ten lessons for the formulation development of monoclonal antibodies...

Ten Lessons for the Formulation Development of

Monoclonal Antibodies from Multimodal Thermal

Unfolding Case Studies

Mark Brader

Protein Pharmaceutical Development

Biogen Idec

PEGS Boston essential protein engineering summit

May 4-5, 2015

Methodology

Differential Scanning Calorimetry

Temperature (oC)

40 50 60 70 80 90

Cp (

cal/

oC

)

CH2 CH3

Fab

Brader PEGS Boston essential protein

engineering summit 5/4/2015

Methodology Optim1000 – simultaneous fluorescence and light scattering

unfolding transition

temperature (Tm)

Intrinsic fluorescence

Static light scattering

aggregation

onset temperature (Tagg)

Thermal ramp



Methodology

DSC + Intrinsic Fluorescence (F350/330)

Temperature (oC)

50 60 70 80 90

As the first domain unfolds a transition is

detected by the fluorescence change Brader PEGS Boston essential protein


Methodology

DSC + Static Light Scattering

Temperature (oC)

50 60 70 80 90

As the second domain unfolds the protein

aggregates



Methodology

DSC – SLS – F350/330 Superimposed

Temperature (oC)

40 50 60 70 80 90

DSC

SLS Fl350/330

mAb7 screening buffer

Initiation of fluorescence change coincides with unfolding of first domain

Initiation of aggregation coincides with unfolding of second domain



In a simple world,

where all proteins behave the same,

and conventional wisdom applies well to mAb formulation development…



…the everything is awesome scenario applies…

Screening buffer

Formulated

Temperature (oC)

40 50 60 70 80 90

SL

S

Temperature (oC)

40 50 60 70 80 90F

350/3

30

Temperature (oC)

40 50 60 70 80 90

CP

(ca

l/m

ole

/oC

)

DSC F350/330

SLS



But unfortunately in the real world of protein product development

it’s a complicated world and everything is not always awesome…



A wide range of thermal unfolding

profiles and conformational stabilities

are observed for developable

molecules

#1



DSC profiles of 8 developable mAbs in citrate buffer

Temperature (oC)

50 60 70 80 90

CP (

cal/m

ole

/oC

)

Tm(1) range 54.6-70.2

Tonset range 47.5-63.1

Large Fab Tm range



Thermal shifts associated with the first

unfolding transition may not correlate

with improved pharmaceutical stability

#2



Thermal shifts of Tm(1)

citrate buffer → pharmaceutically optimized formulations

-12

-10

-8

-6

-4

-2

0

2

4

6

mab1 mab2 mab3 mab4 mab5 mab6 mab7 mab8

DT m

(oC

)

-8

-6

-4

-2

0

2

4

6

8

10


DT m

(oC

)

Tm(1) DSC Tonset DSC

-13

-8

-3

2

7


DT m

(oC

)

Tm(1) F350/330



Improving thermal stability of the Fab

domains may represent a better

general formulation strategy than the

more common approach of focusing

on the first unfolding transition

#3



Thermal shifts of Tm(Fab)

citrate buffer → pharmaceutically optimized formulations

-1

-0.5

0

0.5

1

1.5

2

2.5

3

3.5

4

DT m

(oC

)

(d) Shifts in Fab Tm

6 of 8 mAbs show clear positive shifts



mAb2 formulated under conditions that greatly reduced Tm(1)

but interestingly Fab Tm did not change – importance of the Fab

-10

0

10

20

30

40

50

30 40 50 60 70 80 90

CP (

kcal

/mo

le/

C)

Temperature (C)

Cp(Y)

CpBase(Y)

CpFit(Y)

CpPk1

CpPk2

CpPk3

-5

0

5

10

15

20

25

30

30 40 50 60 70 80 90

CP (

kcal

/mo

le/

C)

Temperature (C)

Cp(Y)

CpBase(Y)

CpFit(Y)

CpPk1

CpPk2

CpPk3

66.5(±1.0) 73.5(±0.1) 82.3(±0.1)

56.5(±0.8) 73.5(±0.3) 82.7(±0.2)



Unusual or anomalous spectroscopic

signatures do not necessarily translate

into poor pharmaceutical stability

#4



Temperature (oC)

40 50 60 70 80 90

F350/3

30

mAb5

Temperature (oC)

40 50 60 70 80 90

F350/3

30

mAb6

Intrinsic fluorescence profiles of 8 mAbs in citrate buffer

Temperature (oC)

40 50 60 70 80 90

F350/3

30

mAb1

Temperature (oC)

40 50 60 70 80 90

F350/3

30

mAb2

Temperature (oC)

