High Throughput PEGylation Process Development – Screening & Optimization Studies

26Oct2015

• PEGylation Background

• Case Study • Reference Standard Target

• OFAT development methodology

• HTPD screening

• HTPD optimization

• Process Improvements using HTPD

2

• Purpose of PEGylation: • Reduce immunogenicity / antigenicity by masking protein from immune system

• Increase hydrodynamic size of protein, increasing circulation time in body

• Improve solubility of protein

• Decrease tendency of molecule to aggregate

• General PEGylation Challenges: • PEGylation chemistry must be carefully selected

• PEG sizes / structures should be tested in vivo to determine most effective form

• PEGylation process must be developed

• Post-PEGylation purification process must be developed » Clearance of residual PEG, removal of undesired PEGylated/unPEGylated species, additional buffer exchange

• Additional analytical methods must be developed

Ref: Veronese, F. “Peptide and protein PEGylation: a review of problems and solutions”. Biomaterials 22, 405-417, May 2010.

3

• PEGylation target chosen by in vitro and in vivo studies

• Generate feasibility PEG:protein conjugates

» Different size PEG molecules

» Different conjugation chemistries

» Different PEG geometries

• Compare different PEGylated products to unPEGylated reference

• Select PEGylated candidates with...

» Improved half-life

» Reduced immunogenicity

» Increased solubility

» Minimized aggregation

4

NHS

Aldehyde

Maleimide

Forked Branched Multi-arm

Ref: NOF America Corporation, Product Catalog

• Target of PEGylation is a purified multimeric enzyme • Lysine PEGylation by NHS ester selection based on…

» 10-24x increase in circulation time

» Substrate is depleted to below LOD for 78 hours by lysine PEGylated enzyme

» Substrate depletion is minimal without PEGylation

• Lysine PEGylation Challenges: • Must match ref standard consistently

» >20 lysines per monomer

» Molecule is naturally a multimer (non-covalent)

• Control extent of PEGylation

» Need to understand PEGylation design space

5

Ref: ThermoFisher Protein Biology Resource Library

• Project capabilities at KBI: • Fast development / optimization of process step(s) to support clinical entry

» For this case study, ~3 months to re-develop entire purification process, including PEGylation step

» < 5months from start of PD to start of downstream manufacturing

• KBI project scope for PEGylation step: • Transfer client process

• Optimize current client process to fit with developed purification process

• Demonstrate control, reproducibility and scalability of PEGylation process

• Scale-up for manufacturing

• Characterize PEGylation process design space

6

• Process Development Objectives: • Identify all critical factors

• Increase understanding of PEGylation design space to enhance process robustness

• Optimize process to target the PEGylated Reference

• Consistently achieve the same PEGylation profile

• Scale-up process for GMP manufacturing

• Starting (reference) conditions: • pH 8.4 conjugation @ 7 – 12mg/mL [protein]

• 20x PEG molar excess addition

• 1 hour incubation at room temperature

• Quench reaction with glycine (0.2M final concentration)

7

• Process Development Approaches (2): • Perform multiple OFAT experiments to identify appropriate conditions for manufacturing

1) Effect of PEG addition on extent of PEGylation 1 week

2) Effect of protein concentration on extent of PEGylation 1 week

3) Effect of other factors on extent of PEGylation 1 week

• Perform screening study using 96 well plate DOE approach

» Single plate execution 2 days

» Analytical assays 2 days 1 week

» DOE analysis 3 days

8

• Analytical Challenges: • Target is not quantitatively well defined (SDS PAGE, SEC HPLC, RP UPLC, PEG:protein)

