145

7

Process Scale Purifi cation of Antibodies, Edited by Uwe GottschalkCopyright © 2009 John Wiley & Sons, Inc.

INTEGRATED POLISHING STEPS FOR MONOCLONAL ANTIBODY PURIFICATION

Sanchayita Ghose , Mi Jin , Jia Liu , and John Hickey

7.1 INTRODUCTION

Monoclonal antibodies (mAbs) have become the most important therapeutic modality in the biotechnology industry (1, 2) . There are 21 Food and Drug Administration (FDA) - approved antibodies currently on the market, with many more under development in pharmaceutical pipelines. Given the high dose requirement and the increasing market potential of this therapeutic class, the primary focus during process development is to reduce manufacturing costs, streamline process development activities, and enable production at very large scales (3, 4) .

Antibodies are typically produced using cultured mammalian cells to ensure proper folding and glycosylation. Over the last decade, signifi cant advances have been made in cell culture technology, including the improvement of production media and feeding strategies, resulting in very high cell culture titers ( > 5 g/L) (5) . The effi cient recovery and purifi cation of antibodies from cell culture media is a critical part of the antibody production process (6) . One of the recent trends in therapeutic antibody downstream processing is the adoption of templated purifi cation schemes to enable shorter development times and multiproduct harmonization at manufacturing scales. This platform approach has been widely adopted by almost all companies with signifi cant numbers of antibody candidates in their development pipelines. The approach

146 INTEGRATED POLISHING STEPS

hinges predominantly on the successful use of Protein A affi nity chromatog-raphy as the capture step, a highly selective process that can result in > 95% purity starting from complex cell culture media (see Chapter 4 ). Following the Protein A step, there typically remain trace levels of process - related contami-nants [such as host cell proteins (HCPs), DNA, leached Protein A, endotoxins, and some cell culture media additives] as well as product - related impurities (such as higher - molecular - weight aggregates and lower - molecular - weight deg-radation products). The subsequent chromatography processes are commonly referred to as the polishing steps since they remove trace - level impurities and serve to reduce these impurities to levels that assure product safety.

Various modes of chromatography including cation exchange (CEX), anion exchange (AEX), hydrophobic interaction chromatography (HIC), and hydroxyapatite (HA) have been used as polishing steps in antibody purifi ca-tion processes (7, 8) . The use of immobilized metal – chelate affi nity chroma-tography (IMAC) and size - exclusion chromatography (SEC), although less common, has also been reported (7, 9) . Genentech has developed a generic purifi cation scheme involving CEX followed by AEX in fl ow - through mode as the polishing steps after Protein A chromatography (10, 11) . On the other hand, Amgen has adopted the use of a fl exible downstream platform to account for biochemical differences seen among various antibodies (12) . In this approach, two polishing steps are typically chosen from the four common modes listed above. Furthermore, Wyeth BioPharma has discussed the possi-bility of a two - step purifi cation platform that employs a single polishing step (13) . Typically, the organization of the polishing steps (number and mode) is case specifi c and is dictated mainly by the predominant impurities found in the respective downstream processes.

This chapter presents an overview of the most frequently used polishing steps in antibody manufacture and discusses the considerations to be kept in mind while integrating these steps into the overall purifi cation scheme.

7.2 POLISHING STEPS IN ANTIBODY PURIFICATION

7.2.1 Ion - Exchange ( IEX ) Chromatography

IEX chromatography is widely used in the biopharmaceutical industry for the process - scale purifi cation of monoclonal antibodies, fusion proteins, and other protein therapeutics (14 – 17) . IEX is predominantly based on electrostatic interactions between surface charges on proteins and charged functional groups on the chromatographic resin. Differential adsorption/desorption sepa-rates the product from impurities (see Chapter 5 ). The protein - binding char-acteristics are determined by net charge of the protein and the charge distribution over the surface as well as the resin type (negatively charged for CEX chromatography or positively charged for AEX chromatography), ligand type (strong or weak ion exchanger), functional group, ligand density, base

POLISHING STEPS IN ANTIBODY PURIFICATION 147

matrix, linker chemistry, and pore size. IEX is the best - characterized chroma-tography mode. The stoichiometric displacement model (SDM) (18) and steric mass action (SMA) model (19, 20) provide a basic understanding of the adsorption phenomena in IEX, while a more complete description of the process has been described by Stahlberg (21) .

7.2.1.1 AEX Chromatography. AEX chromatography is very popular for polishing in antibody manufacture because the high pI of most human anti-bodies prevents them from binding to AEX resins under the pH conditions (pH 7.0 – 8.0) typical for AEX. Higher pH conditions can increase the capacity for antibody binding, but this is usually avoided to minimize the risk of deami-dation and proteolysis (8) . On the other hand, impurities such as DNA, HCP, and endotoxins are negatively charged and thus bind strongly to AEX columns under the same conditions. It has therefore become common practice to operate the AEX step in fl ow - through mode under alkaline pH conditions and low conductivity. AEX fl ow - through chromatography also achieves excellent virus removal and has been validated as a generic step for virus clearance (22) . However, the AEX step does little to eliminate aggregates and leached Protein A, leaving the burden of their removal for other polishing steps. The contami-nants that are present after Protein A affi nity chromatography are in trace amounts (typically ppm or ppb), thereby enabling the AEX fl ow - through step to be carried out at loading capacities of up to 100 mg/mL of resin. Table 7.1 lists some of the commonly used resins for this mode of chromatography.