40 50 60 70 80 90

F350/3

30

mAb3

Temperature (oC)

40 50 60 70 80 90

F350/3

30

mAb4

Temperature (oC)

40 50 60 70 80 90

F350/3

30

mAb7

Temperature (oC)

40 50 60 70 80 90

F350/3

30

mAb8



mAb4’s unusual fluorescence transitions

pre-unfolding transition

Temperature (oC)

40 50 60 70 80 90

blue shift

red shift




blue shift


Temperature (oC)

40 50 60 70 80 90

red shift

% B

ioa

ctivity

% Bioactivity after 6 months at 25oC

and after 3 months at 40oC

Excellent stability




blue shift


Temperature (oC)

40 50 60 70 80 90

red shift % Bioactivity after 6 months at 25oC

and after 3 months at 40oC

Time (months)

0 2 4 6 8

% A

ggre

gate

s b

y S

E-H

PLC

0

5

10

15

20

25

mAb1

mAb2

mAb3

mAb4

mAb5

mAb6

mAb7

mAb8

% Aggregate by SE-HPLC 40oC storage

Excellent stability



Tagg does not appear to be a sensitive

general indicator of improved

formulation stability

#5



Thermal shifts of Tagg by SLS

citrate buffer → pharmaceutically optimized formulation

-7

-2

3

8

13


DT m

(oC

)



Formulation conditions can alter the

cooperativity of domain unfolding

#6



Formulation conditions can alter the cooperativity of

domain unfolding

CP (

cal/m

ole

/oC

)

Temperature (oC)

40 50 60 70 80 90

CP (

cal/m

ole

/oC

)

citrate buffer

pharmaceutically

optimized

formulation



A formulation strategy focused on the first Tm would miss the improved

stabilization conferred by this formulation

CP (

cal/m

ole

/oC

)

Temperature (oC)

40 50 60 70 80 90

CP (

cal/m

ole

/oC

)

citrate buffer

pharmaceutically

optimized

formulation

63.3(±0.1) 70.6(±0.5) 82.6(±0.1)

60.1(±0.2) 66.2(±0.1) 71.8(±0.1) 82.7(±0.1)



Aggregation can be driven by different

domains. The least conformationally

stable domain is not necessarily the

most aggregation-prone

Formulation conditions can alter which

domain drives aggregation

#7 & #8



Temperature (oC)

40 50 60 70 80 90

SLS reveals which domain mediates aggregation Formulation conditions can alter which domain drives aggregation

Temperature (oC)

40 50 60 70 80 90

mAb7

Temperature (oC)

40 50 60 70 80 90

mAb4

citrate buffer

mAb4

formulated

First domain

aggregates

Second domain

aggregates

formulation

optimization

Second domain

aggregates



Stability ranking at high temperature

may not translate to pharmaceutically

relevant temperatures

#9



Poor correlation between Tm(1) and storage stability at 25oC

0

0.05

0.1

0.15

0.2

0.25

0.3

55 60 65 70 75

aggregation rate by SE-HPLC

(%/month)

Tm(1) (oC)

Tm(1) by DSC versus aggregation rate by SE-HPLC at 25oC for optimized formulations



Pharmaceutical stability data

increase in aggregation by SE-HPLC

these developable mAbs

had <3.6%/month

aggregation at 40oC

aggregation kinetics at 5oC may be

non-linear whereas aggregation

kinetics at 25 and 40oC are linear



Prediction of low temperature stability

from high temperature stability is

unreliable

#10



Simultaneous Multiple Sample Light Scattering

monitoring of mAb aggregation

Wayne Reed, Tulane University

Drenski et al. Analytical Biochemistry 437 (2013) 185–197



Conclusions

Significant diversity in the thermal unfolding profiles mAbs possessing a wide range of conformational stabilities and

thermal unfolding profiles can be formulated into stable scalable

products

Formulation conditions shift thermal unfolding transitions Not all excipients confer pharmaceutical stability by increasing

conformational stability

Consideration of first transitions only can be misleading Stabilization of Fab appears to be more important than

stabilization of the first transition



Acknowledgments

• Tia Estey

• Shujun Bai

• Roy Alston

• Karin Lucas

• Steve Lantz

• Kevin Maloney

• Pavel Landsman

Reference

Examination of Thermal Unfolding and Aggregation Profiles of a

Series of Developable Therapeutic Monoclonal Antibodies

Mark L. Brader,* Tia Estey, Shujun Bai, Roy W. Alston, Karin K.

Lucas, Steven Lantz, Pavel Landsman, Kevin M. Maloney

Molecular Pharmaceutics 2015, 12, 1005−1017



ten lessons for the formulation development of monoclonal antibodies...

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