Bio-Rad Q Media User Manual

................................................................................................................................

Nuvia™ S Ion Exchange

Instruction

Catalog

and Q High-Capacity

Media

Manual

numbers 156-0311 156-0411 156-0313 156-0413 156-0315 156-0415 156-0317 156-0417

Please read these instructions before you use Nuvia S and Q High-capacity ion exchange media. If you have any questions or comments regarding these instructions, please contact your Bio-Rad Laboratories representative.

Table of Contents

Section

.

1:

Introduction 1

2 :

Technical Description 1

3 :

Preparation 2

4 :

Column

5 :

Column Packing Evaluation 3

6 :

Operation

7 :

Regeneration

8 :

Cleaning-in-Place

9 :

Storage 8

10:

Regulatory Support

11: Ordering

Packing

and

Maintenance

and

Sanitation 7

(CIP) and

Information 8

5

Sanitation 8

2

8

Section

Nuvia™ S and Q

Introduction

1:

high-capacity

ion

exchange binding capacity chromatography media puriﬁcation process from capture to properties, biomolecule process scale. nucleic acids,

high productivity, and

puriﬁcation

Nuvia

at both

media are

viruses, plasmids, offer high binding capacity at fast backpressure. The unique properties highly effective

If you

have questions

media for biomolecule puriﬁcation.

about

Nuvia media, Rad process chromatography chromatography

Technical

Support department for

ﬁnal polish. Their

exceptional selectivity

laboratory scale

designed

and other

linear

ﬂow

of

sales representative

1-510-741-6563.

:

Section

Technical

2

Description

media are new

designed

for any stage in the

robust

meet the needs for

and

for the

biopharmaceutical

separation

macromolecules. Nuvia

rates

while

Nuvia

media position them as

contact

maintaining

either

your local Bio-

or the Bio-Rad

further assistance

ultra high

physical

of

proteins,

media

low

at

Table 1.

Nuvia S Nuvia Q

Type of ion exchanger Strong cation Strong anion Functional

Total ionic capacity 90–150 µeq/ml 100-170 µeq/ml Dynamic binding capacity* 300

Shipping counterion Na Median Recommended linear Chemical

1.0 N

0.1 N

0.01 N Gel bed

pH stability** 2–14 short term 2–14 short term

Shipping solution 20% Regeneration 1–2 M NaCl 1–2 M NaCl Sanitation 0.5–1.0 N NaOH 0.5–1.0 N NaOH Storage conditions 20%

* 10% NaCl, **Data

NOTE: Recommended elution ﬂow rates are <150 cm/hr to minimize viscosity induced backpressure.

Characteristics

group –SO

particle

size 85 ± 15 µm 85 ± 15 µm

stability NaOH NaOH

NaOH

compression

breakthrough

pH 5.0 for Nuvia S and 5 mg/ml BSA in 20 mM Tris-HCI pH 8.5 for Nuvia Q.

derived

ﬂow rate range 50–600 cm/hr 50–600 cm/hr

(20°C) up to 1 week up to 1 week

(20°C)** up to 5

(20°C)** — up to 5 yr

ratio 1.15–1.18

capacity

under

accelerated

of Nuvia™ media.

-

3

cm/hr

+ Cl-

volume/packed

4–13 long term 4–12 long term

ethanol

determined

with 4.5 mg/ml

conditions at 60°C.

–N(CH3)

>110 mg/ml

yr —

(settled

bed 1.10–1.15

bed volume)

+ 0.1 M NaCl 20%

or 0.1 N NaOH 20%

human

300 cm/hr >170 mg/ml

IgG in 40 mM

+

3

ethanol

NaOAc

+ 1 M NaCl

or 0.01 N NaOH

+ 30 mM

1

Section

Nuvia™ media are a 50% (v/v) is r

ecommended. Small volumes

Büchner funnel cycling

through 3–4

in the

shipping container

Removal particle size range. resuspend sedimentation

of

Section

Nuvia™ media can be packed using pressure, volumetric ﬂow, or vacuum packing methods. To pack operation,

Packing This

slurry

media

in a All buffers slurry

is

required,

1.

Prepare degassed

referred

2.

Nuvia suspended to settle. Decant the shipping

3. Add 50 ml

4.

Seal

the mix with a of

slurry

moderate

5. Add 10 ml packing buffer to the column. Pour in 75 ml

6.

Insert

the column ﬂow adaptor and ﬂow pack at a 300–600 cm/hr with packing buffer for at compressed bed compress the bed 0.1–1.0

7. Attach the column to your chromatography system, and purge the column with starting buffer at bed

compresses,

: Preparation

3

supplied fully hydrated

slurry.

For column packing,

with 4–5

ﬁnes

the

sediment

is complete.

