key formulation challenges of protein (mab) drugs

Key Formulation Challenges of protein

(mAb) drugs

Sandeep Nema, PhD

Pfizer

My Background and About Pfizer Biologics

• Approx 60 biologics at

various stages of

development

• Wide range of modalities

– mAb

– Fusion proteins

– Bispecifics

– Oligonucleotides

– ADC

– Gene therapy

– CAR-T

– Vaccines

• Conjugates

• Viral vaccines

Pfizer Confidential 2

Outline

Part I (Formulation)

• Components of mAb Formulation

• Key considerations

– Buffer choice

– Freezing and thawing (storage)

• Role of crystalline vs amorphous state

– Surfactant benefits (or not)

• Developing high concentration mAb formulation

Part II (Process)

• Basics of Freeze-drying


Typical Formulation of mAb

The active ingredient is typically in the range of 10 to 150 mg/mL (dilute solution)

5

Excipients in formulation

Buffering Agent

pH Adjusters

Cryo or lyo

protector

Sucrose

Trehalose

Sorbitol

Citrate (acid/Na/K)

Phosphate (di/mono)

Histidine

Tris

NaOH

HCl

Bulking Agents

Mannitol

Polymers

• PEG

• Dextran

Surfactants Complexing

Agents

Antioxidants Stabilizers

Polysorbate

Pluronic

EDTA

DTPA

Amino acids like Arg Methionine

Glutathione

Tonicity modifier

NaCl

Mannitol

Dextrose

Preservatives

Phenol

Benzyl alcohol

M cresol

11/16/2017 Parenteral Formulation 6

Buffers (to stabilize pH, hold a drug in solution

and obtain an acceptable shelf life)

• Part of the decision of which buffer to use to control the product pH to

– improve stability

– Improve solubility (of protein)

– modify viscosity (later)

– reduce pain (arginine buffer vs other)

• Depends on the buffer's effective pH range.

– All buffers are not effective at all pH ranges.

• Buffers are the strongest (most buffering capacity) at their pKa's.

• Other considerations in selection of buffer

– Crystallization

– pH shift (pKa change as a function of temperature)

Reaction rates as a function of pH

7

diketopiperazine

β-elimination

racemization

Cys oxidation

Asp-Pro cleavage

fragmentation

isomerization

deamidation

12111098765432Solution pH

diketopiperazine

β-elimination

racemization

Cys oxidation

Asp-Pro cleavage

fragmentation

isomerization

deamidation

12111098765432Solution pH

(Darker color indicates a faster reaction rate at designated pH range)

11/16/2017 Parenteral Formulation 8

Different Buffers At Different pH’s – pKa of

buffer

Buffer Acid Base pH Range

Control

Examples

Phosphate Monosodium

phosphate

Disodium phosphate 5.8–7.8 Elaprase®, Remicade®

Acetate Acetic acid Sodium acetate 3.8–5.8 Avonex®, Neupogen®

Citrate Citric acid Sodium citrate 3.0–7.4 Amevive®, Rituxan®

Succinate Succinic acid Sodium succinate 3.3–6.6 Actimmune®

TRIS TRIS HCl TRIS 7.1–9.1 Wellferon®, Enbrel®

Histidine Histidine HCl Histidine 5.1–7.0 Xolair®, Raptiva®

Carbonate Sodium bicarbonate Sodium carbonate 5.4–7.4, 9.3–

11.3

Fuzeon™

Crystallization potential of some common

buffers on freezing and thawing

9

potassium citrate

sodium citrate

glutarate

lactate

succinate

phosphate

tartrate

malate

2 3 4 5 6 7 8 9

amorphous

crystalline

pH

Buffer

Curve 1-sodium phosphate, pH 7.0; curve 2-sodium citrate, pH 6.2; curve 3-sodium

succinate, pH 5.5; curve 4-sodium acetate, pH 5.6; curve 5-histidine HCl, pH 5.4;

curve 6-histidine acetate, pH 5.5; curve 7-tris HCl, pH 7.4

10

Impact of temperature change on pH of some

common buffers.

