©2013 Waters Corporation 1

Jayesh Kattla, PhD March 2015

Gothenburg & Copenhagen

Biopharmaceuticals - Current FDA & EMAs Regulations on glycan analysis

©2013 Waters Corporation 2

Hope and Risk

©2013 Waters Corporation 3

4,288

6,340

13,600

24,000

45,000 50,000

E. coli Yeast Fruit fly Human Rice Corn

Num

ber

of G

enes

The total number of genes does not define complexity

©2013 Waters Corporation 4

Post translational modifications explains the complexitity of protein function

©2013 Waters Corporation 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 6

EPO glycosylation

©2013 Waters Corporation 7

Glycan micro heterogeneity affects protein function

Asn Desialylation of IVIg abrogates anti-inflammatory properties in K/N mice Kaneko et al (2006). Science; 313(5787): 670-673

Involved with placental transport of IgG IgG galactosylation increased in pregnant women Kibe et al (1996). J Clin Biochem Nutr; 21(1): 57-63

Asn

IgG G0 interacts with MBL to activate complement Malhotra et al (1995). Nat Medicine; 1(3): 237-243

Asn

Loss of core α(1,6) fucose on IgG results in enhanced ADCC activity Okazaki et al (2004). J Mol Biol; 336(5): 1239-1249

Asn

Sialylation increases the half life of protein

Highly mannosylated proteins can be cleared out the system within minutes by mannose binding lectin

©2013 Waters Corporation 8

Glycosylation Functions: Risks and Regulatory Concerns

Structure based on PDB file 1H3Y from Krapp S et al. J Mol Biol, 2003, 325(5): 979-989

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

©2013 Waters Corporation 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 10

The Biologics Market

Blood Proteins

Hormones

Growth Factors

Cytokines

Vaccines

mabs

Bone Proteins

Blood Proteins

Hormones

Growth Factors

Cytokines

Vaccines

Monoclonal Antibodies

Bone Proteins

Other

190 EMA/FDA Approved

Walsh (2010). Nature Biotech; 28(9):917-924

©2013 Waters Corporation 11

The Biologics Market

Blood Proteins

Hormones

Growth Factors

Cytokines

Vaccines

mabs

Bone Proteins

Blood Proteins

Hormones

Growth Factors

Cytokines

Vaccines

Monoclonal Antibodies

Bone Proteins

Other

127 are Glycoproteins (>66%)

Walsh (2010). Nature Biotech; 28(9):917-924

©2013 Waters Corporation 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.

©2013 Waters Corporation 13

First Plant made antibody

©2013 Waters Corporation 14

Negative Attributes to Mammalian Cell Culture Glycosylation

Asn

Asn

Asn

Asn Presence of gal-α(1,3)-gal can induce anaphylaxis Chung et al (2006). N Engl J Med; 358(11): 1109-1117

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): 427-434

N-glycolylneuraminic acid is an oncofetal antigen in humans Muchmore et al (1989). J Biol Chem; 264(34): 20216-20223

©2013 Waters Corporation 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.

©2013 Waters Corporation 16

Bioprocessing Conditions Can Influence Glycosylation

Cell Line Critical in affecting

glycosylation (Raju et al 2000)

Manufacturing Mode Perfusion increases sialylation

over fed-batch (Lipscomb et al 2005)

Dissolved O2 Variable effect, cell line specific

(Restelli et al 2006)

Temperature Low temps (30°C) decrease sialylation

(Trummer et al 2006)

Ammonia High concns affect

terminal glycosylation (Yang and Butler 2000, 2002)

pH Galactosylation, sialylation microheterogeneity vary

(Muthing et al 2003)

©2013 Waters Corporation 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 18

Changes in Bioprocess Can Affect Glycosylation

©2013 Waters Corporation 19

Regulatory Agencies Are Demanding More Detailed Glycan Analysis

©2013 Waters Corporation 20

FDA Drug application pathway

IND BLA Developmental discussion

©2013 Waters Corporation 21 keith webber, FDA

©2013 Waters Corporation 22 keith webber, FDA

©2013 Waters Corporation 23 keith webber, FDA

©2013 Waters Corporation 24 keith webber, FDA

©2013 Waters Corporation 25 keith webber, FDA

©2013 Waters Corporation 26 keith webber, FDA

©2013 Waters Corporation 27 keith webber, FDA

©2013 Waters Corporation 28

Micro & Macro Heterogeniety

keith webber, FDA

©2013 Waters Corporation 29 keith webber, FDA

©2013 Waters Corporation 30 keith webber, FDA

©2013 Waters Corporation 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