4

it is

recommended

Small packing method was

conventional

should be

to

herein

media are shipped as a 50%

slurry

degassed

cylinder

magnetic

may be mixed with a

speed.

volumes

settling

is recommended.

from

Nuvia

If

ﬁnes have

and

:

Column

Columns

column with an

degassed. Because a relatively large volume

it is

recommended

1.0 M

as the packing buffer.

into a 100 ml

and rotate it to

stir bar as damage may occur.

height,

repeat steps 6

of

Nuvia

of packing buffer. For

and

decanting

media is not

been

generated

remove

Repeat several

the opaque supernatant before

Packing

that a 20–50%

designed

that a packing reservoir be used.

NaCl,

20–50 mM buffer salt

graduated cylinder.

solution

packing buffer to the resin.

suspend

low-shear marine impeller

stop the ﬂow, and adjust the ﬂow adaptor to

cm.

linear velocities

and

in 20%

removal

required

columns

internal diameter

slurry. Measure

7.

ethanol + 1 M NaCI

of the

media are

steps using the packing buffer

times.

slurry volume

to pack 25 ml of

away from the

the resin.

least

shipping

easily

large

due to the narrow

during handling,

for highly

50 ml of Allow the

Caution: Larger

linear velocity

10 min. Note the

up to 600 cm/hr. If

buffer

washed in a

volumes,

efﬁcient

be used.

Nuvia

of 5–15 mm.

(see Table

resin

bed.

amounts

at low to

resin

as

of

2)

resin

Do not

slurry.

of

the

bed

2

Packing After

(v/v) with packing buffer recommendations slurry mixing of

1.20 and 1.10 –1.15 respectively height. ﬂow condition the column with fresh packing or 3 column process ﬂow rate. After ﬂow column procedure described in Section 5.

Section

When column packing is complete, 5 CV inject a example, acetone or 1 M a UV have a a conductivity should be 100–200 mM. column velocity used to load and/or

To obtain values theoretical velocities

HETP = L/N N =

L = Bed height (cm) N = Number of Ve =

W Ve and W

Peaks should be possible

Peak asymmetry factor calculation:

Process-Scale

removing

through the packing pump. Use a low-shear

Nuvia

equilibration

absorbance

among columns, the same conditions must be applied. Minimum

5.54(Ve/W½h)

Peak elution volume

=

½h

the storage buffer

or a plastic paddle for

S and Nuvia Q will be obtained with a compression ratio of 1.15–

After the

efﬁciency

sample

salt concentration

volume.

Peak

desired compression

volumes

using your standard

:

5

of a low

monitor),

Column testing should be operated using the same

comparable

plate

values

of 50–500

theoretical

width at peak

should

½h

to 1.

symmetrical

Values

Columns

(see Table

with one major

(CV) in upﬂow followed by 3 CV in downﬂow at the

(Section

manual mixing.

deﬁned

conditioning,

Column Packing

buffer. To test the

molecular

NaCl).

monitor set at 280

then the

height

should be 1,000–3,000 plates/m for linear

cm/hr

2

always

of 0.8–1.8 are acceptable.

If acetone is used as the test

<100 mM. If 1 M

equilibration

The sample volume

elute

equivalent

.

plates

or time

half

height in

be in the same units

and the

3), prepare a 20–50%

2). Follow the column

exception:

do not

The best

as settled bed height /packed bed

is

achieved,

equilibration

it is

recommended

operating procedures

manufacturer’s

recirculate

impeller

overall performance

it is

recommended

Evaluation

equilibrate

effectiveness

weight, unretained compound

nm),

the sample.

asymmetry

the column with up to

of column packing,

the

equilibration

NaCl

is the test

buffer salt

should be 1–3% of the total

to a

theoretical

volume

or time

factor as close as

buffer must

concentration

plate

slurry

the

for automatic

marker (use

Nuvia

buffer for

to

or the

marker (use

(HETP)

to

evaluate

(for

linear

As =

b/a a = Front section of peak width at 10% of peak b = Latter section of peak width at 10% of peak height bisected by line denoting As = 0.8–1.8 is acceptable.

3

height

bisected by line denoting

V

e

V

e

:

Pressure-Flow Characteristics

0

0.5

1

1.5

2

2.5

3

0.0 100.0 200.0 300.0 400.0 500.0 600.0 700.0

Linear ﬂow, cm/hr

Pressure Bar

Water 10% glycerol 30% glycerol

Section

Nuvia™ media are drug per be run at the

operational

Operation and

6

designed

hour per

to

achieve

liter

of

highest linear velocities

the

media) possible. Nuvia

and the column and the chromatography system. A hr and a 20 cm bed is a optimized by buffer,

changing

modifying

recommended

the pH, ﬂow rate,

the

gradient proﬁle,

or

experimenting

Maintenance

highest productivity (grams

loading capacities allowed

linear

starting point.

or

ionic strength of the elution

media should

ﬂow rate of 300 cm/

Puriﬁcation

with

different

salts.

Note: Due to the high binding capacity of Nuvia media, it

is recommended to elute the biomolecule at ﬂow rates <150 cm/hr to minimize

Figure

viscosity

induced backpressure.