Membrane chromatography is becoming a viable alternative to AEX chro-matography in fl ow - through mode (23 – 26) . Traditionally, one of the major disadvantages of membrane chromatography is the low binding capacity refl ecting the lower surface area - to - bed volume ratio (27, 28) . Those hurdles are circumvented by operating in fl ow - through mode, where only trace quanti-ties of contaminants need to bind.

It is sometimes prudent to screen AEX in binding as well as in fl ow - through mode, since surface charge distributions may enable the binding of antibodies to AEX resins under appropriate solution conditions. Recently, it has been reported that the AEX step can be operated in an isocratic mode referred to as weak - partitioning chromatography (WPC) (29) . In this mode, appropriate solution conditions are chosen to allow for signifi cant product binding (1 – 20 g/L of resin). However, since the antibodies are more basic than the other impurities, the impurities bind even more strongly to the resin and act by sample displacement. Despite signifi cant levels of product binding to the resin, high yields ( > 95%) can be achieved by using high loading (up to 250 g/L of resin) and short isocratic washes. Operating the AEX step in this WPC mode preserves the isocratic operation typical of AEX fl ow - through steps while pro-viding greater selectivity for impurity removal. Using this as the only polishing step, signifi cant impurity clearance has been reported including a log reduction value (LRV) of ∼ 4 for HCP, > 2 for leached Protein A, > 3 for nucleic acid, > 5 for retroviruses, as well as a ∼ 20 - fold reduction in product aggregates (13) .

148

TAB

LE

7.1

C

omm

only

Use

d A

EX

Res

ins

Res

in N

ame

Ven

dor

Lig

and

Bas

e M

atri

x A

vera

ge P

arti

cle

Size

, μ m

Po

re S

ize,

Å

Supe

r Q

650

S, M

, an

d C

To

soh

Qua

tern

ary

amm

oniu

m

Met

hacr

ylat

e 35

, 65,

100

10

00

Q S

epha

rose

FF

G

E Hea

lthc

are

Qua

tern

ary

amm

oniu

m

6% c

ross

- lin

ked

agar

ose

90

n/a

Cap

to Q

H

ighl

y cr

oss -

linke

d ag

aros

e w

ith

dext

ran

surf

ace

exte

nder

2000

Q S

epha

rose

XL

6%

cro

ss - l

inke

d ag

aros

e w

ith

dext

ran

surf

ace

exte

nder

n/

a

Frac

toge

l EM

D

TM

AE

(M

), H

icap

M

erck

KG

gA

Trim

ethy

l am

mon

ium

et

hyl

Met

hacr

ylat

e re

sin

wit

h po

lym

eric

“ te

ntac

les ”

65

80

0

Uno

sphe

re Q

B

io - R

ad

Qua

tern

ary

amm

oniu

m

Poly

mer

ic

120

n/a

Q C

eram

ic H

yper

D

Pal

l Q

uate

rnar

y am

mon

ium

C

eram

ic b

ead

fi lle

d w

ith

a hy

drog

el

50

n/a

PO

RO

S H

Q50

A

pplie

d B

iosy

stem

s Q

uate

rniz

ed

poly

ethy

lene

imin

e C

oate

d cr

oss -

linke

d po

ly(s

tyre

nedi

viny

lben

zene

) 50

16

00

n/a

= n

ot a

pplic

able

.

POLISHING STEPS IN ANTIBODY PURIFICATION 149

7.2.1.2 CEX Chromatography. CEX chromatography is also used fre-quently for polishing in antibody manufacture (12, 30, 31) , although in this case, the high pI of most human antibodies means that the step is run in bind - and - elute mode with high loading capacities and good selectivity. CEX has been shown to clear HCP and leached Protein A, as well high - molecular - weight (HMW) aggregates in specifi c cases (7) . CEX is more effective than AEX in reducing leached Protein A levels. Protein A is relatively acidic and is thus retained less strongly than antibodies, leading to removal in the fl ow - through or through an intermediate pH wash. Fragments of leached Protein A that bind to an antibody can also be removed by an intermediate pH wash.

Table 7.2 lists some of the strong CEX resins commonly used in antibody manufacture. Weak cation exchangers such as carboxylmethyl resins, although less frequently used, are also available from the listed vendors. Resins with a polymeric backbone such as methacrylate have greater selectivity for aggre-gate removal due to nonspecifi c hydrophobic interactions with the backbone (32) . Knudsen and colleagues (23) have also evaluated CEX membranes for process - scale antibody purifi cation and concluded that they might not be eco-nomical in a bind - and - elute mode.

The choice of CEX resin and the optimization of operating conditions depend largely on the nature of the impurities to be removed and the antibody of interest. Most antibodies are basic, yet they can vary signifi cantly in their retention on CEX resins (12) . Although one study has argued the possibility of predicting CEX retention behavior of human monoclonal antibodies based on amino acid sequence in the variable heavy chain (VH) region (33) , in practice, process development largely relies on systematic experiments to select the appropriate resin and the best operating conditions.

