Technical Challenges in the Analysis of PEGylated Proteins by Capillary Electrophoresis

Transcription

1 Technical Challenges in the Analysis of PEGylated Proteins by Capillary Electrophoresis Ying-Chen Chen Biologics Process & Product Development Hopewell, NJ

2 Outline PEGylation background information CE-SDS Case review of PEGylated protein A Case review of PEGylated protein B Summary and discussion 2

3 What is Protein PEGylation? Covalent attachment of polyethylene glycol (PEG) chains to proteins IFNα2a PEG-IFNα2a Klaus et al, J Mol Biol. 1997, 274(4), Dhalluin et al, Bioconjugate Chem., 2005, 16,

4 Why PEGylation? Pharmacological advantages: Increase circulation half-life Reduce immunological response Reduce side effects Increase stability against proteases Increase efficacy Commercial advantages: Provide new delivery formats and dosing regimens Extend patent life of previously approved drugs 4

5 PEGylation Chemistry First generation: Amine-specific Lack of specificity Positional isomers/multimers Difficult to purify Approved products (6) Second generation: Most common: N-terminal and cysteine-specific More specific Often engineered free cysteine Potential S-S scrambling Approved products (2 ) Yu et al, Bioorg. Med. Chem. 2007, 15, Kinstler et al, Adv Drug Deliv Rev. 2002, 54,

6 Approved PEGylated Proteins in Biopharmaceuticals Trade name Protein Polymer Indication Company Year to Market * * * ADAGEN Adenosine deaminase 5 kda PEG Oncaspar Asparaginase 5 kda PEG PEG-Intron Interferon α2b 12 kda PEG PEGasys Interferon α2a 40 kda PEG Neulasta Granulocyte colony 20 kda stimulating factor PEG severe combined immunodeficienc y disease acute lymphatic leukemia Enzon 1990 Enzon 1994 hepatitis C Schering- Plough 2001 hepatitis C Hoffman LaRoche neutropenia Amgen 2002 * Somavert GH antagonist 5 kda PEG MIRCERA Erythropoietin 30 kda PEG Cimzia Anti-TNF Fab' 40 kda PEG Simona Jevsevar, Biotechnol.J.2010,5, excessive growth (acromegaly) anemia RA and Crohn's disease Pfizer 2003 Hoffman LaRoche UCB

7 Physical Characterization of PEG and PEGylated Proteins Average molecular weight SDS-PAGE, CE-SDS, SEC Molecular weight distribution: polydispersity DLS, CEX Degree of PEGylation Mass spec, HPLC PEGylation site determination: positional isomers Mass spec 7

8 CE-SDS Automation High-speed separation Enhanced resolution Direct, on-line quantification using UV detection. 8

9 CE-SDS Method Capillary Effective length: 20 cm, total length 30.2 cm Inside diameter: 50 µm Voltage separation 15 kv (reverse polarity), 35 min Cartridge temperature, 25 C Sample Injection 5 kv (reverse polarity), 20 sec, Sample Buffer 100mM Tris-HCl, ph 9.0 with 1 % SDS Gel Buffer Polymer buffer, ph 8.0 with 0.2% SDS Sample preparation 1 mg/ml sample in sample buffer Add 10 kd Internal Standard Add 5% of 2-mercaptoethanol for reduced condition Incubated in water bath at 70 C for 10 min Add 40mM Iodoacetamide (IAM) for non-reduced condition Incubated in water bath at 70 C for 5min Detection UV at 220 nm 9

10 Case Study of Protein A Protein A, ~20 kda PEGylation, N-terminal covalent conjugate of 20 kda linear PEG chain 10

11 CE-SDS: PEGylated Protein A in Non-Reduced and Reduced Conditions Molecular Weight Marker 10 kd 20 kd 35 kd 50 kd 100 kd 150 kd kd Absorbance (220 nm) Internal Standard 10 kd PEGylated protein A Non-Reduced Condition Internal Standard 10 kd PEGylated protein A Reduced Condition 11

12 CE-SDS: PEGylated Protein A and Protein A in Non- Reduced Condition Molecular Weight Marker 10 kd 20 kd 50 kd kd 100 kd 150 kd 225 kd Absorbance (220 nm) 10 kd PEGylated protein A Non-Reduced Condition Non-PEGylated protein A 10 kd Non-Reduced Condition

13 CE-SDS: PEGylated Protein A and Protein A in Reduced Condition Molecular Weight Marker 10 kd 20 kd 50 kd kd 100 kd 150 kd 225 kd Absorbance (220 nm) kd PEGylated protein A Reduced Condition Non-PEGylated protein A 10 kd Reduced Condition

14 CE-SDS: Spike Protein A into PEGylated Protein A in Non-Reduced Condition PEGylated Protein A Protein A Absorbance (220 nm) mv %Protein A + 5.0% Protein A Protein A + 1.0% Protein A + 0.5% Protein A % Protein A %Protein A PEGylated Protein A