• No good quantitative assays were available during development stage

• Process must be developed quickly using existing analytical assays

9

PE

Gyla

ted P

D lot

PE

Gyla

ted G

MP

lot

Un-P

EG

yla

ted

PE

Gyla

ted P

D lot

PE

Gyla

ted G

MP

lot

SEC HPLC

PEGylated GMP lot

SampleName: SW hArgI BDS Lot2

SampleName: SW hArgI BDS Lot2

AU

-0.02

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

Minutes

5.00 10.00 15.00 20.00 25.00 30.00 35.00

RP UPLC

PEGylated GMP lot

PEG:protein ratio = 10±2 mole:mole

• OFAT (One Factor At a Time) experiment #1:

• Investigate effect of molar excess PEG

• Use existing PEGylation conditions, but change molar excess PEG

» 10x, 20x, 30x, and 50x molar excess PEG

• SDS PAGE Results:

» Size of PEGylated molecule increases with increasing amounts of PEG added

» No unPEGylated product detected

» Several PEGylated species apparent in each lane

10

PEG molar excess 10x 20x 30x 50x 0

• Significant factors expected: • pH

• Molar excess PEG

• Reaction Time

• Temperature

• Investigate: • Molar excess PEG

• Reaction Time

• Temperature

• [Protein]

• SDS PAGE Results: • pH – not tested

• Molar excess PEG

• Reaction Time

• Temperature

• Protein Conc.

11

10 min 60 min 60 min

cold

3 hour 18 hour

60

min

60

min

15x 20x 25x

15x 20x 20x 20x 25x

15x 20x 25x

15x 20x 25x

15x 20x 25x

20x 20x 24g/L

Ref std.

(20x)

2 mg/mL

OFAT experiment #2

• When pH and molar excess PEG held constant, protein concentration is the critical factor

• NOTE: Reference PEGylation batch variability may be due to differences in protein concentration at PEGylation step.

• Goal: Match Reference by identifying the appropriate protein concentration for PEGylation

12

SampleName: SW PEG-hArgI Lot2 inj2

SampleName: NB2104p111 PEG 20X 10 min

SampleName: NB2104p111 PEG 20X 60 min CP1



SampleName: NB2104p111 PEG 20X 60 min cold

SampleName: NB2104p111 PEG 20X 3hrs

SampleName: NB2104p111 PEG 20X overnight

SampleName: NB2104p111 PEG 20X 60 min 24g/l

AU

0.00

0.02

0.04

0.06

0.08

Minutes

6.00 8.00 10.00 12.00 14.00 16.00

24 g/L PEG

reaction

PEG’d

reference @

7-12g/L

SEC HPLC

OFAT experiment #2 (continued)

All 20x PEG

reactions @

2g/L

SampleName: SW PEG-hArgI Lot 2

SampleName: NB2104p111 PEG 20X 60 min cold

SampleName: NB2104p111 PEG 20X 3hrs

SampleName: NB2104p111 PEG 20X overnight

SampleName: NB2104p111 PEG 20X 60 min 24g/l




SampleName: NB2104p111 PEG 20X 10 min

AU

0.000

0.010

0.020

0.030

0.040

0.050

0.060

0.070

Minutes

8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00

• Similar qualitative observations made for RP HPLC

• Goal: Match Reference by identifying the appropriate protein concentration for PEGylation

13

PEG’d

Reference @

7-12g/L

All 20x PEG

reactions @

2g/L

RP UPLC

OFAT experiment #2 (continued)

PEG’d @

24g/L

• Confirm protein concentration effect

• Investigate additional factors: • Different PEG lot

• Cold Incubation

• No glycine quenching

• Potential gel artifacts

• Results: • Different PEG lots: No Effect

• Incubation temperature: No Effect

• Glycine quenching: No Effect

• [Protein]: Effect confirmed

• Band migration not an artifact of gel loading

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PEG’n Concentration (g/L) 16 10 5 2 10 10

2ug load

5ug load

0.5

ug load

OFAT experiment #3

PE

G lot#

1

PE

G lot#

2

No g

lycin

e q

uench

Cold

incubation

• Conclusions from OFAT experiments:

• Final PEGylation conditions for manufacturing: • 10g/L (± 1g/L) protein concentration @ pH 8.4 (±0.2)

• 20x molar excess PEG addition with mixing (solid to liquid addition)

• Min 15 minute reaction time after full PEG dissolution

• No glycine quenching

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Critical Factors Non-Critical Factors

Molar Excess PEG

(20x ± ?) Reaction Time

Protein Concentration

(10g/L ± ?) Incubation temperature

Reaction pH

(8.4 ± ?) Reaction Quenching

• Remaining Questions: 1) Could other untested

factors be important?