The two key parameters to consider during resin selection are binding capacity and selectivity. CEX can offer high binding capacities for monoclonal antibodies (up to 100 mg/mL) (30) . Typically, static binding capacity can be measured in a high - throughput format for multiple loading conditions (pH and conductivity). These are indicative of the dynamic binding capacity trends that can then be measured for selected conditions. Intuitively, one would expect maximum CEX capacity to be obtained under lower conductivity and pH conditions since antibodies become increasingly protonated under acidic conditions. However, recent studies have shown that binding capacity for antibodies on CEX resins can increase unexpectedly with increasing conduc-tivity and decreasing protein charge at the lower range of ionic strength condi-tions (34) . This nontraditional behavior can be explained by an exclusion mechanism whereby the antibody can bind to the outer pore region and can electrostatically hinder subsequent antibody molecules from entering. Increas-ing the ionic strength and pH shields the charges on the protein and dampens the exclusion effect, thereby resulting in a higher capacity. As the pH and conductivity increase, the traditional trend of decreased capacity reasserts itself due to reduced interactions between the resin and the antibody. This

150

TAB

LE

7.2

C

omm

only

Use

d C

EX

Res

ins

Res

in N

ame

Ven

dor

Lig

and

Bas

e M

atri

x A

vera

ge P

arti

cle

Size

, μ m

Po

re S

ize,

Å

SP 6

50 S

To

soh

Sulf

opro

pyl

Met

hacr

ylat

e 35

10

00

SP 6

50 M

65

SP

650

C

100

Gig

aCap

S - 6

50M

Su

lfo

75

SP S

epha

rose

FF

G

E Hea

lthc

are

Sulf

opro

pyl

6% c

ross

- lin

ked

agar

ose

90

n/a

Cap

to S

H

ighl

y cr

oss -

linke

d ag

aros

e w

ith

dext

ran

surf

ace

exte

nder

20

00

SP S

epha

rose

XL

6%

cro

ss - l

inke

d ag

aros

e w

ith

dext

ran

surf

ace

exte

nder

n/

a

Frac

toge

l EM

D S

O3

Mer

ck K

GgA

Su

lfoi

sobu

tyl

Met

hacr

ylat

e re

sin

wit

h po

lym

eric

“ t

enta

cles

” 65

80

0 Fr

acto

gel E

MD

SE

(M

), H

icap

Su

lfoe

thyl

Uno

sphe

re S

B

io - R

ad

Sulf

o Po

lym

eric

12

0 70

0 – 20

00

S C

eram

ic H

yper

D

Pal

l Su

lfo

Cer

amic

bea

d fi l

led

wit

h a

hydr

ogel

50

n/

a P

OR

OS

HS5

0 A

pplie

d B

iosy

stem

s Su

lfop

ropy

l C

oate

d cr

oss -

linke

d po

ly(s

tyre

nedi

viny

lben

zene

) 50

16

00

POLISHING STEPS IN ANTIBODY PURIFICATION 151

observation is important to keep in mind during process development while arriving at the optimal conditions for binding capacity.

Linear gradient experiments over a range of mobile phase pH and buffers can be conducted to screen resin selectivity. A plot of cumulative key impurity level vs. cumulative product yield provides an effective means to compare the selectivity of various resins without differences in peak shape and retention biasing the comparison (32) (Fig. 7.1 ). Batch screening carried out in high - throughput mode can also facilitate the screening of a large number of condi-tions, and such platforms have become commercially available. Figure 7.1 shows that the intrinsic selectivity for HMW aggregate removal is greater at lower pH values for both resins. Resin 1 outperforms resin 2 at pH 6.0 and 7.0 but is comparable at pH 5.0. Similar comparisons can be carried out for other impurities to provide guidance on resin selection and operating conditions, to maximize product yield and purity. Although selectivity is tested in linear gradient mode, process operations typically operate in a step gradient mode for manufacturing simplicity and ease of control. Step gradient conditions can be developed based on linear gradient data and some further optimization for yield and impurity removal.

7.2.2 HIC

HIC is based on the interaction between hydrophobic ligands (aliphatic or aromatic) and hydrophobic residues on the protein surface. This mode has

FIGURE 7.1 Selectivity plot for HMW removal. Linear gradient experiments were carried out with moderate loading and NaCl salt gradient at specifi ed pH.

1.0

0.9

0.8

0.7

0.6

0.5

0.4

0.3

0.2

0.1

00 50 100

Cumulative yield (%)

Cu

mu

lati

ve H

MW

(%

) Resin 1—pH 6.0

Resin 1—pH 7.0

Resin 1—pH 5.0

Resin 2—pH 7.0

Resin 2—pH 6.0

Resin 2—pH 5.0

Resin 2—pH 4.5

152 INTEGRATED POLISHING STEPS

been used for protein purifi cation since 1973 (35, 36) and has gained popularity over the years (see Chapter 5 ). In comparison with reversed - phase chroma-tography (RPC), HIC employs milder separation conditions that help to mini-mize protein denaturation. In HIC, protein adsorption on the stationary phase increases with salt concentration and elution is achieved by decreasing salt concentration (37, 38) . HIC and IEX separations are based on completely different mechanisms and are often used as orthogonal polishing steps for protein purifi cation.

