Improving Physicochemical Properties of Biopharmaceutical Drug Candidates
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Improving Physicochemical Properties of Biopharmaceutical Drug Candidates. David Litzinger , PhD Director, Pharmaceutical Sciences Amylin Pharmaceuticals, Inc. PEGS Conference May 20, 2010 Boston, MA. Analogs in Drug Development Comparisons Across Platforms. Immunogenicity Concern.

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Improving physicochemical properties of biopharmaceutical drug candidates

Improving Physicochemical Properties of Biopharmaceutical Drug Candidates

David Litzinger, PhD

Director, Pharmaceutical Sciences

Amylin Pharmaceuticals, Inc.

PEGS Conference

May 20, 2010

Boston, MA


Improving physicochemical properties of biopharmaceutical drug candidates

Analogs in Drug Development

Comparisons Across Platforms

Immunogenicity

Concern

Drug Platform

(typical MW)

Analog Evaluation

in Drug Development

Small

Molecules

<500 Da

Negligible

High

Peptides

1-6 kDa

Slight

Medium

Proteins

15-150 kDa

Significant

Low


Improving physicochemical properties of biopharmaceutical drug candidates

Peptide Analogs as Drug Substances

Examples Related to Aggregation


Improving physicochemical properties of biopharmaceutical drug candidates

Peptide and Protein Optimization

Example Options for Improving Physical Stability

Approaches to Improving

Physical Stability*

Chemical

Modification

Mutational

  • Mutations based on superior

    properties in alternate species

  • Decrease hydrophobicity

  • Increase hydrophilicity

  • Increase net charge

  • Changing the pI

  • Polymer conjugation

* More that one approach can be combined


Improving physicochemical properties of biopharmaceutical drug candidates

Glucose-Dependent Insulinotropic Polypeptide

Example of Analog Evaluation in Drug Development

  • Glucose-dependent Insulinotropic Polypeptide (GIP)

    • 42-amino acid hormone synthesized and secreted from intestinal K-cells

    • Integral role in regulating insulin secretion and response

    • Amylin Pharmaceuticals currently investigating GIP as a possible

      mono- or combination therapy for Type 2 Diabetes Mellitus

  • Development Challenges

  • Native GIP rapidly inactivated by dipeptidyl peptidase-IV (DPP-IV) and has a

    very short half-life

  • Development of GIP analogs challenging due to poor solubility

  • Second Generation Effort (G2)

  • G1 effort addressed DPP-IV metabolism, optimized activity

  • G2 GIP analogs identified and evaluated for improved solubility

    • In Silico modeling used for primary sequences analysis

    • pH-solubility profile, physical and chemical stability were screened

    • CD used to monitor secondary structure


Improving physicochemical properties of biopharmaceutical drug candidates

GIP Drug Development

History and Efforts to Identify Alternative GIP Analogs

Note: Biological Activity- Receptor binding, mouse OGTT, mouse GL, DOA by rat IVGTT, plasma stability, HbA1c in ob/ob mice

Physical Stability- Aggregation, precipitation, solubility

2nd Round of Screening

Native GIP (1-42)

G1 Analogs

G2 Analogs


Improving physicochemical properties of biopharmaceutical drug candidates

GIP Analog Screening

Primary Sequence Ranking by In Silico Tools

Underlined residues denote substitutions; Red - potentially labile residues; Blue – C-terminal end modification

  • Sequences ranked according to In Silico modeling and assessment tools

    • Tango2 – Protein aggregation prediction model based on TANGO algorithm of physico-chemical principles of b-sheet formation

    • In Silico Tool – Primary sequence assessment and pharmaceutical properties predictor

      created in-house

      • GRAVY– Grand average of hydropathicity:  GRAVY value,  hydrophobicity ( solubility)

      • Peptide Charge Calculator – Computes theoretical net charge on peptide from composition of ionizable residues

  • Compounds synthesized and evaluated


Improving physicochemical properties of biopharmaceutical drug candidates

GIP Analog Screening

In Silico Pharmaceutical Property Assessments

Solubility

Chemical Stability

Hydrophilicity

Aggregation

ID #

Calculated

pH 4

pH 7

Net Charge

Net Charge

Overall

(Gravy Score)

(Tango 2 Score)

Potential Labile Residues

pI

Solubility

Solubility

pH 4

pH 7

Stability

Human GIP

Fair

Fair

-0.80

-7.00

7.5

Good

+ 5.68

+ 0.39

D(4), M(1), N(3), Q(4), W (2)

(1-42)