2) What ranges should be set?

3) Can other conditions result in same product quality?

• Develop high throughput method for PEGylation condition screening/characterization

• All reactions in 96-deep well plate

• Less PEG and protein required

• Allows for up to 6 factor CCD experiments with controls (86 runs + 10 controls)

• For smaller factor designs, replicates of important points can be run

• All experiments can be run in parallel

• No randomization required

• Huge amount of data in a short period of time.

• Statistics to back up data, which is absent in OFAT experiments.

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Plate#1: Solid

PEG Addition

Plate#2: Liquid

reagent addition

Add liquids to

solid PEG plate

and mix by plate

inversion.

Test each well for extent

of PEGylation

• Challenges in developing high throughput method: • Converting DOE design into a plate friendly design

• “Qualifying” method for use by replicating known control conditions

• Addition of hygroscopic PEG to small wells

• No high throughput analytical methods, with quantifiable results

» Quantifiable results required for statistical analysis

• Solutions: • Use extra wells in study for controls to qualify plate based PEGylation method

• Let PEG equilibrate to room temp prior to adding PEG to wells

• Adapt analytical methods to provide quantitative output

17

• Problem: No reliable, quantitative assays available for characterization of PEGylation reaction.

• Solution: Make current assays quantitative

• Available assays for characterization of PEGylated protein:

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Method Test Quantative

Result?

Useful in DOE

as is?

Possible

Quantitative Use

SEC HPLC Purity Yes No Retention Time

RP UPLC Quality No No 1st moment

PEG:protein Quality Yes No None*

SDS PAGE Purity No No Band Mobility

Band Migration Range

* Low throughput, poor/fair precision in target range

• DOE Responses (Quantitative Output):

• Analyze all samples by SDS PAGE and SEC

• Perform DOE analysis using Design Expert 9 software

19

0

1

PE

G-R

ef

Ref (n

o P

EG

)

Migration Range

Relative Mobility

SampleName: hArgI BDS demo -5 14Aug2014

15

.2

16

16

.6

85

AU

0.000

0.005

0.010

0.015

0.020

0.025

0.030

Minutes

0.00 5.00 10.00 15.00 20.00 25.00 30.00

SEC Retention Time

1 2 3 4 5 6 7 8 9 10 11 12

A

10x 10x 10x 10x 10x 10x 10x 10x 10x 10x 10x 10x

10 min 10 min 10 min 10 min 10 min 10 min 10 min 10 min 100 min 190 min 190 min 190 min

pH 7.5 pH 7.5 pH 9.5 pH 9.5 pH 7.5 pH 9.5 pH 9.5 pH 7.5 pH 8.5 pH 9.5 pH 9.5 pH 7.5

0 NaCl 0 NaCl 0 NaCl 200 NaCl 200 NaCl 200 NaCl 0 NaCl 200 NaCl 100 NaCl 0 NaCl 200 NaCl 0 NaCl

5 g/L 20 g/L 20 g/L 5 g/L 5 g/L 20 g/L 5 g/L 20 g/L 12.5 g/L 5 g/L 5 g/L 20 g/L

B

10x 10x 10x 10x 10x

190 min 190 min 190 min 190 min 190 min

pH 7.5 pH 7.5 pH 7.5 pH 9.5 pH 9.5

200 NaCl 200 NaCl 0 NaCl 0 NaCl 200 NaCl

5 g/L 20 g/L 5 g/L 20 g/L 20 g/L

C

20x 20x 20x 20x 20x 20x 20x 20x 20x 20x 20x 20x




12.5 g/L 12.5 g/L 20 g/L 12.5 g/L 12.5 g/L 12.5 g/L 12.5 g/L 12.5 g/L 5 g/L 12.5 g/L 12.5 g/L 12.5 g/L