While HIC is not the most common mode of chromatography in antibody purifi cation, it is used as a polishing step when HMW aggregate and HCP removal are primary concerns. Since HIC has an orthogonal mechanism to IEX, it offers distinct selectivity for removing HCP species that are not cleared by that mode of chromatography. HIC is usually very effective for the reduc-tion of HMW aggregates as the latter are generally more hydrophobic and are better retained on HIC resins. HIC is inferior to IEX for the removal of leached Protein A and DNA, the latter tending not to bind under the high - salt conditions used in HIC. HIC can be operated in either fl ow - through or bind - and - elute modes depending on the impurities to be removed. HIC resins usually offer relatively low loading capacities in the bind - and - elute mode, so fl ow - through mode is preferred.

Table 7.3 lists the commonly available HIC resins that can be used in anti-body purifi cation. The choice of the resin is based on resin characteristics, selectivity for impurity removal and binding capacity. A recent product intro-duced specifi cally for antibody purifi cation is the 600 series from Tosoh, in which the pore size has been optimized to maximize binding capacity and recovery.

A series of experiments is needed to screen for the best HIC resin and to develop optimal operating conditions. These typically include the generation of salt precipitation profi les, linear gradient retention studies, linear gradient selectivity studies, and an evaluation of dynamic binding capacities (39) . A salt precipitation curve for the antibody of interest should be prepared in the presence of various lyotropic salts to identify the highest salt concentration that can be tolerated. This is important because higher salt concentrations often translate to higher binding capacities, but this can also lead to antibody precipitation. The salt precipitation curves help to identify a safe operating regime and narrow down loading salt conditions that need to be tested in subsequent development work. Protein precipitation can be evaluated by mea-suring the turbidity of the solution through light scattering or absorbance measurements at 410 or 600 nm. A precipitation profi le is shown in Fig. 7.2 . Since precipitation is a function of protein concentration, contact time, pH, temperature, salt type, and salt concentration, such profi les can be generated for each condition if needed. Along with turbidity measurements, SEC analysis of the samples is recommended since the salt and pH conditions that promote the formation of soluble aggregates can be different from those causing precipitation.

153

TAB

LE

7.3

C

omm

only

Use

d H

IC R

esin

s

Res

in N

ame

Ven

dor

Ave

rage

Par

ticl

e Si

ze, μ

m

Lig

and

Bas

e M

atri

x Po

re S

ize,

Å

But

yl S

epha

rose

HP

G

E H

ealt

hcar

e 34

n -

But

yl

Cro

ss - l

inke

d ag

aros

e n/

a P

heny

l Sep

haro

se H

P

Phe

nyl

n/a

But

yl - S

Sep

haro

se 6

Fas

t F

low

90

B

utyl

- S

6% c

ross

- lin

ked

agar

ose

n/a

But

yl S

epha

rose

4 F

ast

Flo

w

n - B

utyl

4%

cro

ss - l

inke

d ag

aros

e n/

a P

heny

l Sep

haro

se 6

Fas

t F

low

(l

ow s

ub)

Phe

nyl

6% c

ross

- lin

ked

agar

ose

n/a

Phe

nyl S

epha

rose

6 F

ast

Flo

w

(hig

h su

b)

Hig

h su

b ha

s hi

gher

lig

and

dens

ity

than

lo

w s

ub

n/a

Oct

yl S

epha

rose

4 F

ast

Flo

w

n - O

ctyl

4%

cro

ss - l

inke

d ag

aros

e n/

a To

yope

arl E

ther

- 650

To

soh

Bio

scie

nce

100,

C

65, M

35

, S

n - B

utyl

M

etha

cryl

ate

1000

To

yope

arl P

PG

- 600

M

Poly

prop

ylen

e gl

ycol

75

0 To

yope

arl P

heny

l - 65

0 P

heny

l 10

00

Toyo

pear

l But

yl - 6

50

n - B

utyl

10

00

Toyo

pear

l But

yl - 6

00M

n -

But

yl

750

Toyo

pear

l Phe

nyl -

600M

P

heny

l 75

0 To

yope

arl H

exyl

- 650

n -

Hex

yl

1000

154 INTEGRATED POLISHING STEPS

Once the safe operating regime for salt concentrations is defi ned, linear retention experiments can be carried out under decreasing salt gradients on various HIC resins to estimate the affi nity of the protein at different salt and pH combinations. These experiments can help assess the hydrophobicity of the molecule and can guide resin selection accordingly. Resins that cause the antibody to elute too early in the gradient should be avoided in bind - and - elute mode as very high salt concentrations would be required for optimal binding. On the other hand, very hydrophobic resins that cause peak splitting or low recovery should also be avoided as they can denature the product. Following the retention study, linear gradients can be run under preparative loading conditions to generate selectivity plots as described in the previous section. In contrast to the retention study, the selectivity studies compare the resolution of different resins (for the impurity of interest) under a variety of mobile phase conditions. A selectivity plot comparing the ability of two resins to remove HMW aggregates is shown in Fig. 7.3 . The shape of the curves indicates that resin B is more selective than resin A.