1 shows the effect of ﬂow rate on backpressure.

of

by

may be

buffer

Fig. 1. Nuvia S media

bed

height column packed

4

pressure/ﬂow performance

to

compr

ession factor of 1.17

for a 20 cm

.

diameter

x 20 cm

Figure

Effect of Conductivity on Nuvia S Binding Capacity

0

20

40

60

80

100

120

140

160

0 2 4 6 8 10

Conductivity, ms/cm

Binding capacity @ 10% BT, mg/ml

human IgG BC @ 10% BT

Conalbumin BC @ 10% BT

Effect of Conductivity on Nuvia Q Binding Capacity

0

20

40

60

80

100

120

140

160

180

200

0 5 10 15 20 25

Conduct ivity, ms/cm

Binding capacity @ 10% BT, mg/ml

2 shows the effect of conductivity on

Fig 2.

Effect

of

Column: human

Figure

conductivity

1.1 x 9.3 cm; ﬂow rate: 9.75

IgG or 5

mg/ml

3 shows how the effect of conductivity on Nuvia Q media

on Nuvia S media

conalbumin

ml/min

.

Nuvia

S media binding capacity.

binding capacity at pH 5.0.

(600

cm/hr); sample: 5 mg/ml

binding capacity.

5

Effect of Conductivity on human IgG Binding Capacity

0

20

40

60

80

100

120

0 2 4 6 8 10 12 14 16

Conductivity, ms/cm

Binding capacity @ 10% BT, mg/ml

Figure

4 shows how binding capacity for

at higher

Fig. 4. at pH 4.5.

4.5

conductivity and lower load buffer pH.

Effect

of

Column:

mg/ml human

conductivity

IgG in 20 mM

on

0.7 x 5.5 cm; ﬂow rate:

polyclonal

sodium acetate,

human

h.IgG

2.0 ml/min

binding

pH

IgG can be

capacity of

(300

cm/hr); sample:

4.5

.

achieved

Nuvia S media

6

All

buffers used with the same

commonly used for ion Nuvia

media

as the

(see Table

functional

charge

exchange

2). group on the ion

produce the best results.

Table 2.

Buffer

Nuvia S

Acetic acid 4.8–5.2 Citric acid 4.2–5.2 HEPES 6.8–8.2 Lactic acid 3.6–4.3 MES 5.5–6.7 MOPS 6.5–7.9 Phosphate 6.7–7.6 PIPES 6.1–7.5 TES 6.8–8.2 Tricine 7.8–8.9

Nuvia Q

Bicine 7.6–9.0 Bis-Tris 5.8–7.2 Diethanolamine 8.4–8.8 Diethylamine 9.5–11.5 L-histidine 5.5–6.0 Imidazole 6.6–7.1 Pyridine 4.9–5.6 Tricine 7.4–8.8 Triethanolamine 7.3–8.3 Tris 7.5–8.0

Common

buffers for

Buffering Range

ion exchange

The

chromatography can be

use of

buffering

ions

exchanger

chr

omatography

that have

will

.

Section

Regeneration and

7

Sanitation

:

After each run, the packed bed should be washed with 2–6 bed of 1–2 M bound 100 cm/hr; a

NaCl

or until

material.

minimum

7

absorbance

The

column can then be

contact time of 40 min is recommended.

returns to

sanitized

baseline

in 1.0 N

to

remove

NaOH

volumes reversibly

at 50–

Section

8

Sanitation

If a column no thorough contaminants. 1%

Triton

alcohol,

longer yields

CIP

and

Acceptable

X-100, 6 M

1 N

NaOH,

:

Cleaning-in-Place (CIP) and

reproducible

sanitation

and 6 M

after

CIP agents include

potassium thiocyanate,

guanidine

results,

regeneration

hydrochloride.

the media may require

to

remove 25% acetic acid, 8 M urea, 70%

strongly bound

ethanol, 30%

isopr

opyl

Section

For long-term storage, Nuvia™ media should be or

20% ethanol (see Table 1).

Section

Regulatory assistance validating contact your local Bio-Rad representative.

Section

Catalog # Description

156-0311 Nuvia™ S 156-0313 156-0315 156-0317 156-0411 Nuvia Q 156-0413 156-0415 156-0417

Larger volumes available

upon request.

: Storage

9

:

10

support

ﬁles

the use of

: Ordering

11

Nuvia S Media, Nuvia S Media, Nuvia S Media,

Nuvia Q Media, Nuvia Q Media, Nuvia Q Media,

and

special packaging

Regulatory

are

available

for Nuvia™ media.

Nuvia

media in a production process,

Information

Media,

25 ml 100 ml 500 ml 10 L

Media,

25 ml 100 ml 500 ml 10 L

for

Support

industrial applications

equilibrated

If you need

with 0.01– 0.1 N

are

NaOH

Triton is a

trademark

8

of

Union Carbide

Corporation.

Bio-Rad Q Media User Manual

Specifications and Main Features

Frequently Asked Questions

User Manual