Crystallization of dibasic

Tris 7.4

His 5.5

Phosphate 7

Citrate

Distribution of pH in formulations of antibodies, Fc

fusion products, and Fab conjugates

11

Protein products in the pH range of 3.5 to 9.5

Outline




– Buffer choice





Part II (Process)



BioTx Pharmaceutical Sciences 13

Freezing Biologics: Advantages and

Associated Challenges

• Advantages of freezing

protein bulk:

– Minimization of risk of

microbial growth

– Increased product

stability with extended

shelf life

– Elimination of agitation

and foaming during

transportation

– Flexibility for

manufacturing process

• Outcome:

– Long-term stability under

frozen storage

• Freeze concentration

(Cryoconcentration)

• Protein denaturation on

ice-liquid interface

• Tg’ vs Storage

temperature


Passive Freeze/Thaw Active Freeze/Thaw

Celsius System

Cryovessel

Adapted from http://www.sartorius-stedim.com

Plastic Bottles

Adapted from www.nalgene.com

Large Scale Freeze Thaw Systems

BioTx Pharmaceutical Sciences

What happens during freezing?

15


Adapted from http://www.sartorius-stedim.com

Cryovessel

Understanding freezing in a scaled-down model

Active

Cooling

Surface

Active Cooling

Surface

Cryowedge

20 mg/mL mAB in 20 mM

Histidine buffer, 84

mg/mL Trehalose

Dihydrate, 0.2 mg/mL PS

80, pH 5.5


Frozen State Mapping : mAb Solution (Conc)

Bottom Half of frozen solution in Cryowedge

Considerably higher protein concentration values for the bottom half for the cores

Highest concentration observed was for for the position 3 and 5 which represent the greatest distance from active heat transfer surfaces

Approximately more than 3 fold cryoconcentration

Melted portion of frozen mAb

solution


Fraction Analysis of Protein Concentrations in

Cryowedge in Frozen State

1000 - 1200

mOsm/Kg; 2.08%800 - 1000

mOsm/Kg; 3.13%

600 - 800

mOsm/Kg; 7.29%

400 - 600

mOsm/Kg;

18.75%200 - 400

mOsm/Kg;

58.33%

0 - 200 mOsm/Kg;

10.42%

40 - 50 mg/mL;

5.21%

50 - 60 mg/mL;

0.00%

0 - 10 mg/mL;

0.00%

60 - 70 mg/mL;

1.04%

30 - 40 mg/mL;

15.63%

20 - 30 mg/mL;

31.25%

10 - 20mg/mL;

46.88%

Bottom

0 - 200

mOsm/Kg,

83.49%

200 - 400

mOsm/Kg,

16.51% 0 - 10 mg/mL,

36.70%

10 - 20mg/mL,

61.47%

20 - 30

mg/mL, 1.83%

Top Osmolality Protein Concentration


-10°C

Protein Conc (mg/mL)

0 10 20 30 40 50 60

Solu

ble

Aggre

gate

s (

%)

0

1

2

3

4

5

Initial

3 month

6 month

12 month

-20°C


0 10 20 30 40 50 60

So

lub

le A

gg

reg

ate

s (

%)

0

1

2

3

4

5

-40°C


0 10 20 30 40 50 60

So

lub

le A

gg

reg

ate

s (

%)

0

1

2

3

4

5

Stability Under Frozen Conditions

• -20C frozen condition is

detrimental to protein from

aggregates perspective

• At -10 and -40C, no

significant increase in

aggregates compared to -20

C

Stability of protein in cores in frozen

storage (-10, -20, -40C) (SEC data)

Worldwide Pharmaceutical Sciences

Biologics

NaCl-Water

Phase Diagram

20

-21.1°C

0.9 % NaCl

23.3%

Salt Solution

Water NaCl

T2

E

T3

Pure Ice crystals

NaCl.2H2O crystals

< -21C

Protein + Non-crystallizing Buffer salts (Concentrated ~26X)

T1

O°C


Biologics 21

Non-Crystallizing Systems: Solid-Liquid

State Diagram

(Maximally) Freeze Concentrated Liquid

(Cg’ at Tg’)

= Protein + Buffers + Disaccharide (conc ~10X)

Cg’

Pure Ice crystals


Biologics

22

Trehalose / Sucrose Solubility and the

State Diagram

Maximally FCL (~80%)

Sundaramurthi and Sury


Biologics 23

Trehalose

State Diagram

Hold

above

Tg’

(Maximally) Freeze Concentrated Liquid (FCL)

(Cg’ at Tg’)

= Protein + Buffers + Disaccharide (conc ~ 10X)

Cg’

Pure Ice crystals

Trehalose.2H2O crystals


Conclusions

• Trehalose crystallization at -20°C results in loss of cryoprotectant and aggregation over time

• Protein denaturation/unfolding at the ice interface probably contributes to this effect

