis: scfc F:\Xcalibur\...\Remicade+IDES_2mgperml 11/12/214 12:23:15 PM RT: NL: 7.27E8 1 TIC MS Remicade+ IDES_2mgperml NL: 1.65E TIC MS remicade+ides dtt_2mgperml NL: 3.11E6 1 m/z= scfc, 1 Lys MS Remicade+ 7.2 IDES_2mgperml 1 NL: 3.41E6 scfc, Lys m/z= MS Remicade IDES_2mgperml Time (min) Relative Abundance Relative Abundance scfc, 1 Lys scfc, Lys , 1 Lys NL: 3.11E6 Remicade+ IDES_2mgperml#144 RT: 6.92 AV: 1 T: FTMS + p ESI sid=4. Full ms [1.-4.] , Lys m/z NL: 2.92E6 Remicade+ IDES_2mgperml#149 RT: 7.1 AV: 1 T: FTMS + p ESI sid=4. Full ms [1.-4.] 2

21 Separation of mab Fragments with Oxidation Variant 21

22 Oxidized mabs Methionine and Tryptophans are usually oxidized. 22

23 Separation of mab Fragments Containing Oxidation Variant 12. Column: MAbPac RP, 4 µm (a) Oxidized HC HC Format: 3 5 mm Mobile phase A: H 2 O/TFA (99.9 :.1 v/v) LC Mobile phase B: MeCN/ H 75 2 O/TFA (9: 9.9 : v/v/v) Gradient: 5. Time (min) %A %B (b) Fd Temperature: 8 ºC Flow rate:.5 ml/min 6. LC Inj. volume: 2 µl Detection: UV (28 nm) Sample: (a) trastuzumab + DTT (2 mg/ml) 4. scfc (b) trastuzumab + DTT + IdeS (1 mg/ml) 2. Oxidized scfc Retention Time (min) 23

24 LC/MS Analysis: Oxidized HC RT: Relative Abundance Relativ ve Abundance (a) TIC LC 8.33 oxidized HC HC NL: 9.66E7 TIC MS _He rceptin+ H2O2+ DTT_2mgp erml Time (min) NL: 1.6E _Herceptin+H2O2+ DTT_2mgperml#221 RT: 1.17 AV: 1 T: FTMS + p (b) Oxidized HC +31 ESI sid=4. Full ms [1.-4.] (c) HC NL: 9.61E _Herceptin+H2O2+ DTT_2mgperml#227 RT: 1.29 AV: 1 T: FTMS + p ESI sid=4. Full ms [1.-4.] Column: MAbPac RP, 4 µm Format: 3 5 mm Mobile phase A: H 2 O/FA/TFA (99.88 :.1:.2 v/v/v) Mobile phase B: MeCN/ H 2 O/FA/TFA (9: 9.88 :.1:.2 v/v/v/v) Gradient: Time (min) %A %B Temperature: 8 ºC Flow rate:.5 ml/min Inj. volume: 2µL MS Detection: positive-ion mode Mass spec: Q Exactive Plus Sample: oxidized trastuzumab, reduced by DTT (2 mg/ml) m/z 24

25 LC/MS Analysis: Oxidized scfc RT: RelativeAbundance ve Abundance Relativ (a) TIC Oxidizied scfc scfc LC Fd Time (min) (b) Oxidized scfc (c) scfc NL: 7.51E7 TIC MS _He rceptin+ H2O2+ DTT+ IDES_1mgp erml NL: 5.9E _Herceptin+H2O2+ DTT+IDES_1mgperml#149 RT: 7.16 AV: 1 T: FTMS + p ESI sid=4. Full ms [1.-4.] NL: 4.26E _Herceptin+H2O2+ DTT+IDES_1mgperml#16 RT: 7.42 AV: 1 T: FTMS + p ESI sid=4. Full ms [1.-4.] Column: MAbPac RP, 4 µm Format: 3 5 mm Mobile phase A: H 2 O/FA/TFA (99.88 :.1:.2 v/v/v) Mobile phase B: MeCN/ H 2 O/FA/TFA (9: 9.88 :.1:.2 v/v/v/v) Gradient: Time (min) %A %B Temperature: 8 ºC Flow rate:.5 ml/min Inj. volume: 2µL MS Detection: positive-ion mode Mass spec: Q Exactive Plus Sample: Oxidized trastuzumab, reduced by DTT and digested by IdeS (1 mg/ml) m/z 25