The methodology adopted for HIC development can be slightly different according to whether the bind - and - elute or fl ow - through mode is used. For bind - and - elute mode, the selectivity plot allows the developer to choose appropriate loading conditions to maximize capacity without product dena-turation or aggregation, appropriate wash conditions if the impurity is retained, and appropriate elution conditions to maximize product recovery. For fl ow - through mode, weak binding resins can be considered. However, the use of a more hydrophobic resin with salt conditions that favor weak or no retention can often provide greater selectivity. Developing the load salt concentration

FIGURE 7.2 A precipitation profi le with salt concentration vs. antibody absorbance (410 nm) after 1 h, antibody concentration of 4 mg/mL .

0

0.5

1

1.5

2

2.5

3

0.1 0.2 0.3 0.4

Ab

sorb

ance

at

410

nm

Salt concentration (M)

pH 4.0 sodium sulfatepH 5.0 sodium sulfatepH 4.0 potassium phosphatepH 5.0 potassium phosphatepH 5.0 sodium citrate

POLISHING STEPS IN ANTIBODY PURIFICATION 155

that provides best trade - off between product recovery and impurity elimina-tion is very important for fl ow - through steps.

7.2.3 HA Chromatography

HA is a calcium mineral with the formula Ca 10 (PO 4 ) 6 (OH) 2 . In contrast to other adsorbents used in downstream processing, HA is both the ligand and the supporting matrix (40) . There are two types of binding sites on HA — the positively charged calcium referred to as the C site and the negatively charged phosphate referred to as the P site. Depending on the pI of the product and the operational pH, electrostatic interactions with these sites can be either cationic or anionic. These ionic interactions can be disrupted by increasing the salt concentration. Carboxyl groups on the protein surface can also interact with the calcium sites by metal – chelate interaction. The binding is an order of magnitude stronger than normal IEX and increased salt concentration does not disrupt such interactions. Phosphate ions have proven effective for disrupt-ing both metal – chelate and ionic interactions resulting in dissociation (41 – 43) . The relative contributions of these interactions are case specifi c and depend on the surface properties of the protein.

HA chromatography is a very selective polishing step in antibody manu-facture and is able to remove HCP, aggregates, and leached Protein A (44) . Aggregates and Protein A – IgG complexes tend to be better retained than most antibodies in this mode. In addition, DNA usually elutes after most antibodies due to the strong interaction between the calcium sites and the DNA phosphate backbone. Typical operating conditions involve eluting the

FIGURE 7.3 A selectivity plot comparing the ability of two HIC resins to remove HMW aggregates, with sodium citrate buffer pH 4.8.

0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

0 20 40 60 80 100

Cu

mu

lati

ve H

MW

(%

)

Cumulative yield (%)

Resin A Resin B

156 INTEGRATED POLISHING STEPS

antibody in a NaCl gradient with a small amount of additional phosphate (44) . The distribution of impurities in such a gradient is shown in Fig. 7.4 .

HA resins are available from two vendors: (i) a spherical, macroporous form of HA called ceramic HA (CHT Types I and II) from Bio - Rad (Hercules, CA, USA) and (ii) an HA agarose composite called Biosepra HA Ultrogel sorbent available from Pall (St. Petersburg, FL, USA). The former is more frequently employed in industrial applications because it is noncompressible and thus has superior pressure – fl ow properties. The large - scale packing of HA can be challenging and special precautions need to be taken to prevent rapid settling (which can lead to bed heterogeneity) and particle fracturing.

7.2.4 Mixed - Mode and Other Modes of Chromatography

Mixed - mode chromatography, as the name indicates, exploits a combination of interaction mechanisms to enable separation (see Chapter 6 ). These interac-tions can be simultaneous IEX and hydrophobic interactions, CEX and AEX, or IEX with biorecognition. The interplay of multiple interaction mechanisms can provide unique selectivity under conditions more suitable for manufactur-ing process fl ow.

Early research has shown that protein retention and resolution can be increased by incorporating more hydrophobic moieties onto AEX resins (45) . The development and launch of mixed - mode resins has signifi cantly acceler-ated in recent years, driven by a need to achieve greater selectivity in a single processing step. Based on initial high - throughput screening and prototype evaluation (46, 47) , two commercial multimodal resins called Capto adhere and Capto MMC have recently been introduced by GE Healthcare. These combine AEX and CEX with hydrophobic moieties on an agarose backbone.

FIGURE 7.4 Distribution of impurities under typical operating conditions when eluting an antibody in a NaCl gradient with a small amount of additional phosphate (43 ). LPA: leached Protein A. Etox: Endotoxin.