• Mobility at -20°C allows the crystallization and the aggregation to occur (below Tg’ of trehalose) – Prevented by -40C storage

• Sucrose unlikely to crystallize out – Could aggregation still occur due to mobility in the denatured

protein

• Storage at -10°C likely allows trehalose to crystallize but also allows protein to “refold”

BioTx Pharmaceutical Sciences 25 25

Freeze-thaw induced LDH aggregation

• LDH: 35.6 kDa, labile to

F/T stress

• F/T protocol:

– 5 min in liquid N2

– 5 min in water bath

– repeat for multiple

cycles

• LDH Aggregation is

inversely dependent on

protein concentration.


LDH aggregate structure by probe

fluorescence measurement

26

A: Signal increase and blue shift:

exposure of more hydrophobic residues in aggregates.

B: Lack of apparent ThT emission peak:

absence of ordered amyloid-like structure in aggregates.


LDH aggregate structure by intact

molecule HX-MS

27

Fully labeled

• More solvent accessible

to LDH aggregates

molecular unfolding

during aggregation.

• Consistent with Bis-ANS

binding assay

• HX protected structure

exists in LDH aggregates


Identification of intermolecular contact

regions in LDH aggregates

28

HX to native LDH HX to LDH aggregates

0-25% 25-50% 50-75% 75-100%

Solvent accessibility

Pep 13-31

Pep 109-117

Pep 133-143


Conclusions

• HX-MS technique can successfully distinguish the

regions of unfolding and molecular interactions

during aggregation,

F-T of mAb and Fusion Protein

• Drug substance formulation same as that of drug product 20 mM histidine buffer (pH 6.0 for fusion proteins and 5.5 for mAb) 4% mannitol tonicity agent 1% sucrose cryoprotectant

• Drug substance concentration 65 mg/mL for fusion protein and 30 or 50 mg/mL for mAb1

• Both mAb1 and Fusion protein, DS stable to F-T and storage at -80°C for at least 48 mo - Stored in Teflon bottle

• Drug product is lyophilized and stored at 2-8C

• Production facility implemented option for controlled freeze and thaw technology in stainless steel CryoVessels. Storage temperature -50C 10°C

• Process development studies Perform lab scale experiments

Develop profile for freeze and thaw at various CryoVessel fill volumes

The Problem Freeze and Thaw Instability in CryoWedge

0.0

2.0

4.0

6.0

8.0

10.0

0 1 2 3 4 5

Freeze/Thaw Cycle

%H

MW

CW

-80°/37°C

K. Ho, R. Reid, N.Luksha, Li Li

• Freeze and thaw rates were implicated in HMW increase

30 mg/mL

0

1

2

3

4

5

0 1 2 3 4 5

Freeze/Thaw Cycle

% H

MW

.

Cryocassette

-80°C/37°C

65 mg/mL

Freeze and Thaw

Cycling mAb1 Fusion protein1

The Investigation Excipient Effect: Polysorbate 80

D. Sek, N.Luksha, Li Li

0.0

0.3

0.6

0.9

1.2

1.5

0 2 4 6Freeze/Thaw Cycle

%H

MW

0% PS80 0.005% PS80 0.02% PS80

• Polysorbate 80 had no effect on mAb 1HMW

• Rather than stabilize, Polysorbate 80 caused an increase in Fusion

Protein1 HMW, so there is no benefit to adding Polysorbate 80 to

formulation

0

1

2

3

4

5

6

7

8

0% 0.001% 0.01% 0.05%

%Polysorbate 80

%H

MW

In

cre

as

e

3x Freeze/Thaw

Freeze and Thaw

Cycling mAb1 Fusion protein1

mAb1 DS 30 mg/mL with Mannitol

SE-HPLC %HMW

0.0

2.0

4.0

6.0

8.0

10.0

12.0

0 1 2 3 4 5Freeze/Thaw Cycle

%H

MW

0% 2% 4%

The Investigation Excipient Effect: Mannitol

• As mannitol concentration increased, %HMW increased

• Mannitol caused an increase in %HMW

K. Ho

The Investigation Process Scale Effect: 125 L vs. Maximum F/T Rate

K. Ho, R. Reid

• Freeze and thaw rates were also a factor

mAb1 DS CryoPilot 125L vs. Max F/T Rate

SE-HPLC %HMW

0.0

2.0

4.0

6.0

8.0

10.0

12.0

14.0

16.0

0 1 2 3 4 5 6

F/T Cycle

%H

MW

125L Max F/T Linear (125L) Linear (Max F/T)