26 High Resolution MS Relative Abunda ance (a) Oxidized scfc (b) scfc NL: 7.7E _Herceptin+H2O2+DTT+ IDES_1mgperml_ # RT: AV: 18 T: FTMS + p ESI Full ms [1.-4.] NL: 8.74E _Herceptin+H2O2+DTT+ IDES_1mgperml_ # RT: AV: 19 T: FTMS + p ESI Full ms [1.-4.] m/z 26

27 27 Separation of ADC DAR forms

28 Site-selective Antibody-drug Conjugates (ADCs) β-1,4-galactosidase Add in GalT(Y289L) UDP-GalNAz N 3 DIBO-MMAE MMAE MMAE N 3 3 N 3 MMAE MMAE 37 C, 5 hr 37 C, ON N 3 25 C, ON Unlabeled Ab Cleave Terminal Gal Azide-Activated Ab (stable for long-term storage) Antibody drug conjugate (ADC) 28

29 MMAE Modified Trastuzumab ADC Column: MAbPac RP, 4 µm Format: mm Mobile phase A: H 2 O/TFA (99.9 :.1 v/v) Mobile phase B: MeCN/ H 2 O/TFA (9: 9.9 :.1 v/v/v) Gradient: Time (min) %A %B Flow rate:.6 ml/min Temperature: 8 ºC Inj. volume: 2 µl Detection: UV (28 nm) Sample: trastuzumab-mmae Retention Time (min) 29

30 3 Separation of PEGylated Protein

31 PEGylated Protein Analysis (a) (b) Column: MAbPac RP, 4 µm Format: 3. 5 mm Mobile phase A: H 2 O/TFA (99.9 :.1 v/v) Mobile phase B: MeCN/ H 2 O/TFA (9: 9.9 :.1 v/v/v/) Gradient: Time (min) %A %B Temperature: 8 ºC Flow rate:.5 ml/min Inj. volume: 1 µl Detection: UV (28 nm) Sample: (a) PEGylated protein (11 mg/ml) (b)de-pegylated protein (1.24 mg/ml) Retention Time (min) 31

32 Tips and Tricks for RP LC/UV and LC/MS Analysis System QC mix: Ribo A, Cytochrome C, Lysozyme, BSA. Most mab should be analyzed at higher temperature: 7 C to 8 C. To remove carryover, washing column with high h organic and 1 mm NaOH. For better separation resolution, use.1% TFA in mobile phase. For LC/MS analysis, use.1% FA and.2% TFA in mobile phase. 32

33 mabs and ADCs analysis by HIC Shanhua Lin, Ph.D. The world leader in serving science

34 What is Hydrophobic Interaction Chromatography? 34

35 What is Hydrophobic Interaction Chromatography? Chromatographic techniques that separates biomolecules according to differences in their surface hydrophobicity Orthogonal to IEX and SEC Gradient elution: High to Low salt concentration Relatively mild conditions are used; so biomolecules do not become denatured during the separation 35

36 MAbPac HIC family MAbPac HIC-1 MAbPac HIC-2 MAbPac HIC-Butyl Chemistry Proprietary polyamide Proprietary alkylamide Butyl Substrate Spherical, high purity Spherical, high purity Hydrophilic polymer silica particles silica particles Particle Size 5 µm 5 µm 5 µm Pore size 1, Å 1, Å Non-porous *Capacity 28 mg/ml 24 mg/ml 9 mg/ml *Capacity : Amount of Lysozyme / column volume (mg/ml) 36