Average

Average

Fair

Fair

G1-A

-0.37

-7.10

5.8

+ 2.86

- 0.85

Good

D(3), M(1), N(1), Q(3), W (1)

Average

Average

Fair

Fair

G1-B

-0.42

-6.78

5.8

+ 2.86

- 0.85

Good

D(3), M(1), N(2), Q(3), W (1)

Average

Average

Fair

G2-C

-0.41

-13.89

8.6

Good

+ 3.86

+ 0.91

Good

D(1), N(1), Q(3), W (1)

Average

Fair

G2-D

-0.50

-14.57

9.2

Good

+ 4.86

+ 1.91

Good

D(1), N(1), Q(3), W (1)

Average

  • G2 analogs showed improved properties over G1 analogs:

    • Higher pI

    • Good solubility at acidic pH

  • Labile Residues:

    • D – potential aspartic acid isomerization at pH 4

    • M, W – potential site for oxidation

    • N, Q – potential deamidation

  • Fair/Average solubility at neutral pH

  • Highly charged at pH 4 compared to pH 7


Improving physicochemical properties of biopharmaceutical drug candidates

Measured Solubility Results

G2 Analogs Have Improved Solubility

ID #

Solubility at Formulated pH

Hydrophilicity (Gravy Score)

Aggregation (Tango 2 Score)

Measured pI

Calculated pI

Human GIP (1-42)

nd

-0.80

-7.00

6.7

7.5

G1-A

< 1 mg/ml

-0.37

-7.10

5.8

5.8

G1-B

~ 1 mg/ml

-0.42

-6.78

4.7

5.8

G2-C

> 5 mg/ml

-0.41

-13.89

8.4

8.6

G2-D

> 5 mg/ml

-0.50

-14.57

9.0

9.2

Note: nd – not determined

  • G2 analogs show improved solubility profile compared to the G1 analogs


Improving physicochemical properties of biopharmaceutical drug candidates

Clear, Colorless

Slight Precipitation, Aggregation

Moderate to Severe Precipitation, Aggregation

Formulation Screening

G2 Analogs Have Improved Physical Stability

Visual Analysis

  • G2 analogs proved to have the most physically stable profile.

1 mg/mL concentration;

No agitation


Improving physicochemical properties of biopharmaceutical drug candidates

Secondary Structure Analysis

Evaluation of GIP Analogs

Far UV CD

Far UV CD

  • G2 analogs show greater α-helical content

    • Correlates with less aggregation

    • Similar 2° structure at both pH 4 & 7


Improving physicochemical properties of biopharmaceutical drug candidates

GIP Analog Optimization

Conclusions

  • G1 analogs demonstrated improved biological efficacy and longer

    duration of action compared to native GIP, but had poor physical stability

  • G2 analogs showed both improved efficacy and physical stability

    • Experimental results correlated well with their higher net charge and more negative GRAVY scores predicted in silico.

    • At 1 mg/mL concentrations were physically and chemically stable under the tested conditions with little to no visible aggregation. 

    • Secondary structure is predominantly α-helical in liquid state (pH 4.0 and pH 7.0)


Improving physicochemical properties of biopharmaceutical drug candidates

Metreleptin

Compound Properties and Obesity Treatment Approaches

  • 16.2 kd 147 amino acids, (native

  • leptin 146 AA)

  • Isoelectric point 6.1

  • Single disulfide bond

  • No free cysteines

  • Limited solubility at neutral pH, 2-3

  • mg/mL, higher at lower pH

  • Four helix bundle tertiary structure

  • Amgen pursued leptin monotherapy as a treatment for obesity

    • High dose, up to 0.3 mg/kg (~30 mg per injection)

    • Heymsfield et al. (1999) JAMA

  • Amylin is evaluating leptin in combination with pramlintide for

    treatment of obesity

    • Lower dose

    • Roth et al. (2008) PNAS


Improving physicochemical properties of biopharmaceutical drug candidates

Metreleptin

Charge Profile

Net Charge of Metreleptin vs pH

  • Calculated pI= 6.1

  • Suggests high solubility at low pH, and low solubility at neutral pH

Charge calculator/pI finder by Gale Rhodes

http://spdbv.vital-it.ch/TheMolecularLevel/Goodies/Goodies.html


Improving physicochemical properties of biopharmaceutical drug candidates

Metreleptin

Solubility Profile

○leptin solubility

▲reversibility of precipitation

  • Solubility of human leptin

    • At low pH is high

      • > 70 mg/mL at pH 4

    • At neutral pH is low

      • 2-3 mg/mL

  • Precipitation at neutral pH

    is essentially irreversible

  • Murine leptin is more soluble than human leptin at neutral pH

    • 43 mg/mL for murine leptin

    • 31 mg/mL for W100Q/W138Q analog

Ricci, M.S. et al. (2006) in Misbehaving Proteins. New York: Springer.