Slower rate

Faster rate

DS 50 mg/mL with 4% and 0% mannitol

0.0

3.0

6.0

9.0

12.0

15.0

18.0

0 4 8 12 16 20Freeze/Thaw Cycle

%H

MW

4% mannitol

K. Ho

Formulation 2

Formulation 1

0% mannitol

125L (Green)

70L (Blue)

18L (Red)

• As expected, there was no increase in HMW without mannitol

• Process scale effect is demonstrated with 4% mannitol at 50 mg/mL

• Mannitol and freeze and thaw rates are the factors in HMW generation

Verification of Mannitol and Scale Effects mAb1 Drug Substance at 50 mg/mL

Mannitol Crystallization during Freeze and Thaw mDSC: Mannitol Effect with mAb1 at 30mg/mL, 125L rates

• Crystallization increased with mannitol concentration both on freeze

down and thaw

-24.31°C

-22.12°C

9.786J/g

-23.34°C

-22.56°C

1.285J/g

-0.010

-0.005

0.000

0.005

0.010

N

on

rev H

ea

t F

low

(W

/g)

-40

-35

-30

-25

-20

Temperature (°C)

––––––– cryo f-t aab bds 4%man-ps80 r2a.001

– – – – cryo f-t aab bds 2%man-ps80 cooling.001

––––– · cryo f-t aab bds 0%man-ps80 cooling ana.001

Exo Up

Universal V3.9A TA Instruments

0%mann

2%mann

4%mann

Cooling

-28.49°C

-30.40°C

0.6036J/g

-24.63°C

-27.97°C

0.3372J/g

0.002

0.004

0.006

0.008

0.010

0.012

No

nre

v H

ea

t F

low

(W

/g)

-40

-35

-30

-25

-20

-15

Temperature (°C)

––––––– cryo f-t aab bds 4%man-ps80 r2a.001

– – – – cryo f-t aab bds 2%man-ps80 warming ana.001

––––– · cryo f-t aab bds 0%man-ps80.001

Exo Up

Universal V3.9A TA Instruments

2%mann

0%mann

4%mann

Warming

Cooling: 0.13°C/m Warming: 0.3°C/m

K. Ho

• Increase in Fusion protein1 concentration suppresses mannitol

crystallization. Same result seen with mAb1

Frozen Liquid Stability mDSC: Protein Concentration Effect on Mannitol Crystallization

S. Tchessalov

50 mg/ml

68mg/ml

80 mg/ml

89 mg/ml

96 mg/ml

115 mg/ml

-0.02

-0.01

0.00

0.01

0.02

Heat F

low

(W

/g)

180 200 220 240 260 280 300 320 340

Time (min)

––––––– 50TRU015 in HMS.001– – – – 96 mg TRU015HMS.001––––– · 115mgTRU015HMS.001––– – – 80 mg TRU015HMS.003––– ––– 89 mg TRU015HMS.002––––– – 68 mg TRU015HMS.004

Exo Up Universal V3.9A TA Instruments

Fusion protein1

50 mg/mL

68 mg/mL

80 mg/mL

96 mg/mL

115 mg/mL

89 mg/mL

XRD Demonstrates Mannitol Crystallization Mannitol Polymorphs Identified

mAb1 DS (freeze and thaw @0.5°C/min)

00-022-1793 (*) - alpha-D-Mannitol - C6H14O6 - Y: 24.94 % - d x by: 1. - WL: 1.5406 - Orthorhombic - a 8.93900 - b 18.77800 - c 4.8960

00-022-1794 (I) - delta-D-Mannitol - C6H14O6 - Y: 30.14 % - d x by: 1. - WL: 1.5406 - Monoclinic - a 5.09500 - b 18.25400 - c 4.91900 -

00-022-1797 (*) - beta-D-Mannitol - C6H14O6 - Y: 33.25 % - d x by: 1. - WL: 1.5406 - Orthorhombic - a 8.67400 - b 16.89700 - c 5.54900

Y + 52.0 mm - Temp.: -10 °C - 4%man mAb1 bds 50mg/mL fast [004] - File: mAb1 bds50mg 4man -42C hold2 [004].RAW - Type: 2Th/T

Y + 32.0 mm - Temp.: -10 °C – mAb1 30mg 4%man [004] - File: mAb1 bds30mg 4man -42C hold [004].RAW - Type: 2Th/Th locked - St

