Biopharmaceuticals - Current FDA & EMAs Regulations on glycan analysis

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1 Biopharmaceuticals - Current FDA & EMAs Regulations on glycan analysis Jayesh Kattla, PhD March 2015 Gothenburg & Copenhagen 2013 Waters Corporation 1

2 Hope and Risk 2013 Waters Corporation 2

3 The total number of genes does not define complexity 45,000 50,000 Number of Genes 13,600 24,000 4,28 8 6,340 E. coli Yeast Fruit fly Human Rice Corn 2013 Waters Corporation 3

4 Post translational modifications explains the complexitity of protein function 2013 Waters Corporation 4

5 Glycan Biosynthesis is a complex process Complext process No genetic code for glycosylation Glycan sysntiesis occurs in ER and Golgi Main types are N linked and O Linked glycans All proteins need not go thru the whole pathway makes the glycans highly Heterogeneous Macro heterogeniety Micro heterogeniety Size of the glycans can vary Glycan are involved in protein fuction in terms of binding, safety, Efficacy, immune response, half life, 2013 Waters Corporation 5

6 EPO glycosylation 2013 Waters Corporation 6

7 Glycan micro heterogeneity affects protein function Desialylation of IVIg abrogates antiinflammatory properties in K/N mice Kaneko et al (2006). Science; 313(5787): Sialylation increases the half life of protein Involved with placental transport of IgG IgG galactosylation increased in pregnant women Kibe et al (1996). J Clin Biochem Nutr; 21(1): Asn Asn IgG G0 interacts with MBL to activate complement Malhotra et al (1995). Nat Medicine; 1(3): Asn Loss of core α(1,6) fucose on IgG results in enhanced ADCC activity Okazaki et al (2004). J Mol Biol; 336(5): Asn Highly mannosylated proteins can be cleared out the system within minutes by mannose binding lectin 2013 Waters Corporation 7

8 Glycosylation Functions: Risks and Regulatory Concerns Mediates biological activity Glycans impact safety and efficacy Correct and consistent structure of the glycans Obtaining the desired medical effect Avoid adverse immunological reaction Alteration in glycans may eliminate or alter activity Immune response triggered by unrecognized glycans Consistent glycan distribution indicates process stability Structure based on PDB file 1H3Y from Krapp S et al. J Mol Biol, 2003, 325(5): Waters Corporation 8

9 Biopharmaceuticals $163 billion dollar market 20% are biopharmaceuticals 8% Annual Growth 50% of top drugs are biomolecules Was 28% in 2008 Biopharmaceuticals are more complex in structures 2013 Waters Corporation 9

10 The Biologics Market Blood Proteins Blood Proteins Hormones Hormones Growth Factors Growth Factors Cytokines Cytokines Vaccines Monoclonal Antibodies Vaccines Bone Proteins Other mabs Bone Proteins 190 EMA/FDA Approved Walsh (2010). Nature Biotech; 28(9): Waters Corporation 10

11 The Biologics Market Blood Proteins Blood Proteins Hormones Hormones Growth Factors Growth Factors Cytokines Cytokines Vaccines Monoclonal Antibodies Vaccines Bone Proteins Other mabs Bone Proteins 127 are Glycoproteins (>66%) Walsh (2010). Nature Biotech; 28(9): Waters Corporation 11

12 Biopharma Attempts to Replicate Human Glycosylation Struwe WB, Cosgrave EFJ, and Rudd PM. (2011). Glycoproteomics in Health and Disease. Functional and Structural Proteomics of Glycoproteins Waters Corporation 12

13 First Plant made antibody 2013 Waters Corporation 13

14 Negative Attributes to Mammalian Cell Culture Glycosylation Asn Asn 50% of non-allergic blood donors contain antibodies against β(1,2)-xylose and α(1,3)-core fucose Bardor et al (1995). Glycobiology; 13(6): Asn N-glycolylneuraminic acid is an oncofetal antigen in humans Muchmore et al (1989). J Biol Chem; 264(34): Asn Presence of gal-α(1,3)-gal can induce anaphylaxis Chung et al (2006). N Engl J Med; 358(11): Waters Corporation 14

15 Genetically engineered expression system /QBD The concept promotes industry's understanding of the product and manufacturing process starting with product development, basically building quality in, not testing it. Under the concept of QbD, when designing and developing a product, a company needs to define desired product performance and identify CQAs Waters Corporation 15

16 Bioprocessing Conditions Can Influence Glycosylation Cell Line Critical in affecting glycosylation (Raju et al 2000) Dissolved O 2 Variable effect, cell line specific (Restelli et al 2006) Ammonia High conc n s affect terminal glycosylation (Yang and Butler 2000, 2002) Temperature Low temps (30 C) decrease sialylation (Trummer et al 2006) ph Galactosylation, sialylation microheterogeneity vary (Muthing et al 2003) Manufacturing Mode Perfusion increases sialylation over fed-batch (Lipscomb et al 2005) 2013 Waters Corporation 16

17 Analysis of critical quality attributes during the manufacturing process Process analytical technology (PAT) has been defined by the United States Food and Drug Administration (FDA) as a mechanism to design, analyze, and control pharmaceutical manufacturing processes through the measurement of Critical Process Parameters (CPP) which affect Critical Quality Attributes (CQA). It is absolutely essential to tightly control the levels of glycan critical quality attributes during the manufacturing process. Eg your Desired glycoforms that is involved in activitating ADCC Maintain the levels of immunogenenic epitopes Mannose containing glycans and so on 2013 Waters Corporation 17

18 Changes in Bioprocess Can Affect Glycosylation 2013 Waters Corporation 18

19 Regulatory Agencies Are Demanding More Detailed Glycan Analysis 2013 Waters Corporation 19

20 FDA Drug application pathway Developmental discussion IND BLA 2013 Waters Corporation 20

21 keith webber, FDA 2013 Waters Corporation 21

22 keith webber, FDA 2013 Waters Corporation 22

23 keith webber, FDA 2013 Waters Corporation 23

24 keith webber, FDA 2013 Waters Corporation 24

25 keith webber, FDA 2013 Waters Corporation 25

26 keith webber, FDA 2013 Waters Corporation 26

27 keith webber, FDA 2013 Waters Corporation 27

28 Micro & Macro Heterogeniety keith webber, FDA 2013 Waters Corporation 28

29 keith webber, FDA 2013 Waters Corporation 29

30 keith webber, FDA 2013 Waters Corporation 30

31 Summary Biopharmaceuticals are complex molecules Glycan synthesis is a complex process Glycosylation plays a critical role in protein function Glycan complexity increases due to its micro and macro heterogeneity Glycans are in involved in safety and efficacy of the protein Company are genetically engg several expression systems to produce proteins with more human like glycosylation Glycosylation is an important aspect of QbD Glycosylation important in PAT Companies are required to do indepth Glycosylation analysis before regulatory submission 2013 Waters Corporation 31