D

20x 20x 20x 20x

100 min 100 min 100 min 190 min

pH 8.5 pH 7.5 pH 8.5 pH 8.5

100 NaCl 100 NaCl 100 NaCl 100 NaCl

12.5 g/L 12.5 g/L 12.5 g/L 12.5 g/L

E

30x 30x 30x 30x 30x 30x 30x 30x 30x 30x 30x 30x




5 g/L 5 g/L 20 g/L 20 g/L 20 g/L 5 g/L 5 g/L 20 g/L 12.5 g/L 5 g/L 20 g/L 20 g/L

F

30x 30x 30x 30x 30x

190 min 190 min 190 min 190 min 190 min

pH 7.5 pH 9.5 pH 7.5 pH 9.5 pH 7.5

200 NaCl 200 NaCl 200 NaCl 0 NaCl 0 NaCl

5 g/L 5 g/L 20 g/L 20 g/L 5 g/L

G

20x 20x 20x 20x 20x 20x 0x 0x

10 min 10 min 10 min 10 min 10 min 10 min N/A N/A

pH 8.4 pH 8.4 Tris pH 8.4 Tris pH 8.4 pH 8.4 pH 8.4 pH 8.4 pH 8.4

0 NaCl 0 NaCl 0 NaCl 0 NaCl 0 NaCl 0 NaCl 0 NaCl 0 NaCl

10 g/L 10 g/L 10 g/L 10 g/L 0 g/L 0 g/L 10 g/L 10 g/L

H

20x 20x 20x 20x 20x 20x 20x 20x

premix premix 10 min 10 min 10 min 10 min 10 min 10 min

pH 8.4 pH 8.4 pH 8.4 pH 8.4 pH 5.0 pH 5.0 pH 8.4 pH 8.4

0 NaCl 0 NaCl 0 NaCl 0 NaCl 0 NaCl 0 NaCl 1000 NaCl 1000 NaCl

10 g/L 10 g/L 10 g/L 10 g/L 10 g/L 10 g/L 10 g/L 10 g/L

20

• HT Study Design: • 5 factor RS CCD

• pH (7.5 – 9.5)

• [Protein](5 – 20 mg/mL)

• Molar excess PEG (10 – 30 x)

• Salt concentration (0 – 200 mM)

• PEG air exposure time (10 – 180 min)

• Ranges set so some center points are not current processing conditions

• Additional controls: • No PEG

• No protein

• Quenching agent in well

• SDS PAGE: relative mobility, DOE Results: • Relative mobility of “0” will match Reference

• Quadratic model summary » Adj R2 = 0.933, Pred R2 = 0.852

» P-value <0.0001

» Lack of fit p-value = 0.1106

» Statistically significant

• P-values for… » Model <0.0001

» pH <0.0001

» Protein Conc (B2) <0.0001

» Excess PEG <0.0001

» 2FI between Protein Conc and Excess PEG = 0.0598

• Conclusion: » Mobility of main band in SDS PAGE is affected by pH, protein

concentration, and molar excess PEG

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Design-Expert® Software

Factor Coding: Actual

SDS PAGE (Rel.Mob.)

Design Points

0.2296

-0.0649

X1 = B: Protein Conc.

X2 = D: Excess PEG

Actual Factors

A: pH = 8.50

C: NaCl Conc. = 100.00

E: Air Exposure = 95.00

5.00 8.00 11.00 14.00 17.00 20.00

10.00

15.00

20.00

25.00

30.00

SDS PAGE (Rel.Mob.)