Y + 12.0 mm - Temp.: -10 °C – mAb1 30mg 2%mann [004] - File: mAb1 bds30mg 2man -42C hold [004].RAW - Type: 2Th/Th locked - S

Lin

(C

ou

nts

)

50

100

200

2-Theta - Scale

8 10 20

δ-mannitol δ-mannitol

Mannitol

hydrate 50mg/mL, 4% mannitol

30mg/mL, 4% mannitol

30mg/mL, 2% mannitol

Scans at -10°C during warming

• Magnitude of mannitol crystallization likely below XRD detection limit

during freeze, but polymorphs observed during thaw

• Crystallization increased with mannitol concentration

• Higher protein concentration suppressed mannitol crystallization

• XRD less sensitive than mDSC for observing/quantitating mannitol

crystallization K. Ho

Freeze and Thaw of mAb1 (30 mg/mL mAb with 4% mannitol) Protein Characterization – Near UV CD and FTIR

D. Luisi, K.Ho

Circular Dichroism

(Near UV)

Wavelength nm

240 260 280 300 320 340 360

[]

(deg c

m2 d

mol-1

)

-1.4e+5

-1.2e+5

-1.0e+5

-8.0e+4

-6.0e+4

-4.0e+4

-2.0e+4

0.0

2.0e+4

4.0e+4

Start

5x F/T

• No change to tertiary structure detected by near UV CD

• No change in secondary structure detected

• Sensitivity of method or reality?

~12% HMW after 5x F/T

FTIR

mAb1 30 mg/mL

160016201640166016801700

Wavenumber, 1/cm

5xF/T

St

Subambient DSC - Mannitol vs Sorbitol

250 mM Mannitol Buffer 250 mM Sorbitol Buffer

1

3

5

7

9

11

13

Rev C

p (

J/g

/°C

)

-0.05

0.00

0.05

0.10

0.15

Nonre

v H

eat F

low

(W

/g)

-16.0 -15.5 -15.0 -14.5 -14.0

Temperature (°C)

Sample: 250 mM Mann bufferSize: 11.3940 mgMethod: liq mDSCComment: 250 mM mann buffer

DSCFile: S:...\250 mM mann buffer.001Operator: danRun Date: 10-Jan-07 13:23Instrument: DSC Q1000 V9.6 Build 290

Exo Up Universal V3.9A TA Instruments

Mannitol

Crystallization

D. Sek

• No crystallization was observed in the sorbitol buffer • Sorbitol could provide an option for reformulation

0

5

10

15

Re

v C

p (

J/(

g·°

C))

-0.01

0.00

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

0.09

0.10

No

nre

v H

ea

t F

low

(W

/g)

-45 -35 -25 -15

Temperature (°C)

Sample: sorbitol bufferSize: 10.1060 mgMethod: liq mDSCComment: 250 mM sorbitol buffer

DSCFile: S:...\sorb buff.010Operator: daveRun Date: 13-Jan-2007 07:03Instrument: DSC Q1000 V9.6 Build 290

Exo Up Universal V4.2E TA Instruments

No Sorbitol

Crystallization

Mannitol - Sucrose Combinations

Sucrose in solution helps control HMW

0

2

4

6

8

10

12

0 50 100 150 200 250 300

Ch

an

ge

in

HM

W o

ve

r 5

x F

T

[Mannitol] 0(0) 75(1.4) 150(2.7) 225(4.0) 260(4.6) 300(5.5)

[Sucrose] 300(10.3) 225(7.7) 150(5.1) 75(2.6) 40(1.4) 0(0)

Excipient concentration mM(%)

mAb3

mAb2

mAb1

mAb2 without Sucrose

Mannitol:Sucrose molar ratio of 3:1 eliminated aggregation in three mAbs

Solution at RT

F/T

Summary

• Freeze and thaw rates are important in protein frozen storage

• Mannitol-dependent HMW generation depends on Processing scale and resulting rates of freeze/thaw Mannitol crystallization Protein concentration

• mDSC and XRD revealed crystallization events

• No changes to secondary or tertiary structure and thermal transitions were detected

• High concentration protein ( 80mg/mL) could contain mannitol without an increase in HMW during freeze and thaw of protein (not a universal number)

• Use of Mannitol- sucrose combination (>3:1 molar ratio) or sorbitol are alternate options to minimize freeze-thaw induced aggregation

Outline




– Buffer choice





Part II (Process)