5 mM PO4

0.5 MPO4

LPADNAEtox

Aggregates

Native IgG

NaCl gradient

POLISHING STEPS IN ANTIBODY PURIFICATION 157

These resins enable IEX chromatography loading under high - salt conditions, which can simplify process fl ow by eliminating dilution or diafi ltration require-ments. These multimodal resins can also offer unique selectivity for the removal of impurities such as HMW aggregates or leached Protein A compared to traditional IEX. The process development methodology for mixed - mode resins is usually similar to their AEX and CEX counterparts if used under conditions where the IEX mechanism dominates. It is noteworthy that when operating these multimodal resins in bind - and - elute mode, elevated salt concentrations may not always be an effective strategy for protein elution due to secondary hydrophobic interactions. It has been shown that modifying the pH or adding chaotropic reagents can be necessary to facilitate complete recovery (48) . Capto adhere should be used in fl ow - through mode much like traditional AEX resins for typical antibodies (GE Healthcare application note 28 - 9078 - 89 AA), with pH, salt concentration, and loading as the main operating variables that need to be optimized to achieve the best trade - off between yield and purity. The vendors note that the use of these mixed - mode resins can result in a two - step antibody purifi cation process (GE Healthcare application note 28 - 9078 - 92 AA). However, more comprehensive evaluations need to be conducted for this strategy to fi nd wider acceptance.

Another type of mixed - mode chromatography introduced for antibody purifi cation is hydrophobic charge induction chromatography (HCIC). This employs a heterocyclic ligand such as 4 - mercaptoethyl pyridine (MEP), which becomes positively charged at low pH values (49) . While adsorption at neutral pH is based on hydrophobic interactions, elution is facilitated at low pH by charge repulsion between the ionized ligand and charged residues on the protein. Initial HCIC studies have focused on its use as a capture step in anti-body processing (50, 51) . However, recent studies have revealed its limited selectivity as a capture step compared to Protein A (52) . HCIC might have more potential as a polishing step in place of traditional HIC since it operates under low salt conditions (53) . Moreover, the combination of hydrophobic interactions along with electrostatic repulsion could also provide unique selec-tivity for impurity removal compared to traditional HIC matrices. More recently, two new resins based on hexylamine (HEA) and phenylpropylamine (PPA) ligands have also been introduced. Binding on these resins occurs through a combination of hydrophobic interactions (aliphatic hexyl group for HEA or aromatic phenyl group for PPA) and electrostatic interactions (through the amine group), while desorption is facilitated through electrostatic repulsion as described above (54) . There is as yet no comprehensive under-standing of how these two resins perform in mAb purifi cation. Table 7.4 sum-marizes some of the mixed - mode resins that are commercially available and that can be employed as a polishing step in antibody manufacturing.

Recently, dye - ligand affi nity chromatography has been used for the purifi -cation of a mAb from cell culture (55) . Cibacron Blue resin contains a syn-thetic polycylic dye ligand that can bind to a variety of proteins by hydrogen bonding, van der Waals forces, hydrophobic and ionic interaction, and in some

158 INTEGRATED POLISHING STEPS

TABLE 7.4 Commercially Available Mixed - Mode Resins

Resin Name

Vendor Ligand Base Matrix Average Particle Size, μ m

Interactions

Capto MMC

GE Healthcare

Multimodal ligand containing carboxylic and phenyl group

Highly cross - linked agarose

75 CEX and hydrophobic interaction

Capto adhere

N - benzyl - N - Methyl ethanolamine

Highly cross - linked agarose

AEX and hydrophobic interaction

HEA HyperCel

Pall n - Hexylamine Cross - linked cellulose

80 – 100 AEX and hydrophobic interaction

PPA HyperCel

PPA AEX and hydrophobic interaction

MEP HyperCel

4 - Mercapto - ethylpyridine

Hydrophobic charge induction

Blue Trisacryl M

Cibacron Blue F3GA

Polymeric 40 – 80 Electrostatic and hydrophobic interaction

cases bioaffi nity (the ligand is a NAD cofactor mimic) (56, 57) . It has been used in earlier commercial processes for the production of albumin and thyroid - stimulating hormone, but only recently has it been evaluated as a potential mAb polishing step. This resin has been used as the second polishing step (in fl ow - through mode) following the capture for an IgG4 by Protein A chromatography. The resin has a high loading capacity and has signifi cantly reduced the levels of bovine serum albumin (BSA), HCP, HMW aggregates, and degradation products. Several Protein A mimetic ligands have also been developed based on triazine dye chemistry (58, 59) . While their perfor-mance as the antibody capture step has not been very promising (52) , the selectivity afforded by their unique chemistries could be employed as a polish-ing step.

7.2.5 Dedicated Virus Removal Steps

In addition to virus clearance achieved by polishing, the ICH Q5A guidance document requires two dedicated virus clearance steps with orthogonal mech-anisms (see Chapter 8 ). The two most frequently used processes are low - pH virus inactivation and fi ltration. The low - pH inactivation step is typically

placed after the Protein A step since the Protein A elution pool is already at a relatively low pH. Monoclonal antibodies tend to endure exposure to low pH conditions with no ill effects. However, if the antibody is unstable at low pH, other techniques such as solvent – detergent treatment may be used instead (60) .

