Capillary Electrophoresis–Compendial Applications

Anita Szajek, Ph.D. Principal Scientific Liaison, Biologics & Biotechnology

14th Symposium on the Practical Applications for the Analysis of Proteins, Nucleotides and Small Molecules

1. USP Introduction & Overview: Who we are and what we do 2. Capillary Electrophoresis in the USP-NF today 3. Recent development experiences with CE for compendial

purposes: <212> Other USP chapters General Considerations for the development of a compendial CE

procedure

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Presentation Outline

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• Scientific, independent, volunteer- driven, nonprofit organization

– Established in 1820: Headquartered in Rockville, MD – Facilities in India, China, Brazil, Switzerland

• Sets public quality standards for prescription and over-the-counter medicines, excipients, dietary supplements, and food ingredients

• Work closely with FDA and predecessors for >100 years, developing and revising drug quality standards

• Standards recognized and used

in over 140 countries.

U.S. Pharmacopeia – Who We Are

Recognition of USP Standards in Federal Food Drug and Cosmetic Act (FDCA)

• Section 501(b) - Adulterated Drugs and Devices – A drug or device shall be deemed to be adulterated if it purports to be or

is represented as a drug the name of which is recognized in an official compendium, and its strength differs from, or its quality or purity falls below, the standards set forth in such compendium.

– Such determination as to strength, quality, or purity shall be made in accordance with the tests or methods of assay set forth in such compendium…

• Section 502(g) Misbranded Drugs and Devices – A drug or device shall be deemed to be misbranded if it purports to be a

drug the name of which is recognized in an official compendium, unless it is packaged and labeled as prescribed therein.

Biologics and Biotechnology Council of Experts Structure

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Biologics and Biotechnology

General Chapters Biological Analysis

<1050> Viral Clearance

<1240> Viral Testing for Human

Plasma

Glycoprotein and Glycan Analysis

Residual DNA Measurement

<1102-1105> Immunological Test

Methods Cryopreservation

Immunogenicity Total Protein

<1239> Vaccines for Human Use

Glyconjugate Vaccine Product Class Chapters

Residual Host Cell Proteins (To be

formed)

Recombinant Therapeutic

Monoclonal Abs

Monographs 1

Glucagon

Pharmaceutical Enzyme

Preparations

Unfractionated Heparin

Low Molecular Weight Heparin

Insulin

Epoetin

Monographs 2

Tissue and Tissue-Based Products

Plasma Protein Analytical

Plasma Derived and Recombinant

Coagulation Factors

Expert Committees

Expert Panels

Importance of Glycosylation in Pharmaceutical Products

• Protein glycosylation can affect biological activity – Directly, modulating binding to receptors or by changes in

equilibrium between conformational forms (e.g. Factor IX) – By direct glycan-receptor binding – Indirectly, by modulating in vivo half life (clearance - e.g. rhEPO) – Indirectly, by modulating degradation in vivo – By activating downstream activity, affecting ADCC* (in mAbs) – Through immunogenicity (eg. Neu5Gc and Galα1,3Gal epitopes)

• Protein glycosylation can also affect products – By modulating in vitro stability (and product shelf life) – By influencing isolation and purification steps (process

consistency)

• Glycosylation can often be a critical quality attribute for a biopharmaceutical product

* ADCC = Antibody-dependent cell-mediated cytotoxicity

Why it Deserves Pharmacopeial Recognition

• Protein glycosylation is not template-directed – It is a post-translational modification requiring the interaction of

many enzymes and substrates

• It depends on the cell line factors and culture conditions – Cell type, culture components, cell age, stresses

• It can be affected by purification procedures – Selective separation depending on glycosylation

• This allows the possibility of significant variability – Batch-to-batch (Lot release: product potency or process

consistency) – If manufacturing changes occur (comparability) – Between nominally similar products (biosimilars)

Direct impact on product quality

USP: Chapters and Monographs

• Monographs are product-specific, contain specifications and are linked to Reference Standards (RSs)