15 CE-SDS: Effects of Matrix on PEGylated Protein A in Non-Reduced Condition Absorbance (220 nm) mv Mix with DP Placebo Mix with arginine Mix with sucrose Mix with histidine Desalt Lyo DP PEGylated Protein A

16 CE-SDS: PEGylated Protein A and Stressed Samples in Non-Reduced Condition Absorbance (220 nm) Fraction 4 Fraction 3 Fraction 2 Fraction PEGylated Protein A Time 16

17 Case Study of Protein B c N Protein B, <20 kda PEGylation: C-terminal covalent conjugate of 40 kda branched PEG chain 17

18 UV - 214nm MWM CE-SDS: Protein B and PEGylated Protein B in Non-Reduced Condition Molecular Weight Marker 10 kd 50 kd 35 kd 20 kd 100 kd 225 kd 150 kd Absorbance (220 nm) Protein B PEGylated Protein B Time 18

19 UV - 214nm MWM CE-SDS: PEGylated Protein B in Non-Reduced and Reduced Conditions Molecular Weight Marker 10 kd 50 kd 35 kd 20 kd 100 kd 225 kd 150 kd Absorbance (220 nm) kd Reduced Non-Reduced Time 19

20 CE-SDS: Method Development for PEGylated Protein B Sample Buffer SDS Concentration Buffer ph Reducing reagent Mercaptoethanol vs TCEP 20

21 CE-SDS: Effect of SDS Concentration on PEGylated Protein B % Absorbance (220 nm) % 3% 2% % Time 21

22 CE-SDS- Effect of Sample Buffer on PEGylated Protein B in Non-Reduced Condition Citrate Buffer Absorbance (220 nm) Citrate Buffer 6.7 Citrate Buffer 6.3 Citrate Buffer 5.2 Citrate Buffer Citrate Buffer Time 22

23 CE-SDS- Effect of Sample Buffer on PEGylated Protein B in Non-Reduced Condition Tris Buffer Absorbance (220 nm) Tris Buffer 8.6 Tris Buffer 7.8 Succinate Buffer Succinate Buffer Time 23

24 CE-SDS- Effect of Sample Buffer on PEGylated Protein B in Reduced Condition Citrate Buffer Absorbance (220 nm) Citrate Buffer 6.7 Citrate Buffer 6.3 Citrate Buffer 5.2 Citrate Buffer Citrate Buffer Time 24

25 CE-SDS- Effect of Sample Buffer on PEGylated Protein B in Reduced Condition Absorbance (220 nm) Tris Buffer 9.0 Tris Buffer 8.6 Tris Buffer 7.8 Succinate Buffer Succinate Buffer Time 25

26 CE-SDS: Effect of Reducing Reagent on PEGylated Protein B Absorbance (220 nm) R: TCEP 100oC R: TCEP 70oC R: TCEP room temp R: Mercaptoethanol 70oC NR Time 26

27 SEC : PEGylated Protein B Absorbance (280 nm) Peak 1 Peak shoulder Time 27

28 SDS-PAGE: PEGylated Protein B in Non-Reduced Condition 200,000 D - 116,300 D - 97,400 D - 66,300 D - 55,400 D Band 1 Band 2 36,500 D - 31,000 D - 21,500 D - 14,400 D - 6,000 D - 3,500 D - 2,500 D % Bis-Tris Gel, 35 min at constant Voltage 200V 28

29 RT: Relative Abundance Mass Spec : PEGylated Protein B Time (min) NL: 1.46E7 m/z= F: FTMS + p ESI Full ms [ ] MS high2 Band 1 NL: 1.26E7 m/z= F: FTMS + p ESI Full ms [ ] MS middle2 Mix band 1and band 2 NL: 1.56E7 m/z= F: FTMS + p ESI Full ms [ ] MS low2 Band 2 29

30 Summary PEGylated Protein A Linear PEG (20kDa) Molecular weight ratio Protein A : PEG= 1: 1 Single peak in both NR and R conditions Challenge: Matrics: signal suppression by Arginine 30

31 Summary (Continue) PEGylated Protein B Branch PEG (40kDa) Molecular weight ratio, Protein B : PEG= 1: 4 Challenges: Three peaks in CE-SDS: not buffer, SDS concentration and ph dependent Two smear bands in SDS: Identical peptide mapping (in gel digestion) Two peaks in SEC-HPLC 31

32 Discussion and future work Higher PEG:Protein molecular weight ratio (4:1) in PEGylated protein B may cause: Conformational change when interact with SDS Different interaction in forming SDS/protein complex during separation CZE: No SDS/protein complex Bioanalyzer: Reduce the injection amount of protein 32

33 Acknowledgement Ming Zeng Richard Ludwig Wei Wu Vicky Romero Kunnel Babu Yunping Huang Jinping Liu Reb Russell Michael Grace 33