Filtration involves the size - based removal of virus particles by passage through a membrane with a small pore size. Filters are available in two catego-ries based on their pore size ratings — retroviral ( < 50 nm) or parvoviral ( < 20 nm) (61) . For more recent antibody products, the industry is favoring the use of parvoviral - grade fi lters to satisfy heightened stringency in regulatory expecta-tions. Parvoviral - grade fi lters typically need a larger surface area because they tend to clog even in the presence of low levels of particulates and aggregates in process streams. As a result, virus fi ltration can be the second most expen-sive step in the process after Protein A chromatography, and the optimization of this process step and its integration into the overall purifi cation sequence are equally important. The virus fi ltration step is typically placed after at least one of the polishing chromatography steps. The choice is usually based on product stream volume considerations as well as the volume that can be fea-sibly fi ltered for that process intermediate (62) .

7.3 INTEGRATION OF POLISHING STEPS

The previous sections briefl y described the nature of the polishing steps in antibody manufacture and provided several key considerations concerning process development. Additional considerations need to be kept in mind while integrating the above - mentioned polishing steps into a downstream process. One key factor is that of overall process productivity. Tugcu and colleagues (63) have recently described a two - stage resin screening approach that inte-grates chromatographic steps with a view toward maximizing productivity. Apart from productivity, other factors that infl uence the choice and number of polishing steps, their mode of operation, and their placement include (i) key impurities that need to be removed, (ii) how diffi cult they are to separate, (iii) fi nal product purity constraints, (iv) optimal process fl ow with the fewest operational steps, and (v) how easy they are to fi t in the manufacturing facility. Some of the philosophies concerning the integration of polishing steps into a manufacturing process are illustrated through the following two case studies.

7.3.1 Case Study I : Selection and Placement of Polishing Steps

The following is a case study concerning the sequence of polishing steps chosen for a mAb purifi cation process. Based on initial development, three process sequences were proposed for further evaluation as shown in Fig. 7.5 . All sequences started with the same Protein A capture step and a low - pH hold for viral inactivation. Following that, Sequence I adjusted the Protein A elution

INTEGRATION OF POLISHING STEPS 159

160 INTEGRATED POLISHING STEPS

pool to an alkaline pH, then used AEX in fl ow - through mode followed by CEX in bind - and - elute mode. Note that the AEX step was placed before the CEX step to facilitate process fl ow because the Protein A elution pool had a lower conductivity than the CEX elution pool, so placing AEX as the second step eliminated the requirement for load dilution or buffer exchange. More-over, the AEX fl ow - through pool was alkaline and could be loaded directly onto the CEX column. However, adjusting the pH of the Protein A pool to the neutral range prior to the AEX step caused a large amount of impurities to precipitate. Although the precipitate could be removed by fi ltration, evalu-ation of the fi ltration process suggested that it posed a signifi cant manufactur-ing challenge due to the inherent variability in the precipitation process and the large fi lter area required to achieve adequate clarifi cation.

Sequences II and III both used CEX as the second step. This was operated in bind - and - elute mode at a lower pH to avoid the precipitation that occurred in Sequence I, at the same time improving the selectivity of the CEX step for HMW aggregate reduction. The third step in Sequence II was HIC in the fl ow - through mode, which provided additional HMW aggregate removal but had somewhat limited DNA clearance capability. A key consideration in this scheme was the load preparation for the HIC step. The elevation in conductiv-ity required for the HIC load was achieved by mixing the CEX elution pool with a high - salt buffer. The resulting product pool volume expansion intro-duced additional constraints on tank volume capacities for storing the HIC load and the product pool after HIC.

The third step in Sequence III was mixed - mode Capto adhere chromatog-raphy operated in fl ow - through mode. As mentioned before, this resin has both AEX and hydrophobic properties, allowing the antibody to bind at high con-ductivities. As a result, the CEX elution pool could be pH adjusted and loaded directly onto the mixed - mode column without further dilution or buffer exchange steps. Development data also revealed good DNA, HCP, and leached Protein A clearance for this sequence (data not shown). This sequence was

FIGURE 7.5 Proposed process sequence choices for mAb purifi cation .

Protein A

AEX

CEX HIC

CEX(at lower pH)

Capto adhere(mixed mode)

Sequence I Sequence II Sequence III

found to fi t well into an existing manufacturing facility with the fl exibility to accommodate increased cell culture titers in the future.

The pros and cons of each sequence are summarized in Table 7.5 . This was based on data concerning the clearance of impurities, facility - fi t analysis, and an overall economic evaluation. A fi nal decision on the appropriate process sequence needs to take into account multiple factors including the nature and level of the major impurities, manufacturing convenience, facility fi t, and cost considerations. As a case in point, if the dominant consideration is HMW aggregate levels and if tank capacity is not the governing issue , Sequence II might be a better choice than Sequence III. However, if DNA clearance is a concern, then Sequence III would a better choice and this would also circum-vent the limitations on tank capacity. If the issues surrounding precipitation were not dominant, Sequence I could also be a good choice as it provides superior DNA clearance.