• Chapters are general and cover broader topics and more widely applicable methods. These are not linked to individual products unless referenced in a monograph. – Chapters with numbers above <1000> are advisory and contain

general information and recommendations – Chapters with numbers below <1000> are enforceable, often

describe procedures and are linked to Reference Standards (RSs)

CE in the USP-NF Today: Chapters

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Official: •<727> Capillary Electrophoresis

was proposed for omission in PF36(1) because of redundancy. The content of this chapter is covered completely in the more current and inclusive harmonized chapter Biotechnology-Derived Articles— Capillary Electrophoresis <1053> , which will be renamed Capillary Electrophoresis

•Chapter <1053> will be the sole guidance for the capillary electrophoresis method in USP–NF. •<1084> Glycoprotein and Glycan Analysis-General Considerations

New Chapters in development:

•<212> Oligosaccharide Analysis •<210> Monosaccharide Analysis • USP Chapters on Monoclonal Antibodies

From General to Specific - Procedures

<1084> Glycoprotein and

Glycan Analysis – Introduction and Choice of Analysis Methods

<212> Oligosaccharide Analysis

<210> Monosaccharide Analysis

Monosaccharide Mixes 1–4

Oligosaccharide mixtures from -Human α acid Glycoprotein -Fetuin -RNAse B -Human IgG

Single Monosaccharides

Guidance &Information

Procedures &Performance

Criteria

Procedural (Horizontal)

Standards for System

Suitability & Validation

Glycoprotein Analysis Strategies: CE

Reference: <1084> Glycoprotein and Glycan Analysis-General Considerations

Intact Glycoproteins: Product Specific Methods

• Depends on protein as much as the glycans • Will form part or individual product-specific monographs • For example (from European Pharmacopoeia)

– sialylation profile of rhEPO by capillary electrophoresis

– 2- and 3-glycan chain variants of alteplase by PAGE gels

<1084> Goals

• To be a simple introduction to glycoprotein glycosylation – To describe different approaches to analysis of glycoprotein

glycosylation • Intact glycoprotein, cleaved glycans and monosaccharide analysis

– To summarise method for cleavage of the glycan chains

• To guide analysts in the choice of appropriate procedures for glycan analysis

• To provide a framework within which more detailed analytical chapters can be written

• To provide links to other analytical chapters relevant to the analysis of glycosylation, either at the level of intact glycoprotein or cleaved glycan chain

<210> Goals

• Procedure focused: not discursive • Quantitative analysis of monosaccharide components

present in glycoprotein glycans – Isolation/cleavage from glycoprotein through enzymatic or

hydrolytic release – Quantitation methods – chromatography or colorimetric – Understand specificity of the methods – Linked to quantitative reference standards

• Focus initially on sialic acid, expanding to neutral sugars • Will not include quantification of other saccharides (e.g.

bacterial polysaccharides used in vaccine manufacture)

<212> Goals

• Method focused: not discursive • Qualitative analysis of glycosylation through profiling of

released glycans – Relative quantification

• Monograph highlights minimum expectations for a “fit-for-purpose” glycan analysis study

• Linked to system suitability standards – Use of the Reference Standards in the compendial methods will

produce a reference profile. – Acceptance criteria being developed

• Allows comparison of compendial and other analytical approaches

Proposed <212> Structure

Scope This chapter refers only to Asn-linked glycan (N-glycan) chains, deals with chromatographic

separation of oligosaccharides and provides established procedures as well as performance criteria for the analysis of N-glycans found in recombinant therapeutic proteins.

• Sample preparation (desalting etc.) • Glycan cleavage (PNGase F) • Fluorophore labelling, if required • Separations appropriate to different types of chain

– HPAEC for charged chains – HPLC for neutral and low-charge chains – CE methods

• Method acceptance criteria – What does a good separation look like?

• Use of Reference Standards

Reference Standards for <212>

• Glycans prepared by digestion of commercial glycoproteins – Ribonuclease b high mannose chains – Human polyclonal IgG biantennary chains – Human α1 glycoprotein moderate sialylation, more branching – Bovine fetuin high sialylation, extensive branching

• Not using conventional USP approach – 20 µL (ca. 20 µg total saccharide) per vial, excipient free. – Use plastic vials in 96-vial trays and freeze dry – Soft push-on caps in 96-vial sheets.

