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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2

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

8

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