Improving physicochemical properties of biopharmaceutical drug candidates

Human and Murine Leptin

Amino Acid Sequence Comparison

  • Comparison of human and murine leptin sequences

0

10

20

30

HUMAN:

MURINE:

MVPIQKVQDD

MVPIQKVQDD

VSSKQKVTGL

VSAKQRVTGL

TKTLIKTIVT

TKTLIKTIVT

RINDISHTQS

RINDISHTQS

40

50

60

70

HUMAN:

MURINE:

DFIPGLHPIL

DFIPGLHPIL

SRNVIQISND

SQNVLQIAND

TLSKMDQTLA

SLSKMDQTLA

VYQQILTSMP

VYQQVLTSLP

80

90

100

110

HUMAN:

MURINE:

LENLRDLLHV

LENLRDLLHL

LGGVLEASGY

LDGVLEASLY

LAFSKSCHLP

LAFSKSCSLP

WASGLETLDS

QTSGLQKPES

120

130

140

HUMAN:

MURINE:

STEVVALSRL

STEVVALSRL

QGSLQDMLWQ

QGSLQD I LQQ

LDLSPGC

LDVSPEC

Residues that differ between the human and murine sequences are in red.

Note that the first methionine residue associated with E. coli production is not counted.

  • Differ at 22 sites

  • Sequence differences of particular significance in

    solubility/aggregation properties


Improving physicochemical properties of biopharmaceutical drug candidates

Metreleptin

Surface Modeling

Hydrophobicity SurfaceBrown = Lipophilic Blue = Hydrophilic, charged

Electrostatic SurfaceRed = Basic (+) Blue = Acidic(-)

Trp 138

  • Surface modeling shows region around Trp 138 has potential role in aggregation

    • Low electrostatic potential

    • High lipophilicity

Benchware3DExplorer (Tripos)


Improving physicochemical properties of biopharmaceutical drug candidates

Human Leptin

Evidence for Leptin Conformational Transition with pH Change

● human

○ murine

  • Increased ANS fluorescence

    at pH 4 to 5

    • Not observed for murine

      leptin

  • Suggests a folding intermediate with increased hydrophobicity

    populated at pH 4-5

  • May result in the formation of soluble multimeric species under

    acidic conditions

Ricci, M.S. et al. (2006) in Misbehaving Proteins. New York: Springer.


Improving physicochemical properties of biopharmaceutical drug candidates

Human Leptin

Low pH Aggregation and Relation to Neutral pH Precipitation

▲ human, % aggregates, pH 4

●human, % precipitation, pH 7

∆murine, % aggregates, pH 4

○murine, % precipitation, pH 7

  • Initial concentration at low pH varied

  • Precipitation induced by diluting into

    neutral pH buffer

    Inset: human leptin multimers formed

    at 50 mg/mL, pH 4:

    • diluted to 5 mg/mL, pH 4

    • diluted again into pH 7

Human leptin

  • Forms multimers at low pH

  • Precipitation correlates with multimer formation

  • Multimers formed at acidic pH dissociate upon dilution in acid pH

  • Precipitation at pH 7 decreases with multimer dissociation

  • Did NOT form multimers and did not precipitate

Murine leptin

Ricci, M.S. et al. (2006) in Misbehaving Proteins. New York: Springer.


Improving physicochemical properties of biopharmaceutical drug candidates

N

I

U

Iassoc

precipitation

Human Leptin

Proposed Aggregation Mechanism

Increased hydrophobicity at

acidic pH not observed**

Murine Leptin

Precipitation not

observed*

Multimers not

observed*

,

* Under conditions in which human leptin precipitated

and formed multimers.

N: native state

I: folding intermediate

U: unfolded conformer

Iassoc: folding intermediate self associated into a soluble multimer

** As observed for human leptin in ANS studies.

Ricci, M.S. et al. (2006) in Misbehaving Proteins. New York: Springer.


