Capillary Electrophoresis Sodium Hexadecyl Sulfate (CE-SHS): A Novel Approach to Evaluate the Purity of Therapeutic Proteins

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1 Capillary Electrophoresis Sodium Hexadecyl Sulfate (CE-SHS): A Novel Approach to Evaluate the Purity of Therapeutic Proteins Jeff Beckman Bristol Myers Squibb 1

2 OUTLINE 1.CE-SDS: Improvement Opportunities Background Challenges in applying CE-SDS to certain protein types CE-SHS : Hydrophobic Detergents to Improve Data Quality 2. CE-SHS Method Development Challenges Solutions 2

3 CE-SDS: Capillary Gel Electrophoresis using Sodium Dodecyl Sulfate Electrophoretic separation of analytes by size through a sieving (gel) matrix within a capillary: Computer Detector Typical Reduced mab - At Bristol Myers Squibb it is used to: Quantify protein fragments/covalent aggregates

4 CE-SDS Method Principles Proteins are denatured using SDS: + Why SDS? Rooted deep in method history; Developed around SDS (Tubes of gels Slab gels Capillaries) Uniform coating: 1.4 g SDS per g of Protein Harsh conditions sometimes necessary (70+ C Heat) Artifactual Fragmentation Reducing/Non-Reducing Size separation through a sieving matrix Uniform m/z crucial Different sized molecules essentially migrate at equal speeds Sieving: Larger molecules move slower than smaller ones

5 CE-SDS: When the Data Looks Poor Limited development options: Sample optimization: no problem Limited Running Buffer (Gel) commercial options Intimidating: HPLC Analogy is that the Gel = Solid/Mobile phase combined LC HC Reduced mab-1 Post-HC Unknowns Relative % Post-HC No Assay Range: No Assay! % Nominal Concentration % Broad HC + Post-HC unknowns = poor denaturation? % Unknowns highly dependent on [Sample]

6 CE-SDS: When the Data Looks Poor - Another Example Fusion Protein ( RTP-1 ):? How Much Is There? Impurity: Potential CQA Impurity Peak Area Signal Expect LINEAR Relative % Area of Impurity Total Peak Area Signal No Assay Range: No Assay! Total Peak Area Signal

7 CE-SDS: When the Data Looks Poor Try Changing the Detergent Replacement or Addition of a Longer- Chain Detergent: SDS (C12) SHS (C16) Absorbance (AU) RTP-1 Impurity CE-SHS Main protein Peak CE-SDS Impurity Signal CE-SHS CE-SDS Total Peak Area Signal Relative % Area of Impurity We Now Have an Assay! CE-SDS CE-SHS Total Peak Area Signal Migration Time (Minutes) 3-fold increase in resolution 8-fold increase in plate count Sensitivity Potential > 3-Fold 7

8 Detergent Tail Length Improved Peak Efficiency C11 C16 Samples/Sieving Gel buffers contain only the detergent shown: RTP-1 8

9 Peak Efficiency: SHS >> SDS Regardless of [SDS] RTP-1 Main Peak Shape Dependence on Gel Buffer Detergent Concentration: SHS 5 X SDS 10 X SDS SDS Improved Resolution 9

10 CE-SDS CE-SHS : Another Example Reduced mab-1 : Recovery of Expected Profile: LC HC CE-SHS CE-SDS 10

11 CE-SDS CE-SHS : Non-Reduced Example Non-Reduced mab-2 Improvements: Typical mab-2 NR CE-SDS Profile We Now Have an Assay! 0.5% Zoom-in 2.5% CE-SHS Improved Resolution Intact mab Reduction of Artifactual Species CE-SDS No Assay Range

12 Further Improvement: Detergents in the Sample* Adding a Hydrophobic Detergent to the SAMPLE as well as the gel buffer further improved results by reducing post-hc artifacts under reducing conditions STS (C14) was used due to SHS solubility issues mab-1 HC LC Absorbance Custom SHS gel + SDS sample buffer Custom SHS gel + STS sample buffer Time (Min) * Qian Guan, Poster P-110-T 12

13 Principles Behind SHS Performance 13

14 Hypothesis Some Proteins can be Resistant to SDS binding* Add a hydrophobic detergent (SHS): Detector Protein:SHS More uniformly denatured = better peak efficiency** Important: More Hydrophobic = Higher Affinity * Colon et al. Biochemistry (2004) 43 (3612), ** Pozzo D et al. Langmuir 2011, 27, ; Otzen et al. (2009), J Phys Chem B, 113, 42, ; J. Am. Chem. Soc. 133, 44,

