Capillary Electrophoresis Compendial Applications

Transcription

1 Capillary Electrophoresis Compendial Applications Anita Szajek, Ph.D. Principal Scientific Liaison, Biologics & Biotechnology 14 th Symposium on the Practical Applications for the Analysis of Proteins, Nucleotides and Small Molecules

2 Presentation Outline 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 2

3 U.S. Pharmacopeia Who We Are 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-thecounter 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. 3

4 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.

5 Biologics and Biotechnology Council of Experts Structure Biologics and Biotechnology Expert Committees General Chapters Biological Analysis Monographs 1 Monographs 2 <1050> Viral Clearance <1240> Viral Testing for Human Plasma Glucagon Tissue and Tissue- Based Products Glycoprotein and Glycan Analysis Residual DNA Measurement Pharmaceutical Enzyme Preparations Plasma Protein Analytical Expert Panels < > Immunological Test Methods Cryopreservation Unfractionated Heparin Plasma Derived and Recombinant Coagulation Factors Immunogenicity Total Protein Low Molecular Weight Heparin <1239> Vaccines for Human Use Glyconjugate Vaccine Product Class Chapters Insulin Residual Host Cell Proteins (To be formed) Recombinant Therapeutic Monoclonal Abs Epoetin 5

6 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

7 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

8 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)

9 CE in the USP-NF Today: Chapters 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 9

10 From General to Specific - Procedures <1084> Glycoprotein and Glycan Analysis Introduction and Choice of Analysis Methods Guidance &Information <212> Oligosaccharide Analysis <210> Monosaccharide Analysis Procedures &Performance Criteria Oligosaccharide mixtures from -Human α acid Glycoprotein -Fetuin -RNAse B -Human IgG Monosaccharide Mixes 1 4 Single Monosaccharides Procedural (Horizontal) Standards for System Suitability & Validation

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

12 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

13 <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

14 <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)

15 <212> Goals Method focused: not discursive Qualitative analysis of glycosylation through profiling of released glycans Relative quantification Monograph highlights minimum expectations for a fitfor-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

16 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

17 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

18 Procedures in <212> and MAb Chapter N-linked glycans <212> Oligosaccharide Analysis & Associated RSs Normal phase/hilic High-pH AEC with fluorescence (HPAEC) USP Oligosaccharide Mixture A USP Oligosaccharide Mixture B USP Oligosaccharide Mixture C USP Oligosaccharide Mixture D <129> Mab chapter in development LC with fluorescence detection 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, anionexchange 18

19 <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

20 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. 13

21 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: Main peak of HC and peak of non-glycosylated HC can be clearly identified. Resolution between nonglycosylated 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 nonglycosylated to total HC by dividing relative amount of nonglycosylated 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%. 21

22 CE-SDS: Suitability Requirements for Non-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. 22

23 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

24 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)

25 Acknowledgements USP Glycan Expert Panel USP Recombinant Therapeutic Monoclonal Antibodies Expert Panel USP Lab: Dr. David Parmelee NIBSC: Dr. Chris Jones, Dr. CT Yuen

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