Improving physicochemical properties of biopharmaceutical drug candidates

O

O

Chemical Modification Example

Succinylation

O

+

Protein-N-C-CH2-CH2-C-O-

Protein-NH2

O

H2

O

  • Reaction at pH 7.0

    • 5-fold molar excess of succinic anhydride

    • 2-16 hours at 4oC

  • Purification by ion exchange chromatography

    • 45-47% final yield

  • Site-specific conjugation to N-terminus

    • Endoproteinase Lys-C

    • Peptides resolved by RP-HPLC

  • M1-K6: N-terminal peptide

  • Succ-(M1-K6): Succinylated

    N-terminal peptide

From Gegg et al. US Patent 6,420,340


Improving physicochemical properties of biopharmaceutical drug candidates

Two Related Examples

DTPA and EDTA

O

O

Diethylenetriamine-

pentaacetic acid (DTPA)

O

N-R-N

O

COOH

O

O

CH2

H2O (1) orH2N-Protein (2)

H2N-Protein

-CH2-CH2-N-CH2CH2-

R =

H+

(1)

(2)

O

O

O

O

OH

HO

Ethylenediaminetetra-

acetic acid (EDTA)

HO

OH

N-R-N

N-R-N

OH

-CH2-CH2-

R =

O

O

O

O

N-Protein

N-Protein

N-Protein

H

H

H

Dimer conjugate

Monomer conjugate

From Gegg et al. US Patent 6,420,340


Improving physicochemical properties of biopharmaceutical drug candidates

Succinylation and Related Modifications

Impact on pI and Solubility of Metreleptin

Sample

Maximum Solubility in PBS* (mg/mL)

Change in pI

Unmodified leptin

Succinyl-leptin

DTPA-leptin monomer

EDTA-leptin monomer

3.2

8.4

31.6

59.9

N/A **

-0.7 **

Not reported

Not reported

* pH = 7.0

** Leptin pI = 6.1; succinyl-leptin estimated to be 5.4

  • Conjugations with acidic moieties to the N-terminus lower pI and increase

    solubility at neutral pH

From Gegg et al. US Patent 6,420,340


Improving physicochemical properties of biopharmaceutical drug candidates

Succinylation

Reduces Injection Site Precipitation of Metreleptin

  • Three mice dosed per sample

  • Tissues sections from the injection sites examined histologically

Concentration

(mg/mL)

Injection volume

(mL)

Sample

Precipitation

Acetate buffer, pH 4.0

Unmodified leptin

(in acetate buffer, pH 4.0)

PBS buffer, pH 7.5

Succinyl-leptin

(in PBS, pH 7.5)

0

0

0

50

50

50

0

0

0

50

50

50

20

20

20

20

20

20

20

20

20

20

20

20

0

0

0

4

4

1.5

0

0

0

0

0.5

0

Score system: 0 Normal, 0.5-1 minimal change, 1.5-2 mild change,

2.5-3 moderate change, 3.5-4 marked change, 4.5-5 massive change

From Gegg et al. US Patent 6,420,340


Improving physicochemical properties of biopharmaceutical drug candidates

Succinylated and Related Metreleptin Conjugates

Retain In Vivo Activity

  • Similar activity in vivo for conjugates relative to unmodified leptin

  • Normal mice dosed s.c. daily, 10 mg/kg

  • Results shown as % weight-loss relative to buffer control

From Gegg et al. US Patent 6,420,340


Improving physicochemical properties of biopharmaceutical drug candidates

Polymer Conjugation Example

PEGylation

  • What is PEGylation?

  • Covalent attachment of poly(ethylene glycol) (PEG)

  • Example PEGylation reagent:

CH3O-(CH2-CH2-O)n-CH2-CH2-X

Reactive group

Methoxy cap

  • Why PEGylation?

  • Slow clearance/maintain circulating concentrations/reduce dose frequency

  • Increase solubility

  • Reduce aggregation

  • Reduce proteolysis

  • Reduce immunogenicity

  • In several approved products


Improving physicochemical properties of biopharmaceutical drug candidates

NeH3+

O

NeH3+

H-C-PEG

-OOC

Protein

NH2

-OOC

Protein

NH-CH2-PEG

NaCNBH3

NeH3+

NeH3+

Site-Directed PEGylation

N-Terminal Site-Specific Example

  • Low pH selectively protonates lysine e-amino groups

  • N-terminal amino group remains unprotonated and reactive

  • Reductive alkylation specific to the N-terminus

Example: Neulasta® (20kDa PEG-rhGCSF)

  • Why site-directed PEGylation?