The Need for High Concentration Formulations

44

Need to develop High protein concentration formulations to

allow home use

~2 mL dose for sc home injection

dose of mAbs are high

Goal is to develop 100-200 mg/mL solution

Pre-filled syringes can be loaded into auto-injector

Key Issue – increase in viscosity. Prefer to keep viscosity Manufacturability (~50 cp or less)

Injectability (~20 cp or less)

Patient Convenience

44

Protein concentrations (mg/mL) in formulations of antibodies, Fc fusion products, and Fab conjugates

45

Viscosity of mAbs Show an Exponential

Dependence on Concentration

Figure adapted from Li et al. “Concentration Dependent Viscosity of Monoclonal Antibody

Solutions: Explaining Experimental Behavior in Terms of Molecular Properties” Pharm Res (2014) 31:3161–3178

mAbB: The Case for Electrostatics

Blue = positive charge

Red = negative charge

Surface Electrostatic Map for mAbB Fv

Region

0

2

4

6

8

10

12

14

Vis

co

sit

y (

cp

)

High Ionic Strength Formulations Reduce mAbB Viscosity at low pH

Low ionic strengthformulation

High ionic strengthformulation

76%

Reduction

pH Modification Does Not Ameliorate

Viscosity

47

BioTherapeutics Pharmaceutical Sciences

Why Does Viscosity Increase at Low pH?

ANS Fluorescence mAb A

mAb B mAb A

Self Association and Aggregation Observed

49

• Aggregates and clumping were seen at pH 3.5

• The pH 5.8 sample was better dispersed and did not show signs of aggregation

or significant self association

• Molecule is known to have unfavorable electrostatic interactions at pH 5.8

Low pH Middle pH

Shifting to Hydrophobically Driven

Interactions at Low pH

50

Viscosity (cp)

Control 28.3

200 mM NaCl 74.5

200 mM Arg 15.8

Viscosities of Low pH

Formulations at 90 mg/mL mAb B

• Elevated viscosity on addition of NaCl is

consistent with a hydrophobic self

association

• Hydrophobic characteristics of arginine

allow it to lower viscosity at low pH

Turbidity observed on addition of

NaCl

pH 4.6 pH 4.6

150 mM

NaCl


mAb B: Impact of Excipients on Viscosity

pH Concentratio

n (mg/mL)

Viscosity

(cp)

+ Excipient 1 + Excipient 2

(cp) (cp)

Low

(4.6) 85 18.8

25.5

(36% increase)

8.8

(53% decrease)

Mid

(5.9) 70 11.7

3.6

(69% decrease)

2.8

(76% decrease)

High

(7.0) 120

Phase

Separation

9.8

(N/A)

5

(N/A)


mAb A: Impact of pH on Viscosity


mAb A: Viscosity “Lowering” Excipients

pH Concentration

(mg/mL)

Viscosity

(cp)

+ Excipient 1

(cp)

+ Excipient 2

(cp)

Low (3.3) 200 110 84

(24% decrease)

95

(14% decrease)

Optimal

(5.1) 300 70 -

78

(11% increase)

Optimal

(5.1) 250 19

40

(111% increase) -

High

(8.0) 250 75

94

(25% increase)

47

(37% decrease)


Why Should Space and Shape Matter?

mAb concentration

(mg/mL)

Distance between center of molecules*

(nm)

100 13.6

150 11.8

200 10.8

250 10.0

300 9.4

350 8.9

*Assumes MW of 150 kDa

Conclusion

• Elevated viscosities remain a challenge for the delivery of high concentration

formulations

• mAb B has high viscosity even at relatively low concentrations

pH adjustment did not ameliorate viscosity issue

Excipients, both of which raise ionic strength, improve viscosity.

Improvement most pronounced around the pI of the molecule

• mAb A

Despite already having a desirable viscosity profile, further

improvement through formulation is possible

Optimal pH range was found to be 4.6-5.6

Viscosity increases at higher pH values

Viscosity increases sharply at pH values below 4.0

Excipients were not beneficial compared to optimized pH

• TEM with image analysis was a useful tool for exploring molecular dynamics

• Self association was observed to shift from being driven by electrostatic

interactions to hydrophobic interactions as a result of moving to low pH

values

mAb Morphology

56

Low pH Mid pH

• Antibody monomers appear as expected

• Approximately 15 nm particles

• Fab, Fc regions approximately 7 nm

key formulation challenges of protein (mab) drugs

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