CE in biopharma science: it s more than GCE and cief Unexploited applications from the sewing box

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1 CE in biopharma science: it s more than GCE and cief Unexploited applications from the sewing box CEPharm 2018, PD Dr. Maria A. Schwarz and Dr. Samuel Bader

2 Introduction It s more than GCE and cief Feasibility 1 Characterization of PEGylated protein by CZE. ACE MEKC CE Feasibilty 2 Site specific identification of deamidation site using deamidation specific digest and CZE. Feasibility 3 Separation of glycated from non-glycated proteins by affinity CE. CZE cief RP HIC A HPLC SEC IEC/HILIC cgce (SDS) Rough estimate of projects at Solvias 2

3 Feasibility 1 PEGylation Introduction Non-PEGylated protein (Lys-C digest) PEGylated protein (Lys-C digest) p3 p4 p4 p1 p2 p3 p4 p1 p1 p2 p3 p4 p2 p1 p2+p3 300 mau 200 Disappearing/decreasing signals (in peptide map of PEGylated protein) indicate that the respective peptides are PEGylated completely/partially The number of new signals correlates with the number of PEGylation sites Problem: New signals not observed in HPLC peptide mapping WVL:214 nm PEGylated protein min Lys-C peptide map (HPLC) of a PEGylated (top trace) and non-pegylated protein (bottom trace) non-pegylated protein 1

4 Feasibility 1 PEGylation Peptide map of a PEGylated protein by CZE p3 p6 p1 p9 p7 p5 p6 px p4 p4 Non-PEGylated protein 2 p4 p4 PEGylated peptides p7/1/5 p3 p9 p6 p6 PEGylated Protein Da > Da p6, p7 = glycosylated, non-glycosylated 4

5 Feasibility 1 PEGylation Study heterogeneity on PEGylated peptides p6/p2 dg p6 p4 dg p6 ds p4 ds deglycosylated p2 ds desialylated p6 p6 p4 p4 p2/3 p1 p3/4 Native PEGylated 5

6 Feasibility 1 PEGylation Summary Summary The separation of both PEGylated and non-pegylated peptides can be achieved within one CE run ranging from to > Da. PEGylation site localization possible. Detection of PEGylated peptide allows additional characterization of glycans and sialylation on those peptides. Estimation of the overall PEGylation based on the A% of PEGylated signals to non- PEGylated signals. Outlook Distinguish between PEGylation site on the same peptide. Further investigation will be needed in order to precise the individual PEGylation sites/degree. 6

7 Feasibility 2 Deamidation Current methods Other charge variants Why does deamidation occur: One of the degradation pathway of biopharmaceuticals. Occurs under thermal and ph stress. Changes mass only minimally and charge by 1, but unspecifically. Can significantly affect structure and function of the protein. Analytical problem: Determine the deamidation degree site specifically? Current methods: ISOQUANT detection kit: Quantification of isoaspartate on protein level. isoasp specific. Peptide mapping: relative and site specific quantification of deamidation Prone to sample preparation artifacts due to digestion at neutral or basic ph. Charge based methods: quantification of deamidation on protein level. Charge variants not related to deamidation might interfere. 7

8 Feasibility 2 Deamidation Specific digest approach (Asp-N) Asp-N cleave before Asp and sometimes before Glu. Deamidation on an Asn generates an new cleavage site -> two new peptides Asp-N does not cleave at isoasp sites so the deamidated peptide is still present. Asp-N also cleaves under acidic conditions allowing to minimize sample preparation artifacts due to digestion at neutral or basic ph. 8

9 Feasibility 2 Deamidation Specific digest (Asp-N) p3 p2 p1 p4 high ph stress low ph stress unstressed p2 p1 RT, 4w, 80 C, 3d 80 C, 5d p1 p2 p3 p4 Significant degradation of p1 and p2. Detection of peptides expected to occur after Asn deamidation. Minor deamidation under high ph or low ph conditions at RT within 6d. 9

10 Feasibility 2 Deamidation Outlook Summary Asp-N can be used as a deamidation specific digest. Peptides specific for each deamidation site are created. Can distinguish between closely positioned deamidation sites. Deamidation specific peptides increase when stressed. Based on the deamidation specific signals either an overall or a site-specific deamidation degree can be calculated. Outlook Study more complex analytes. Develop a combined method to study Asn deamidation and Asp isomerization. Establish workflow with Glu-C to monitor glutamine deamidation. 10

11 Feasibility 3 Glycation Boronate affinity chromatography (BAC) What is glycation: Non-enzymatic glycosylation on protein amine groups. Occurs when protein is incubated with reducing sugars (e.g. glucose, galactose, fructose). Can further degrade to advanced glycation end product (AGE), which are highly reactive and toxic to some cell types. Consequently, glycation is a critical quality attribute. It may also happened during storage when reducing sugars are present in the formulation buffer. Boronate affinity chromatography (BAC): Non-specific interaction have to be minimized by a shielding reagent. Problem: Method has to be fine tune for each new analyte. Retained peak is not pure The retained peak purity has to be assessed by an orthogonal method. Alternatively methods: charge based separation and LC-MS. 11

12 Feasibility 3 Glycation CE Approach-Principle + H + Boric acid interactions with vicinal diols of sugar: BGE ph > 8 (strong interaction, if ph > pi protein, negatively charged proteins 12

13 Feasibility 3 mobility (µ e = [m 2 /Vs] ) Width at 50% Glycation Results mab 1 (Glucose) d0 mab1-glucose Characteristic for individual protein 0.3 d Glycation degree Days saturation Days Impact on CE profile: Width of the peak correlates with the width of the glycation distribution. Reactivity (reaction enthalpy) and glycation rate Number of potential glycation sites not sterically hindered 13

14 Feasibility 3 Glycation Results mab2-glucose mab2-fructose Day 0 Day 1 d0 Glycation degree mab1-glucose mab1-glucose Day 4 d8 Day 8 Changed µ eff and profile of protein signal upon glycation stress Different behavior between glucose and fructose due to different dissociation constant. Up to 14 hexoses per mab molecule detected by LC-MS upon stress. 14

15 Feasibility 3 Glycation Conclusion Summary: Transferred BAC from a HPLC setup to a CE setup. Separation of glycated peak proportionally to the degree of stress. Separated two different molecules with the same method. Separated the same molecule with glycated with two different sugars. Outlook: Focus on separation on only minimally glycated biopharmaceuticals. Assess the separation of different sugars. Boronate CE on a digested sample be it subunits or peptides. 15

16 Conclusion It doesn t always have to be gel CE or cief. CZE allows to monitor very small and very large peptides simultaneously. AspN is an interesting approach identify site specific deamidation sites. Boronate affinity chromatography can adapted to CE settings. 16

17 Thank you Thanks to the Solvias CE Team especially Maria Schwarz, Dora Boylan, Claudia Michael, Cristina Montealegre and Angelina Rafai. Thanks to Christian Neusüss and Oliver Höcker for the CE-MS measurements. Specials thanks to head of Biopharma Happy to serve you! Dr. Alex Beck Thank you for your attention! 17

18 Solvias AG Römerpark 2, 4303 Kaiseraugst, Switzerland Samuel Bader,