7.3.2 Case Study II : Selection of Operational Mode and Infl uence of the Previous Polishing Step

This case study shows the rationale for the selection of bind - and - elute mode versus fl ow - through mode for HIC in an antibody purifi cation process. Figure 7.6 shows the two process sequences that were developed and Table 7.6 lists the operating parameters for the HIC steps. For this case study, the Protein A capture step and the fi rst polishing step had removed the majority of the process - related impurities such as DNA, HCP, and leached Protein A. The main purpose for the HIC step was to reduce HMW aggregate levels. Both the modes were optimized to provide similar HMW aggregate reduction.

On comparing the operating parameters in Table 7.6 , it can be seen that a signifi cantly higher loading capacity was possible in fl ow - through mode, and the mobile phase salt concentration was also lower. This can have several operational advantages such as lower salt disposal costs and reduced tank corrosion. The lower salt concentration also enabled the use of a higher protein concentration in the load. The mAb concentration reached 8 – 10 g/L with no sign of precipitation in a mobile phase comprising 120 mM sodium citrate, whereas the highest mAb concentration achieved in the 500 mM citrate was ∼ 2 g/L. The higher protein concentration translated into a lower load volume and a reduced loading time. This can be important from a facility - fi t point of view if tank volumes in the load preparation area are the limiting factor. Under these circumstances, it would be desirable to operate the HIC step in fl ow - through mode.

On the other hand, fl ow - through also has some drawbacks and, in certain cases, the bind - and - elute mode can be more suitable. In an earlier version of the above - mentioned process, there was a specifi c HCP (referred to as impu-rity X) that was not removed by the previous polishing step. It was necessary to remove X for comparability purposes, and the subsequent HIC step had to

INTEGRATION OF POLISHING STEPS 161

162 INTEGRATED POLISHING STEPS

TABLE 7.5 Analysis of Pros and Cons for Each Sequence Choice

Sequence I Protein

A – AEX – CEX

Sequence II Protein

A – CEX – HIC

Sequence III Protein A – CEX – mixed

mode

Pros Good DNA and HCP clearance

Selectivity of the CEX step for HMW was greater at lower pH

Selectivity of the CEX step for HMW was greater at lower pH

No load adjustment required for the AEX or CEX step

Robust HMW clearance provided by the HIC step

Good HCP, DNA, and HMW clearance

Minimal load adjustment for all the steps

Cons Precipitation can pose a signifi cant fi ltration challenge

CEX elution pool had to be diluted for the subsequent HIC step

Mixed - mode resin is more expensive than traditional ion exchangers or HIC resins

Limited HMW clearance capability

Limited DNA clearance capability for the HIC step

FIGURE 7.6 Process fl ow diagram for Case Study II.

Protein A capture step

Second polishing step

Flow-through HIC

Final concentration/diafiltration

Bind-and-elute HIC

Final concentration/diafiltration

be optimized to remove both HMW aggregates and this specifi c HCP. X was less hydrophobic than the antibody and was therefore coeluted with the anti-body in fl ow - through mode, making it an unfavorable option. However, for the bind - and - elute step, the salt concentration could be chosen carefully to elute X while the monomer and HMW aggregates bound to the resin during the loading step. Suitable wash conditions were also developed to wash impu-rity X through the column prior to elution. Then, during the elution step, the

salt concentration was optimized to elute the antibody preferentially leaving the HMW aggregates bound to the resin.

For the case described above, the fi rst polishing step was further optimized to remove impurity X during subsequent development. Thus, in the fi nal process, the HIC step could be operated in the fl ow - through mode and could take advantage of the operational benefi ts mentioned in the previous paragraph . This also emphasizes the importance of looking into the down-stream process as an integrated system rather than discrete chromatographic steps to achieve at the best combination of polishing steps.

7.4 CONCLUSIONS

It is not yet practical to use a single generic polishing step for antibody purifi -cation. The presence of the common Fc moiety in all antibodies allows for a generic capture step using Protein A chromatography, but the development of a polishing strategy is still case specifi c and needs to be tailored toward specifi c impurity removal requirements (which depend on the upstream process) and unique properties of each antibody, refl ecting variable charge distribution and hydrophobic characteristics. Current best practice involves the use of two polishing steps following Protein A chromatography, but the industry is trying to move toward a single polishing step. Some of the chromatography strategies discussed in this chapter, such as AEX in weak - partitioning mode, the use of mixed - mode resins, or the use of membrane chromatography might help to achieve that vision. Several attempts have also been made to purify antibodies without a Protein A step (see Chapters 5 and 14 ). In such cases, the demand on the polishing steps would be even greater and signifi cantly more method development time would be required.

7.5 ACKNOWLEDGMENTS

The authors would like to acknowledge the analytical group at Bristol - Myers Suibb for analytical results.

TABLE 7.6 Operating Parameters for the HIC Bind - and - Elute and Flow - Through Steps

Operating Condition Bind - and - Elute Mode Flow - Through Mode

Loading capacity, mg antibody/mL resin

15 – 20 50 – 60

Loading salt concentration, mM citrate

500 – 600 100 – 120

Load protein concentration, mg/mL < 2 8 – 10

ACKNOWLEDGMENTS 163

164 INTEGRATED POLISHING STEPS

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