B: Protein Conc. (mg/mL)

D:

Ex

ce

ss

PE

G

-0.05

0

0.05

0.10.15

0.15

5



SDS PAGE (Rel.Mob.)

Design Points

0.2296

-0.0649

X1 = A: pH


Actual Factors


D: Excess PEG = 20.00


7.50 8.00 8.50 9.00 9.50

5.00

8.00

11.00

14.00

17.00

20.00

SDS PAGE (Rel.Mob.)

A: pH

B:

Pro

tein

Co

nc

. (m

g/m

L)

-0.020

0.02

0.02

0.04

0.04

0.06

0.06

5

= Control sample

NOTE: Control BDS = 0,

Control BI = 1

pH = 8.4

Protein Conc = 10 g/L

NaCl conc = 0mM

Air Exposure = 10 min

Excess PEG = 20x

0

0

pH

Protein Concentration (g/L)

Pro

tein

Con

ce

ntr

ation

(g/L

) M

ola

r E

xce

ss P

EG

(x)

Design-Expert® SoftwareFactor Coding: ActualOriginal ScaleSEC mig range

Design Points436

145

X1 = B: Protein Conc.X2 = D: Excess PEG

Actual FactorsA: pH = 8.50C: NaCl Conc. = 100.00E: Air Exposure = 95.00

5.00 10.00 15.00 20.00

10.00

15.00

20.00

25.00

30.00

SEC mig range


D: E

xc

es

s P

EG

200

250

300

350400

5

• SDS PAGE Migration Range, DOE Results: • Quadratic model summary

» Adj R2 = 0.8909, Pred R2 = 0.7792

» P-value <0.0001


» Model statistically significant

• P-values for… » pH = 0.2504

» Protein Conc = 0.0135


» 2FI, Protein Concentration

and Excess PEG= 0.0049

• Conclusion: » The number of detected lower MW bands by SDS PAGE is

affected by molar excess PEG (major) and protein concentration (minor)

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= Control sample

SDS PAGE Migration Range

• SEC HPLC, DOE Results: • Quadratic model summary

» Adj R2 = 0.8663, Pred R2 = 0.7112

» P-value <0.0001


» Statistically significant

• P-values for… » Model <0.0001

» pH <0.0001

» Protein Conc <0.0001


» 2FI, pH and Protein Conc = 0.0004

• Conclusion: » Size of molecule detected by SEC HPLC retention

time is affected by pH, protein concentration, and molar excess PEG.

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SEC RT (min)

Design Points

18.096

16.431

X1 = A: pH


Actual Factors




7.50 8.00 8.50 9.00 9.50

5.00

8.00

11.00

14.00

17.00

20.00

SEC RT (min)

A: pH

B:

Pro

tein

Co

nc

. (m

g/m

L)

16.6

16.7

16.8

16.9

17

17.1

5

NOTE: Control BDS (batch)

RT = 16.83min

%RSD = 0.9%

Control BDS (plate)

RT = 16.82min

pH = 8.4

Protein Conc = 10 g/L

NaCl conc = 0mM

Air Exposure = 10 min

Excess PEG = 20x

= Control sample

pH

Pro

tein

Con

ce

ntr

ation

(g/L

)



SEC RT (min)

Design Points

95% CI Bands

X1 = D: Excess PEG

Actual Factors

A: pH = 8.50

B: Protein Conc. = 12.50



D: Excess PEG

10.00 15.00 20.00 25.00 30.00

SE

C R

T (

min

)

16

16.5

17

17.5

18

18.5

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One Factor

• HT DOE Study Results:

• SDS PAGE mobility can measure the migration distance of the main band indicating size of the main PEGylated species.

• SDS PAGE migration range can provide data on the polydispersity of PEGylated species, if lower MW species are present.

• SEC HPLC can measure the overall size of the molecule indicating the extent of PEGylation.