15 What Type of Protein Requires SHS for High Peak Efficiency? Since Hydrophobicity of SHS >> SDS: Increase protein hydrophobicity, increase need for SHS? Spacial Aggregation Propensity (SAP) Score: Measures surface hydrophobicity* SAP scores for select BMS mabs shown below: SAP Score (CDR region) 70.0 SAP Score (CDR Region) mab-1 Higher SAP score, more surface hydrophobic patches Did not need SHS mab-2 *mabs (2009) 1, Overall Protein Surface Hydrophobicity Not a Primary Determinant Naresh Chennamsetty, Stanley Krystek (BMS) 15

16 What Type of Protein Requires SHS for High Peak Efficiency? Is Surface Charge Important? RTP-1 Modeling: High - charge on the surface (Red) Charge surface profile of the Variable Domain equivalent of RTP-1:? SDS: electrostatic repulsion too great? SHS: Binding of more hydrophobic tail to the hydrophobic core overcomes the repulsion? Surface Charge Appears to be an Important Determinant * Chen, Z. et al (2018) Protein Cell 9(1), 3-14; D. Lipovsek (2011) Protein Engineering, Design and Selection, 24(1-2),

17 What Type of Protein Requires SHS for High Peak Efficiency? Is Thermal Stability Important? RTP-1: Thermophilic* E 2 Tm value > 80 C High energy of unfolding (DSC) Cp (Cal/mole/ C) SHS: Much less energy at lower concentration needed to denature Domain (E 2 ) Thermostability Appears to be an Important Determinant H 2 (cal/mol) E 1 Temperature, C E 2 mab-1 and mab-2 : High Tm (> 85 C) [Detergent] * Chen, Z. et al (2018) Protein Cell 9(1),

18 What Type of Protein Requires SHS for High Peak Efficiency? Recent Industry Trend: Higher thermostability to extend expiries* As T m rises industry-wide, can we expect an increase in the need for SDS alternatives? Atypical (Includes alternative modalities: non-mab; fusions, PEGylated, etc) examples**: 80:20 Alternative Detergent May Be the Solution for Some of These Modalities * Maloney, K. M. et al. Mol. Pharmaceutics 2015, 12, ** Schwartz, M. Facilitator, CASSS CE-Pharm 2017, Table 8 Notes

19 OUTLINE 1.CE-SDS: Improvement Opportunities Background Challenges in applying CE-SDS to certain protein types CE-SHS : Hydrophobic Detergents to Improve Data Quality 2. CE-SHS Method Development Challenges Solutions 19

20 CE-SHS Method Development Challenges Primary Challenge: SHS-containing sieving gel matrix buffer Recall HPLC Analogy: Gel = Solid/Mobile phase combined Can we make this from scratch??? Quality Control groups were nervous Reproducible? Supply risk? GOAL: Provide robust Gel Buffer solution for multiple programs (mabs 1 and 2, RTP-1) 20

21 CE-SHS Method Development Solutions Developed from scratch Production Protocol Control over gel buffer components Better understanding of component robustness/performance Identified two vendors for each component Performed Multivariate DOEs Around Critical Parameters Responses: Resolution, plate count, baseline, relative migration time Reproducible: 13 lots, 4 sites, 10 analysts Responses: same as above Stable: Two year expiry MESSAGE: Don t be afraid to open the gel box - High method optimization potential

22 Summary When CE-SDS doesn t work: CE-SHS Replacement/Addition of Sulfate detergents with longer alkyl chains Better for thermophilic proteins? Don t be afraid to open the gel box Industry trend: Increasing thermophilicity Expect more use of our CE-SHS method? When to use SHS? When to use SDS? Hypothesis: peak efficiency depends on the state of denaturation 22

23 Acknowledgements BMS Devens Yuanli Song Qian Guan Jennifer Atsma Sergey Voronov Rekha Tulsan Yan Gu Jay West Julia Ding Nesredin Mussa BMS New Jersey Ming Zeng Ya Fu Abbie Esterman Tara Enda Naresh Chennamsetty Stanley Krystek BMS Zymogenetics Devi Visone Jeff Meyer 23