  • Optimally preserve biological activity

  • Homogenous product/consistent lot-to-lot activity


Improving physicochemical properties of biopharmaceutical drug candidates

Effect of PEGylation on Solubility

PEG-GCSF Has Improved Solubility

  • Under conditions in which GCSF rapidly precipitated, 20kDa PEG-GCSF

    remained completely soluble

Samples formulated at 5 mg/mL in phosphate buffer, pH 6.9 and incubated at 37oC

  • PEG-GCSF remained clear and showed no turbidity, unlike GCSF

  • Free PEG was unable to prevent GCSF precipitation

From Rajan, R.S. et al. (2006) Protein Science


Improving physicochemical properties of biopharmaceutical drug candidates

PEG-GCSF Forms Soluble Aggregates

Analysis by Size-Exclusion Chromatography

  • Significant loss of GCSF monomer due

    to conversion into insoluble forms

  • 20K PEG-GCSF accumulated into soluble,

    higher order multimeric forms eluting in

    the void volume

* Aliquots analyzed after 72 h of incubation at neutral pH and 37oC

From Rajan, R.S. et al. (2006) Protein Science


Improving physicochemical properties of biopharmaceutical drug candidates

PEGylation and Aggregation

Mechanism Findings

  • PEGylation does not alter the linkages or heterogeneity of the aggregates

  • Resolubilized GCSF and PEG-GCSF soluble aggregates comparison

    • Both included a mixture of monomer, dimers, trimers, and higher order multimers

    • Multimers in both cases were covalent, disulfide-linked

    • Similar extent of covalent formation

  • PEGylation does not alter the helix-to-sheet transition that accompanies

    aggregation

  • GCSF and PEG-GCSF showed similar starting FTIR spectral profiles as well

    as temperature-induced conversion to b-sheet

  • The GCSF precipitate and the PEG-GCSF soluble aggregate showed similar

    extent of b-sheet content by FTIR analysis

  • PEGylation confers improved solvation by water molecules

    • In phase partition studies, GCSF aggregates partitioned to octanol while

      PEG-GCSF aggregates remained in the aqueous phase

From Rajan, R.S. et al. (2006) Protein Science


Improving physicochemical properties of biopharmaceutical drug candidates

Aggregation and Drug Development

Improving the Drug Compound

  • Identify potential issues early

    • Dose level, dose concentration

    • Solubility at physiological pH

    • Manufacturing, shipping and handling

  • Consider strategy to reduce aggregation

    • Remove aggregates during manufacture

    • Formulate to prevent aggregate formation

    • Modify the compound to reduce/remove aggregation potential

  • Generally, testing compounds early is preferred

    • Logistical benefit, test compounds while in vitro and in vivo screens are

      in process (rather than restarting assays)

    • Opportunity to solve before Candidate Nomination


Improving physicochemical properties of biopharmaceutical drug candidates

Early Pharmaceutical Development

Opportunities to identify and solve aggregation issues

during SAR development

Stage 1

  • Analytical method

    development

  • Early screening

Stage 3

  • IND enabling

  • Phase I enabling

Stage 2

  • Analytical method

    optimization

  • Late screening

  • Developability risk

    assessment

Compound screening

Team formation

IND

Candidate nomination

Pre-project activities

  • In silico modeling

Phase I activities

  • Monitor

  • Address questions/issues


Improving physicochemical properties of biopharmaceutical drug candidates

Acknowledgments and References

Acknowledgments

Pharmaceutical Sciences

Steven Ren

Derrick Katayama

Ellen Padrique

Johnny Gonzales

Jenny Jin

Biology, cont’d

Pam Smith

Christine Villescaz

Tina Whisenant

Lynn Jodka

Kim DeConzo

Julie Hoyt

Jenne Pierce

Amy Carroll

Aung Lwin

Informatics

Eugene Coats

Robert Feinstein

Paul Nelson

Research Chemistry

Odile Levy

Ramina Nazarbaghi

Lawrence D’Souza

John Ahn

Biology

Diane Hargrove

Eric Kendall

Augustine Cho

Krystyna Tatarkiewicz

Slave Gedulin

Bioanalytical Chemistry

Swati Gupta

Kristine De Dios

Liying Jiang

References

M.S. Ricci et al. (2006) Mutational Approach to Improve Physical Stability of Protein

Therapeutics Susceptible to Aggregation. In Misbehaving Proteins (Murphy RM and

Tsai AM, ed) pp331-350. New York: Springer.

Gegg, C. and Kinstler, O. (2002) Chemical modification of proteins to improve

biocompatibility and bioactivity. US Patent 6,420,340

Rajan, R.S. et al. (2006) Modulation of protein aggregation by polyethylene glycol

Conjugation: GCSF as a case study. Protein Science 15: 1063-1075.


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