SWEDISH EXCELLENCE IN NANOPORUS SILICA

CONTENT

Nanologica’s Vision 3

NLAB Saga™ 4

About NLAB Saga™ 5

NLAB Saga™ Physical Properties 6

Chemical Stability 7

Mechanical Stability 8

Example of Insulin Purification 10

3Nanologica |

NANOLOGICA’S VISION

“Better and cheaper medicine through porous silica”

4 | Nanologica

NLAB SAGA™• Perfectly spherical, fully porous silica• Tightly controlled particle size • High purity silica - very low metal content• High carbon content• Exceptional chemical stability at high and low pH• High loading

5Nanologica |

ABOUT NLAB SAGA™Nanologica’s core competency lies in developing nanoporous silica. NLAB Saga™ hasbeen specifically developed to meet the expectations of industrial scale purificationby chromatography. An optimized combination of pore volume, high surface area andhigh carbon load results in a robust and reliable product with stellar performance.

Nanologica has full control of the entire manufacturing process, from raw material tofinished product. NLAB Saga™ has been tested and used by pharmaceuticalcompanies worldwide manufacturing APIs at industrial scale where quality,performance and durability are uncompromisable.

Excellent productcombined withtechnical support

Proprietarymanufacturing ofhigh purity silica

Controlled particle size and particle size distribution

Bonded phases usingproprietary methods

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NLAB SAGA™ PHYSICAL PROPERTIES

• Surface area ~ 300 m2/g

• Pore volume ~ 0.85 ml/g

• Pore size ~ 110 Å

• d90/d10 ≤1.7

• Low metal content

• Carbon load

o C18 19%

o C8 11%

High surface area

Pore volume 0.85 ml/g

Controlled particle sizeand distribution

High loading capacity

High mechanical stability

Easy to pack and lowbackpressure

The large surface area and high pore volume combined with controlled particle sizeand distribution results in a silica with high loading capacity, high chemical stabilityand low backpressure, that is easy to pack. NLAB Saga™ is available in a variety ofparticles sizes from 10 to 15 µm.

7Nanologica |

CHEMICAL STABILITYIn large scale purification of proteins and peptides there will be depositions in the packingmaterial. To avoid time-consuming packing and unpacking of the columns it is important torun cleaning in process. These cleaning steps are usually performed at a very high pH. Suchconditions are harsh for the silica, therefore it is of great importance that the silica surviveunder these conditions in order to have a long-lasting product.

Test conditions:

• Wash: 95/5 MeOH/H2O, 10 CV

• Eluent: 60/40 MeOH/100mM NaOH, pH 13.0, 12 CV

• Temperature: Ambient cycle 1-30. From cycle 30 and onwards 50oC

• Neutralization: 90/10 MeOH/1%AcOH, 10CV

• Flow rate: 1ml/min

• Chromatographic evaluation

Illustration of one cycle

Alkaline regeneration test designed to mimic wash cycles

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Comparison of NLAB Saga™ 13 µm C8, Competitor A 13 µm C8 and Competitor B 10 µm C8in terms of alkaline stability shows that NLAB Saga™ and Competitor A performsconsistently well and withstand the harsh experimental conditions, while Competitor B isfalling behind.

Chromatographic test conditions:

Mobile phase: 80/20 MeOH/25 mM K-phosphate, pH 7.0

Flow rate: 1 ml/min

UV: 210 nm

Temperature: 30oC, after 30 cycles the temperature was raised to 50oC to speed up the experiment

Analytes: Uracil, Toluene

0

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0 10 20 30 40

% o

f in

itia

l eff

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ncy

, to

luen

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Cycle number

NLAB Saga™Competitor ACompetitor B

Columns break

NLAB Saga™Competitor ACompetitor B

Agilent 1100 system used for chromatographic evaluations and Schimadzu LC-20AD stand alone pump was used for

regeneration simulation. The tests were performed at Nanologica’s lab in Södertälje, Sweden.

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MECHANICAL STABILITYNLAB Saga™ has a high mechanical stability. Backpressure stays the same meaningthere is no mechanical degradation of the silica. This is also shown by sizedistribution staying the same for native silica and used silica.

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rop

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ar)

Packing number

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Vo

lum

e %

Particle diameter (µm)

native silica

middlefrit

A DAC column with 5 cm internal diameter was used. 5 packings/unpackings to 100 bars weremade with the same silica slurry.

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EXAMPLE OF INSULIN PURIFICATIONThe target for the experiment was to reach the purity threshold of 99.2% as set per theUSP. Insulin purity was measured before and after one step of reversed-phase purification.

