Capillary Electrophoresis Sodium Hexadecyl Sulfate (CE-SHS ... · Capillary Electrophoresis Sodium...
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Capillary Electrophoresis Sodium Hexadecyl Sulfate (CE-SHS): A Novel Approach to Evaluate the Purity of Therapeutic Proteins
Jeff BeckmanBristol Myers Squibb
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OUTLINE
1.CE-SDS: Improvement OpportunitiesBackgroundChallenges in applying CE-SDS to certain protein
types“CE-SHS”: Hydrophobic Detergents to Improve Data
Quality2. “CE-SHS” Method Development Challenges Solutions
CE-SDS: Capillary Gel Electrophoresis using Sodium Dodecyl Sulfate
TypicalReduced mAb
At Bristol Myers Squibb it is used to:Quantify protein fragments/covalent aggregates
Electrophoretic separation of analytes by size through a sieving (gel) matrix within a capillary:
-
Detector
Computer
Proteins are denatured using SDS:
Why SDS? Rooted deep in method history; Developed aroundSDS (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 crucialDifferent sized molecules essentially migrate at equal speeds…
Sieving: Larger molecules move slower than smaller ones
CE-SDS Method Principles
+
%“Broad HC” + Post-HC unknowns = poor denaturation?
CE-SDS: When the Data Looks Poor
HC
LC
Post-HC Unknowns
Reduced “mAb-1”
Limited development options: Sample optimization: no problem Limited “Running Buffer” (Gel) commercial options Intimidating: HPLC Analogy is that the Gel = Solid/Mobile phase combined
02468
101214
0 100 200
Rel
ativ
e %
Pos
t-HC
% Nominal Concentration
No Assay Range:No Assay!
% Unknowns highly dependent on [Sample]
Impurity:Potential
CQA
Fusion Protein (“RTP-1”):
1.00
1.50
2.00
2.50
3.00
3.50
0 10000 20000 30000
Rel
ativ
e %
Are
a of
Impu
rity
Total Peak Area Signal0.0
50.0100.0150.0200.0250.0300.0350.0400.0450.0
0 10000 20000 30000
Impu
rity
Peak
Are
a Si
gnal
Total Peak Area Signal
?
Expect LINEAR
CE-SDS: When the Data Looks Poor -Another Example
No Assay Range:No Assay!
How Much Is There?
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CE-SDS: When the Data Looks Poor –Try Changing the Detergent
CE-SDS
“CE-SHS”
Abso
rban
ce (A
U)
Migration Time (Minutes)
“RTP-1”
Impurity
Main protein Peak
Replacement or Addition of a Longer-Chain Detergent: SDS (C12)
SHS (C16)
0.0100.0200.0300.0400.0500.0600.0700.0800.0900.0
0 10000 20000 30000 40000
Impu
rity
Sign
al
Total Peak Area Signal
1.00
1.50
2.00
2.50
3.00
3.50
0 20000 40000
Rel
ativ
e %
Are
a of
Im
purit
y
Total Peak Area Signal
CE-SDS
“CE-SHS”
We Now Have an Assay!
CE-SDS
“CE-SHS”
3-fold increase in resolution8-fold increase in plate countSensitivity Potential > 3-Fold
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Detergent Tail Length Improved Peak EfficiencyC11 – C16Samples/Sieving Gel buffers contain only the detergent shown:
“RTP-1”
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Peak Efficiency: SHS >> SDS Regardless of [SDS] “RTP-1” Main Peak Shape Dependence on Gel Buffer Detergent
Concentration:
SDS
5 X SDS
10 X SDS
SHS
Improved Resolution
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CE-SDS
“CE-SHS”
Reduced “mAb-1”: Recovery of Expected Profile:
CE-SDS “CE-SHS”: Another Example
HC
LC
Typical “mAb-2” NR CE-SDS Profile
Zoom-in
IntactmAb
Improved Resolution
Non-Reduced “mAb-2” Improvements:
CE-SDS
“CE-SHS”
CE-SDS “CE-SHS”: Non-Reduced Example
0.5%
2.5%
Reduction of Artifactual Species
We Now Have an Assay!