24

Critical Factors Non-Critical Factors

Molar Excess PEG Sodium chloride content

Protein Concentration PEG exposure to air

Reaction pH -

• HT DOE Study Results: • Shaded area on contour plots is current manufacturing range.

• Red dot is scale up PEGylation sample

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SDS PAGE (Rel.Mob.)

Design Points

0.2296

-0.0649

X1 = A: pH


Actual Factors




7.50 8.00 8.50 9.00 9.50

5.00

8.00

11.00

14.00

17.00

20.00

SDS PAGE (Rel.Mob.)

A: pH

B:

Pro

tein

Co

nc

. (m

g/m

L)

-0.020

0.02

0.02

0.04

0.04

0.06

0.06

5



SEC RT (min)

Design Points

18.096

16.431

X1 = A: pH


Actual Factors




7.50 8.00 8.50 9.00 9.50

5.00

8.00

11.00

14.00

17.00

20.00

SEC RT (min)

A: pH

B:

Pro

tein

Co

nc

. (m

g/m

L)

16.6

16.7

16.8

16.9

17

17.1

5

SEC HPLC SDS PAGE

pH pH

[Pro

tein

]

[Pro

tein

]

26

• Process optimizations: • Use analyzed data to present optimal process solutions

depending on need (desirability):

• Main targets for all optimizations are:

» Relative mobility, 0 ± 0.2

» Migration range ≤ 250

» SEC retention time 16.7 min – 16.9 min

• Solutions (based on hypothetical requirements):

» pH (less than 8.0 to increase buffering capacity) pH 7.5, 13.5mg/mL [protein], 22.3x molar excess PEG (CONFIRMED)

» PEG (minimize amount to minimize raw material cost) pH 9.5, 7.8mg/mL [protein], 19.2x molar excess PEG

» [protein] (minimize to limit HMW formation, if applicable) pH 9.5, 5mg/mL [protein], 25.4x molar excess PEG

Design-Expert® SoftwareFactor Coding: ActualDesirability

1.000

0.000



5.00 8.75 12.50 16.25 20.00

10.00

15.00

20.00

25.00

30.00

Desirability


D:

Ex

ce

ss

PE

G

00 0

0 00 0

00 0

0

0.1

0.1

0.2

0.2

0.3

0.3

0.4

0.4

0.5

0.6

Prediction 0.638


1.000

0.000



5.00 8.75 12.50 16.25 20.00

10.00

15.00

20.00

25.00

30.00

Desirability


D:

Ex

ce

ss

PE

G

00 0

0 00 0

00 0

0

0.2

0.4

0.6

0.8

Prediction 0.826


1.000

0.000



5.00 8.75 12.50 16.25 20.00

10.00

15.00

20.00

25.00

30.00

Desirability


D:

Ex

ce

ss

PE

G

00

00

0

0

0

0.2

0.2

0.4

0.4

0.6

0.6

Prediction 0.795

Desirability Plots

X-axis = [protein]

Y-axis = PEG molar excess

#1

#1

#2

#2

#3 #3

pH = 7.5

pH = 9.5

pH = 9.5

• HT DOE Screening Study Conclusions: • Scale-down high throughput model is a good representation of larger scale process

• SEC retention time can be used to monitor PEGylation extent

• SDS PAGE mobility can be used to monitor size of main PEGylated species

• SDS PAGE migration range can be used to detect less PEGylated species

• Product quality targets can be met under a variety of different conditions

» Contour plot and desirability plot can be used to identify different acceptable PEGylation conditions based on need.

» Perform process optimization predictions using DOE software

• ≥ 2.5x savings on protein and PEG raw material requirements

• > 3x development time savings

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• KBI Analytical Development Group

• KBI Downstream Process Development Group

High Throughput PEGylation Process Development – Screening & Optimization Studies

Health & Medicine

Transcript of High Throughput PEGylation Process Development – Screening & Optimization Studies