37 MAbPac HIC Family MAbPac HIC-1 MAbPac HIC-2 MAbPac HIC-Butyl ph Range Temperature Limit ( C) Organic Compatibility -1% -1% -5% Flow Rate.5-1. ml/min (recommended) 1.5 ml/min (max) Maximum Pressure 6, psi (4.6 1 mm) 8, psi ( mm) 6, psi (4.6 1 mm) 8, psi ( mm) 4, psi (4.6 1 mm) 37

38 Separation of Aggregates on MAbPac HIC-1 Absorbance (ma AU) mab variants Monomer Aggregate Column: MAbPac HIC-1, 5 µm Format: mm Mobile phase A: 2 M ammonium sulfate, 1 mm sodium phosphate, ph 7 7. Mobile phase B: 1 mm sodium phosphate, ph 7. Gradient: Time (min) %A %B Temperature: 3 ºC Flow rate: 5.5 ml/min Inj. volume: 1 µl (4 mg/ml) Detection: UV (28 nm) Sample: Monoclonal antibody Retention Time (min) 38

39 Oxidized mabs Methionine and Tryptophans are usually oxidized. 39

40 Separation of Oxidized mab on MAbPac HIC-2 Absorbance (ma AU) 6 Native mab Column: MAbPac HIC-2, 5 µm Format: mm 5 Mobile phase A: 2 M ammonium sulfate, 1 mm sodium phosphate, ph 7. Mobile phase B: 1 mm sodium phosphate, ph 7. Gradient: 4 Time (min) %A %B Oxidized variants Temperature: 3 ºC Flow rate:.5 5mL/min Inj. volume: Untreated mab: 2 µl (1.25 mg/ml) Oxidized mab: 2 µl (1.25 mg/ml) Detection: UV (28 nm) Sample: Untreated mab H 2 O 2 oxidized mab Retention Time (min) 4

41 Antibody-Drug Conjugates (ADCs) 41

42 Heterogeneity of Cys-Linked ADC 42

43 Separation of Cys-Linked ADC on MAbPac HIC-Butyl 4 Absorbance (mau) 3 2 DAR 2 DAR 4 Column: MAbPacHIC-Butyl, 5 µm Format: mm Mobile phase A: 1.5 M ammonium sulfate, 5 mm sodium phosphate, ph 7. / isopropanol (95:5 v/v) Mobile phase B: 5 mm sodium phosphate, ph 7. / isopropanol (8:2 v/v) Gradient: Time (min) %A %B DAR DAR 6 DAR 8 Temperature: 25 ºC Flow rate: 1. ml/min Inj. volume: 5 µl (5 mg/ml) Detection: UV (28 nm) Sample: Cys-conjugated ADC mimic Retention Time (min) 43

44 Separation of Bispecific mab Absorbance (mau) Column: MAbPac HIC-2, 5 µm Format: mm Mobile phase A: 2 M ammonium sulfate, 1 mm sodium phosphate, ph 7. Mobile phase B: 1 mm sodium phosphate, p ph 7. Gradient: Time (min) %A %B Temperature: 3 ºC Flow rate: 1. ml/min Inj. volume: A+B: 2 µl A+D: 12 µl C+B: 1 µl C+D: 2 µl Detection: UV (28 nm) Sample: A+B (.87 mg/ml) A+D (1.4 mg/ml) C+B (1.72 mg/ml) C+D (1. mg/ml) Retention Time (min) 44

45 Method Development Type of salt (lyotropic salt, salting-out agents) Ammonium sulfate Sodium chloride ph of the mobile phase ph 7: sodium phosphate ph 5: sodium acetate Starting salt concentration Addition of organic solvent (acetonitrile or isopropanol) p Sample matrix Flow rate Temperature 45