No Assay Range
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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
Custom SHS gel + SDS sample buffer
Custom SHS gel + STS sample buffer
Time (Min)
Abso
rban
ce
* Qian Guan, Poster P-110-T
“mAb-1”HC
LC
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Principles Behind SHS Performance
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Protein:SHS
Detector
More uniformly denatured = better peak efficiency**
* Colon et al. Biochemistry (2004) 43 (3612), 11248-11254** Pozzo D et al. Langmuir 2011, 27, 928-935; Otzen et al. (2009), J Phys Chem B, 113, 42, 13942-13952; J. Am. Chem. Soc. 133, 44, 17681-17695
Some Proteins can be “Resistant” to SDS binding*
Add a hydrophobic detergent (SHS):
Hypothesis
Important: More Hydrophobic = Higher Affinity
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What Type of Protein Requires SHS for High Peak Efficiency?
Naresh Chennamsetty, Stanley Krystek (BMS)
*mAbs (2009) 1, 580-582
0.010.020.030.040.050.060.070.0
SAP
Sco
re (
CDR
regi
on)
“mAb
-1”
“mAb
-2”
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:
Higher SAP score, more surface hydrophobic patches
Did not need SHS
Overall Protein Surface Hydrophobicity Not a Primary Determinant
SAP
Scor
e (C
DR
Reg
ion)
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Is Surface Charge Important? RTP-1 Modeling: High “-”
charge on the surface (Red)
* Chen, Z. et al (2018) Protein Cell 9(1), 3-14; D. Lipovsek (2011) Protein Engineering, Design and Selection, 24(1-2), 3-9
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
?
What Type of Protein Requires SHS for High Peak Efficiency?
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Is Thermal Stability Important?RTP-1: Thermophilic* E2 Tm value > 80˚C High energy of unfolding (DSC)
SHS: Much less energy at lower concentration needed to denature Domain (E2)
∆H
2(c
al/m
ol)
Temperature, ˚C
[Detergent]
E1
* Chen, Z. et al (2018) Protein Cell 9(1), 3-14
Thermostability Appears to be an Important Determinant
Cp
(Cal
/mol
e/˚C
)E2
“mAb-1” and “mAb-2”: High Tm (> 85˚C)
What Type of Protein Requires SHS for High Peak Efficiency?
Recent Industry Trend: Higher thermostability to extend expiries*
As Tm 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
* Maloney, K. M. et al. Mol. Pharmaceutics 2015, 12,1005−1017
** Schwartz, M. Facilitator, CASSS CE-Pharm 2017, Table 8 Notes
Alternative Detergent May Be the Solution for Some of These Modalities
What Type of Protein Requires SHS for High Peak Efficiency?
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OUTLINE
1.CE-SDS: Improvement OpportunitiesBackgroundChallenges in applying CE-SDS to certain protein
types“CE-SHS”: Hydrophobic Detergents to Improve Data
Quality2. “CE-SHS” Method Development Challenges Solutions
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“CE-SHS” Method Development ChallengesPrimary Challenge: SHS-containing sieving gel matrix bufferRecall 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)
Developed “from scratch” Production ProtocolControl over gel buffer components Better understanding of component robustness/performanceIdentified two vendors for each component
Performed Multivariate DOEs Around Critical ParametersResponses: Resolution, plate count, baseline, relative migration
time
“CE-SHS” Method Development Solutions
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
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SummaryWhen CE-SDS doesn’t work: “CE-SHS”Replacement/Addition of Sulfate detergents with longer
alkyl chainsBetter 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
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AcknowledgementsBMS Devens• Yuanli Song• Qian Guan• Jennifer Atsma• Sergey Voronov• Rekha Tulsan• Yan Gu• Jay West• Julia Ding• Nesredin Mussa
BMS Zymogenetics• Devi Visone• Jeff Meyer
BMS New Jersey• Ming Zeng• Ya Fu• Abbie Esterman• Tara Enda• Naresh Chennamsetty• Stanley Krystek