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Glycan Analysis During Biosimilar Development

HILIC methodology is an important tool during biosimilar development.  It has previously been described that glycosylation is not only a CQA in terms of protein biopharmaceutical safety and efficacy, but it is one of the most variable.  This variability is still experienced when the exact same cell line is used as shown below.

Herceptin Glycoprofiles from 4 Different Production Batches

Figure 1: Herceptin glycoprofiles from 4 different production batches

Glycan

Relative Intensity (%)

Batch 1

Batch 2

Batch 3

Batch 4

G0-GlcNAC

0.63

0.79

0.86

0.78

G0F-GlcNAc

0.63

2.09

2.65

2.14

G0

4.70

4.43

4.02

4.24

G1-GlcNAc

0.80

1.55

1.05

0.87

G0F

35.19

44.72

49.67

48.00

Man5

2.41

1.76

1.77

1.64

G1a + G1F-GlcNAc

2.37

3.03

2.60

2.45

G1b

0.97

0.69

0.51

0.58

G1Fa

32.32

26.74

24.34

25.82

G1Fb

10.41

9.01

8.36

8.74

G1F

42.72

35.75

32.70

34.56

G2F

9.56

5.19

4.17

4.74

Table 1: Glycan relative intensities from 4 different production batches

It has also been discussed that the cell line is specific to the glycosylation profile and the cell line used in expression of the originator will be available to manufacturer the biosimilar or clone.  This is demonstrated in Figure 1 where markedly differing glycoprofiles are generated for the trastuzumab clone in development and the originator Herceptin.  Whilst a similar profile over all is observed, there is a clear undergalactosylation produced by the clone cell line.

Herceptin Glycoprofile vs. a Biosimilar in Development

Figure 2: Herceptin glycoprofile vs. a biosimilar in development

In order to address the (under)galactosylation of the clone, differing amounts of activators and substrate (uridine, galactose, and manganese chloride) have been added and the resulting chromatograms shown in Figure 2 with the associated relative intensities in Table1.

Figure 17 – Biosimilar Glycoprofile during Cell Culture Optimization

Figure 3: Biosimilar glycoprofile during cell culture optimization

Glycan

Relative Intensity (%)

Clone

Clone 4x

Clone 8x

Clone 16x

Clone 24x

Originator specifications

% G0-GlcNAC

0.55

0.49

0.47

0.53

0.61

0.77 +/- 0.09

% G0F-GlcNAc

5.35

2.72

2.15

1.78

1.67

1.88 +/- 0.87

% G0

1.57

2.67

1.98

2.35

2.02

4.35 +/- 0.29

% G1-GlcNAc

0.39

0.32

0.35

0.45

0.51

1.07 +/- 0.34

% G0F

70.80

48.91

46.39

44.19

43.72

44.39 +/-6.47

% Man5

8.22

5.79

4.95

6.21

7.14

1.89 +/- 0.35

% G1a + %
G1F-GlcNAc

0.55

1.35

1.34

1.51

1.44

2.61 +/- 0.30

% G1b

0.17

0.32

0.29

0.37

0.38

0.69 +/- 0.20

% G1Fa

7.76

22.66

25.58

25.83

25.91

27.30 +/- 3.48

% G1Fb

3.62

8.11

8.70

8.75

8.62

9.13 +/- 0.89

% G1F

11.38

30.77

34.28

34.57

34.53

36.44 +/- 4.38

% G2F

1.01

6.66

7.80

8.03

7.98

5.92 +/- 2.47

Table 2: Glycan relative intensities during cell culture optimization

The main problematical glycans have been highlighted, and it can be seen that after increasing the amount of substrate and activators added by a factor of 8 or more brings the resulting glycans into line and specification with the originator biopharmaceutical.
[2] G.R. Guile, P.M. Rudd, D.R. Wing, S.B. Prime, R.A. Dwek, Anal. Biochem. 240, 210-226 (1996)

 
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