• Store at -20°C • Most stability data now available

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Procedures in <212> and MAb Chapter

<212> Oligosaccharide Analysis & Associated RSs

<129> Mab chapter in development

N-linked glycans

Normal phase/HILIC

USP Oligosaccharide Mixture A USP Oligosaccharide Mixture B

LC with fluorescence detection

High-pH AEC with fluorescence (HPAEC)

USP Oligosaccharide Mixture C USP Oligosaccharide Mixture D

N/A

CE separation of APTS-labeled glycans

USP Oligosaccharide Mixture A USP Oligosaccharide Mixture B

CE with LIF

Sialic acid analysis

N/A N/A LC with electrochemical detection, anion-exchange

<129> Round Robin Study – Overview and Status •Proposal: Multi-participant study for the analysis of relevant IgG mAb samples by USP <129> size variant and purity (CE-SDS; reduced and non-reduced) methods

•Objectives: To evaluate the proposed procedures intended for inclusion in chapter <129>. To test organization specific or any additional MAb samples to further increase understanding of the appropriateness of the test methods across antibody preparations.

•Participants: Open to all relevant interested stakeholders

•Submitted blinded data is for scientific information purposes only, not to be used to define ‘general’ acceptance criteria 12

CE-SDS Method for MAb Round Robin Study

• USP Monoclonal IgG System Suitability Reference Standard for system suitability for proposed SEC and CE-SDS USP methods

• It allows the quantitative estimation of product related impurities such as non-glycosylated molecules, half antibodies, and fragments and is thus also useful as stability indicating test procedure.

• After denaturation with SDS the method allows the analysis of the complete antibody under non reducing conditions and the analysis of light and heavy chains under reducing conditions.

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Electropherogram: The electropherogram of the System suitability solution is consistent with the typical example provided in the Certificate of USP Monoclonal IgG SS RS .

Resolution: Main peak of HC and peak of non-glycosylated HC can be clearly identified. Resolution between non-glycosylated HC and intact HC is NLT 1.3.

Ratio of non-glycosylated to total heavy chain: Use time corrected peak areas for calculation. Calculate the ratio of non-glycosylated to total HC by dividing relative amount of non-glycosylated HC through the sum of all HCs and multiply with 100. The ratio in the reference should be within the limits of 0.8 to 1.3%.

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CE-SDS: Suitability Requirements for Reducing Conditions

Electropherogram: The electropherogram of the System suitability solution is consistent with the typical example provided in the Certificate of USP Monoclonal IgG SS RS .

Resolution: IgG main peak can be clearly identified; Resolution between IgG main peak and Fragment 1 (F1) is NLT 1.3.

Amount of main peak: Calculate the relative amount of main peak by dividing the sum of the time corrected peak area of the main IgG-Peak through the sum of all time corrected peak areas after the internal standard and multiply with 100. The relative amount of the main IgG peak of the system suitability solution should be within the limits of 70-80%.

Blank: Relative standard deviation of migration time of internal standard in bracketing Blank solution is NMT 2%. No other peaks migrating after the internal standard are detectable.

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CE-SDS: Suitability Requirements for Non-Reducing Conditions

Challenges in Establishing a Compendial CE Test

• Platform-to-platform variability • Migration time drift and variability:

– How to set performance and acceptance criteria? • Assay and sample suitability: particularly in impurity

assessment

Summary

• USP is developing a series of chapters on analysis of glycans cleaved from glycoproteins – CE applications captured in <212> and others

• Product-specific methods in product monographs – Transition from gel-based methods to CE

• Generic approaches: initial work – <1084> overview chapter, official – <210> on quantitative monosaccharide analysis – <212> on oligosaccharide profiling – Linked to Reference Standards

• Upcoming MAb round robin study (size variant and CE-SDS) – Contact Anita Szajek (aey@usp.org)

Acknowledgements

• USP Glycan Expert Panel • USP Recombinant Therapeutic Monoclonal Antibodies

Expert Panel • USP Lab: Dr. David Parmelee • NIBSC: Dr. Chris Jones, Dr. CT Yuen