46 Mobile Phase Recommendation Mobile phase formula 1 Mobile phase A: 2 M ammonium sulfate, 1 mm sodium phosphate, ph 7. Mobile phase B: 1 mm sodium phosphate, p ph 7. Mobile phase formula 2 Mobile phase A: 1.5 M ammonium sulfate, 5 mm sodium phosphate, ph 7. / isopropanol (95:5 v/v) Mobile phase B: 5 mm sodium phosphate, ph 7. / isopropanol (8:2 v/v) Mobile phase formula 3 Mobile phase A: 2 M ammonium sulfate, 2 mm sodium acetate, ph 5. Mobile phase B: 2 mm sodium acetate, ph 5. 46

47 Optimization of the Starting Salt Concentration Absorbance (m mau) Column: MAbPac HIC-2, 5 µm Dimension: mm 3 Mobile phase A: 2 M ammonium sulfate, 1 mm sodium phosphate, ph 7. Mobile phase B: 1 mm sodium phosphate, ph Gradient 1: Time (min) %A %B Gradient 2: Time (min) %A %B Gradient 3: Time (min) %A %B 1.2 M M M Temperature: 3 ºC Flow rate: 1. ml/min Inj. volume: 1 µl Retention Time (min) Detection: UV (28 nm) Sample: Trypsin inhibitor (5 mg/ml) 47

48 Addition of Organic Solvent in Mobile Phase B Absorbance (m mau) Column: MAbPac HIC-2, 5 µm Dimension: mm 1) Mobile phase A: Mobile phase B: 2) Mobile phase A: Mobile phase B: 3) Mobile phase A: Mobile phase B: 2 M ammonium sulfate, 1 mm sodium phosphate, ph 7. 1 mm sodium phosphate, ph 7. 2 M ammonium sulfate, 1 mm sodium phosphate, ph 7. 1 mm sodium phosphate, ph 7./isopropanol (9:1 v/v) 2 M ammonium sulfate, 1 mm sodium phosphate, ph 7. 1 mm sodium phosphate, ph 7. / isopropanol (8:2 v/v) % Isopropanol 1% Isopropanol No solvent Gradient: Time (min) %A %B Temperature: 3 ºC Flow rate: 1. ml/min Inj. volume: 5 µl Detection: UV (28 nm) Retention Time (min) Sample: Trypsin inhibitor (5 mg/ml) 48

49 Effect of Matrix on Peak Shape Absorbance (ma AU) a b Column: MAbPac HIC-2, 5 µm Format: mm Mobile phase A: 2 M ammonium sulfate, 1 mm sodium phosphate, ph 7. Mobile phase B: 1 mm sodium phosphate, ph 7. Gradient: Time (min) %A %B Temperature: 3 ºC Flow rate: 1. ml/min Inj. volume: 1 µl Detection: UV (28 nm) Sample: Protein mixture Retention Time (min) Sample matrix: Peaks: a) Water b) 1 M ammonium sulfate, 5 mm sodium phosphate, h ph 7. 1) Myoglobin 2) Ribonuclease A 3) Lysozyme 4) α-chymotrypsinogen A 49

50 Tips and Tricks for HIC Analysis Wash the system thoroughly before and after analysis using DI water. Make sure the ammonium sulfate does not cause high background signal. If the protein is less hydrophobic (indicated by broader fronting peak and short retention time), increase the salt concentration (lyotropic agent) of mobile phase A or you will need a more hydrophobic column. If the protein is less hydrophobic try increasing the run temperature. If the protein is very hydrophobic, try adding organic solvent to obtain better efficiency and resolution. 5

51 MAbPac HIC-2 Conditioning This column requires conditioning by injecting 75 µg of protein. Ovalbumin or BSA can be used for this purpose. After storing the column storage solution, you may need to re-condition the column. 51

52 Protein Standard Myoglobin, RNase A, Lysozyme, α-chymotrypsinogen A Make 1 mg/ml of each protein Then mix these proteins with the following ratio Myoglobin : RNase A : Lysozyme : α-chymotrypsinogen A = 2:4:1:2 52

53 Thank you! 53