3,0 7,5 10,0 12,5 15,0 17,5 20,0 22,5 25,0 27,5 30,0 32,5 35,0 37,5 40,0 42,8

-29

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116INS NANO #13 [modified by gxp-pem] Human,100mg, IEC, # 3-6 UV_VIS_1mAU

min

1 -

8,6

47

2 -

11,3

07

3 -

12,3

55

4 -

13,9

13

5 -

17,7

88

6 -

22,0

67

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22,6

83

8 -

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63

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23,5

75

10 -

24,1

65

11 -

24,5

57

12 -

26,0

12

13 -

34,5

68

WVL:215 nm

Insulin purity measured to 87.9% BEFORE reversed-phase purification

Insulin purity: 87.9%

USP analytical method conditions

Flow rate: 1.2 ml/min

UV: 214 nm

Temperature: 40oC

Eluent: A: 200mM Na2SO4/H3PO4 (pH=2.3)

B: Acetonitrile/ 200mM Na2SO4/H3PO4 (pH=2.3) 45/55

Gradient: 0-30 min / 57%B; 30-44 min /57-89%B; 44-50 min / 89%B

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0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 106

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1 - INS.NAN,PREP #15 [modified by gxp-pem, normalized] RPC1, (efter IEC), 200mM NH4Ac, pH 4.3, 24-29 % / 60 min., Human, 250 mm, Nano UV_VIS_1

2 - INS.NAN,PREP #16 [modified by gxp-pem] RPC1, (efter IEC), 200mM NH4Ac, pH 4.3, 24-29 % / 60 min., Human, 250 mm, 15 um,YMC- triart UV_VIS_1mAU

min

21

1 -

71,7

53

2 -

72,9

27

3 -

81,4

47

WVL:280 nm

Inhbit =

On

Mass overload -the exact same fraction from the insulin purification for both Competitor A and NLAB Saga™

Blue: Competitor ABlack: NLAB SagaTM

Process parameters which were kept constant:

• Loading: 0.37 g/cm2 column crossectional area (15 g/liter)

• Yield: 90 % ± 1 % in all steps

• Slope of the gradient: (0.083 % / minute, pH= 4.3)

• Linear flow rate: 180 cm/h

• Organic solvent: Acetonitrile

• Feed: Insulin with 87.9 % purity

Experimental set upThe mass overload experiments were performed under the same conditions for NLAB Saga™ and the competitor.

Product pool

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NLAB Saga™ surpasses the set USP target by reaching a purity of 99.5% after one step ofreversed-phase purification. The Competitors A, B and C do not reach the USP target,meaning they would need one more step of purification to pass the USP threshold.

Thus, NLAB Saga™ needs fewer steps to reach the USP purity target leading to a lowermanufacturing cost for the insulin manufacturer.

2,8 4,0 5,0 6,0 7,0 8,0 9,0 10,0 11,0 12,0 13,0 14,0 15,0 16,0 17,0 18,0 19,0 20,0 21,0 22,0 23,0 24,0 25,0 26,0 27,0 28,0 29,0 30,0 31,0 32,6

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1 - INS NANO #59 [modified by gxp-pem] YMC Triart, USP UV_VIS_1

2 - INS NANO #61 [modified by gxp-pem, normalized] Nano, USP UV_VIS_1mAU

min

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1

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03

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6,2

30

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58

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7,8

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WVL:214 nm

Insulin purity measured to 99.5% AFTER one step of reversed-phase purification

Blue: NLAB SagaTM

Black: Competitor A

Company PhaseUSP method

Purity (%)Backpressure *

(Bar)

Nanologica NLAB Saga™ C8 99.50 15.5

Competitor A C8 97.08 10.2

Competitor B C8 98.56 19.8

Competitor C C8 98.82 21.5

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EXAMPLE OF LIRAGLUTIDE PURIFICATION

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DAC 50X250mm, packed with NLAB Saga™, 13 µm, C8

Samples Purity (%) Max. Impurity (%) Loading (g) Yield (%)

Crude 85.54 2.32

1st purify 95.6 1.45 2 88

2nd purify 99.7 0.11 1 80

3rd purify 99.91 0.05 1.1 82

Data kindly provided by end customer

A DAC 50X250mm, packed with NLAB Saga™ 13 µm C8 was used for the purification ofliraglutide.

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CONTACT DETAILS AND

ORDER INFORMATION

Nanologica AB

Forskargatan 20 G

SE-151 36 Södertälje

Sweden

+46 8 410 749 49

info@nanologica.com

www.nanologica.com