efficientaggregateremoval

2024年3月16日发(作者：宝马750最便宜多少钱)

B

io

P

rocess

Technical

Efficient Aggregate Removal

from Impure Pharmaceutical

Active Antibodies

Sybille Ebert and Stefan Fischer-Frühholz

P

olishing with membrane

chromatography (MC) has

toxicity and immunogenicity. Because

achieved acceptance as state-of-

of their toxic potential, such

aggregates can cause an unwanted

impurities. Traditionally, anion-

the-art technology for charged response or even overreaction of a

exchange (AEX) and cation-exchange

patient’s immune system (anaphylaxis).

(CEX) membrane chromatography

have been used to remove charged

are monitored using size-exclusion

Typically, product aggregate levels

contaminants such as host-cell proteins

chromatography (SEC). Removal of

(HCPs), recombinant DNA, protein

aggregates from a protein solution,

A, endotoxins, and viruses. In

however, is typically performed using

monoclonal antibody (MAb) processes,

HIC because monomeric proteins

polishing steps usually follow a protein

display less hydrophobicity than

A affinity column step. In some cases,

aggregates do. Because they form at

CEX capture is applied, either with at

lower concentrations, flow-through

least one AEX or a combined AEX

mode is most favorable for modern

and CEX step. The latter may be

MC, which is primarily driven by

replaced by a hydrophobic-interaction

volume rather than mass capacity.

chromatography (HIC) step. Ceramic

This is reasonable because a flow-

hydroxyapatite is also used, though less

through approach significantly reduces

frequently.

buffer consumption and allows

application of disposable devices. Until

formed during MAb manufacturing

Hydrophobic antibody aggregates recently, however, HIC has been

are frequent process-related impurities

applied only in a bead/column format

that must be removed during

and bind-and-elute mode. Trace

downstream processing because they

contaminants can be efficiently

can cause loss of activity as well as

removed, particularly HCPs,

recombinant DNA, leached protein A,

and product-related impurities such as

P

roduct

F

ocus

: P

roteins

(

antibodies

)

soluble aggregates.

P

rocess

F

ocus

: d

ownstream

capabilities for high flow rates and

To make use of membrane

Processing

convective flow, Sartorius Stedim

W

ho

s

hould

r

ead

: P

rocess

Biotech addressed the limitation of

develoPment

engineers

,

analysts

conventional beads and developed a

hydrophobic membrane adsorber

K

eyWords

: H

ydroPHobic

-

interaction

carrying a phenyl ligand to efficiently

cHromatograPHy

,

PolisHing

,

remove product aggregates (

disPosables

,

laboratory

scale

novel phenyl membrane adsorber has

1

). The

proven useful for aggregate removal in

l

evel

: i

ntermediate

a MAb purification process.

36 BioProcess International F

ebruary

2011

)

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Table 1: examples for reduction of aggregate

levels in one step during downstream

processing

From (%)To (%)

Protein 1 (non-IgG)15.0 %≤1.0 %

Protein 2 (non-IgG)30.0 %≤0.1 %

Protein 3 (IgG)6.0 %0.8 %

Protein 4 (IgG) 7.0 %1.0 %

d

eveloPment

oF

hic m

the

emBrane

Flow rate and diffusion limitations

with packed-bed resins can lengthen

process times, which may increase the

risk of protein unfolding and

denaturation, leading to product loss

(

create a hydrophobic adsorber that

2

). The developer’s intention was to

shows hydrophobic interaction at high

salt concentrations but keeps mass

transfer limitation as small as possible.

That would circumvent a number

of disadvantages seen with

of five bed volumes per minute.

traditional resins.

Binding sites for proteins are

accessible by convection rather than

membrane adsorber has a pore size of

The new macroporous phenyl diffusion. That minimizes the effect

>3 ?m with a recommended flow rate

of decreased binding capacity at high

flow rates (

3

). The mechanism for

Table 2: ammonium sulfate concentrations (mmol/L) applied in twelve semichromatographic

batch experiments

Equilibration, Loading,

Conditionand WashingElution 1Elution 2Elution 3Elution 4

100000

25025000

375502500

41007550250

5

6

72

83

94

10

118

121

Table 3: buffers and chromatographic parameters applied in laboratory-scale experiment for

aggregate removal (transfer from batch to dynamic conditions)

Volume Flow Rate

StepBuffer(mL)(mL/min )

Equilibration50 mmol/L sodium phosphate buffer at pH 7.0 with 275

480 mmol/L ammonium sulfate (78.8 mS/cm)

Load30.9 mg MAb in 50 mmol/L sodium phosphate 105

buffer at pH 7.0 with 480 mmol/L ammonium

sulfate (78.8 mS/cm)

Washing50 mmol/L sodium phosphate buffer at pH 7.0 with 95

480 mmol/L ammonium sulfate (78.8 mS/cm)

Elution 150 mmol/L sodium phosphate buffer at pH 7.0 with 95

430 mmol/L ammonium sulfate (70.5 mS/cm)

Elution 250 mmol/L sodium phosphate buffer at pH 7.0 with 95

330 mmol/L ammonium sulfate (57.5 mS/cm)

Elution 350 mmol/L sodium phosphate buffer at pH 7.0 with 95

230 mmol/L ammonium sulfate (43.6 mS/cm)

Elution 450 mmol/L sodium phosphate buffer at pH 7.0 95

(6.03 mS/cm)

Table 4: buffers and chromatographic parameters applied in laboratory-scale experiment for

aggregate removal (optimized dynamic conditions)

Volume Flow Rate

StepBuffer(mL)(mL/min)

Equilibration50 mmol/L sodium phosphate buffer at pH 7.0 275

with 430 mmol/L ammonium sulfate (70.5 mS/cm)

Load31.4 mg MAb in 50 mmol/L sodium phosphate 225

buffer at pH 7.0 with 480 mmol/L ammonium

sulfate (78.8 mS/cm)

Washing50 mmol/L sodium phosphate buffer at pH 7.0 295

with 430 mmol/L ammonium sulfate (70.5 mS/cm)

Regeneration 150 mmol/L sodium phosphate buffer at pH 7.0 155

(6.03 mS/cm)

Regeneration 220% isopropanol155

Regeneration 3Purified water405

Storage20% ethanol125

Unique selectivities

increase

separation options

HyperCelHyperCel

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IEX Sorbents IEX Sorbents

Q and S HyperCel high productivity Q and S HyperCel high productivity

ion exchange (IEX) sorbents offer ion exchange (IEX) sorbents offer

unique selectivities that can improveunique selectivities that can improve

separation of target molecules from separation of target molecules from

closely-related contaminants, enhancingclosely-related contaminants, enhancing

separation performance and tion performance and economics.

These sorbents demonstrate high flow These sorbents demonstrate high flow

rates and high dynamic binding capacity rates and high dynamic binding capacity

at short (2 minutes) residence short (2 minutes) residence time.

Differentiated Selectivities Help

Achieve Specific Separation Goals

mAUmAU

S HyperCel SorbentS HyperCel Sorbent

mS/cmmS/cm

100100

Rigid Agarose SRigid Agarose S

5050

8080

mm

4040

nn

00

6060

88

3030

22

DD

4040

OO

2020

2020

1010

00

0.0 10.0 20.0 30.0 40.0 50.0 60.00.0 10.0 20.0 30.0 40.0 50.0 60.0

00

Elution Time (min.)Elution Time (min.)

The separation achieved with four model

proteins on S HyperCel sorbent differs from a

competitor sorbent under the same conditions.

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e:

*****************

Defining Process Economics

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