winter process chemistry conference - almacgroup.com · cold storage formulation development...

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Winter Process Chemistry Conference

December 14th 2016

2

Almac overview- contract services supporting pharmaceutical development

4th Winter Process Chemistry Conference 14 December 2016 [email protected]

• Founded in 1968

• Global HQ in Craigavon, Northern

Ireland, US HQ in Pennsylvania

• ~4,600 personnel globally

• Unique ownership – charitable

foundation

• All profit re-invested

PHARMA SERVICES

Pharmaceutical development and DP

Commercial services

CLINICAL TECHNOLOGIES

Technology solutions for clinical trial

design and data management

DIAGNOSTICS & BIOMARKERS

Biomarker discovery

Biomarker development

CLINICAL SERVICES

Clinical trial supply – labeling,

packaging, distribution.

SCIENCES

Peptide and small molecule APIs

Analytical development

Physical sciences

3

Almac’s global presence


• Facilities with ~4,600 personnel in:

– Europe– Northern Ireland– Republic of Ireland– Scotland– England

– US– Pensylvania– North Carolina– California

– Asia– Japan– Singapore

4

Craigavon – global HQ


Document

archive

Secondary

manufacture

API

Drug Product

Clinical Trial Supply

Cold Storage

Formulation

development

Analytical

development

GMP API

Manufacture

Micronisation

Radiolabelling

Biocatalysis

Process & Analytical development,

Physical Sciences

Small Scale GMP API manufacture

GMP Peptide manufacture

Formulation &

Tablet & capsule

manufacture.

Drug reconciliation

& destructionWarehouse

Engineering

ICH Stability

5

Almac Sciences – API services


Sciences

Peptide Manufacture

Small Molecule API Manufacture

Biocatalysis

14C radio-labelling

Analytical services

Solid state services

6

Peptide capabilities at Almac


Non-GMP Custom Synthesis

• High throughput parallel manufacture; >9,000 peptides

• Long sequences – routinely >100 amino acids (longest 276 amino acids)

• Complex products, route investigation, 14C radiolabelling

Process and Analytical Development

• Process design and development

• Small scale models representative of manufacturing equipment

• Extensive analytical capability

cGMP Manufacture

• Parallel manufacturing streams

• Full analytical support and stability testing

• Strong embedded project management culture HQ, Craigavon

Edinburgh Technopole

7

Peptides compared with other therapeutic classes


Molecular weight

Small

molecule

Peptide Protein Monoclonal

antibody

Advantages of peptides:

High levels of specificity

Low toxicity

High potency

Modification to impart favourable properties

Disadvantages of peptides:

Easily metabolised by proteases

Poor oral bioavailability

Tend to be injectables

<500 1,000-4,000 10,000 150,000

8

Peptide market


30

20

10

Year

20202015201020052000

$Bn

Per year

Source:

Kaspar, and Reichart, Drug Discovery Today (2013) 18, 807-17

• Currently approximately 70 approved

peptide drugs on the market.

• Approximately half approved since 2000

• Approval rate 2-3 per year – fairly steady in

that 15 year period

• Several hundred products in clinical

pipeline

9

Manufacturing sources of peptides

Extraction from natural

sourcesRecombinant

Chemical synthesis

Semi-synthetic


• Liquid phase synthesis

• Solid phase synthesis

• “Third generation” hybrid

• Solid/liquid fragment strategies

• Native chemical ligation

• Scorpion, snake, snail venom

• Bacteria derived

• Proteins

• Antibodies

• Engineered proteins

10

Manufacturing sources of peptides


Extraction from natural

sourcesRecombinant

Chemical synthesis

Semi-synthetic

• Liquid phase synthesis

• Solid phase synthesis

• “Third generation” hybrid

• Solid/liquid fragment strategies

• Native chemical ligation

• Scorpion, snake, snail venom

• Bacteria derived

• Proteins

• Antibodies

• Engineered proteins

11

General manufacturing principles

1. Synthesis (solid phase)

2. Cleavage/deprotection

3. Purification

4. Counterion exchange

4-ish manufacturing steps


Solution phase

modification

12

Solid phase peptide synthesis (SPPS)

• Bruce Merrifield, 1963

• Building up the peptide on an insoluble polymeric solid support

• Intermediates are easily filtered

• Allows use of excess reagents, and amenable to automation


Basic principles

Site of

attachment of

peptide C-

terminus

Insoluble polymer

“Cleavable”

linker

13

Peptide manufacture – general challenges


Making peptides is easy! It’s just amide bonds??!!

Chemical protection required for carboxyl, amine, and side chain

14

Natural amino acids


All natural amino acids are

(S) / L configuration

15

SPPS – protecting groups


H2N

O

O OH

Glutamic acid immobilisedon SPPS resin

H2N

O

OH

SH

Cysteine

+

16

Fmoc-SPPS


Base cleavable

Acid cleavable

Acid cleavable

Acid cleavable

17

SPPS – repeated cycle


• A small amount of incomplete chemistry soon adds up…

• 70mer peptide

– 97% yield – 0.97140 = 1.4% overall yield

– 99% yield – 0.99140 = 24% overall yield

– 99.5% yield - 0.995140 = 50% overall yield

• Can also see major failure points

18

SPPS - automation


19

Process development fundamentals


190 sub unit

operations

described

8 Families defined

Process Best View

• Scale models of manufacturing equipment

• Representative reagent addition, agitation

methods, temperature control

• Reproduction of manufacturing process times

and hold points

20

Nature of resin


Polystyrene, cross-

linked with 1%

divinlybenzne

Polyethlene glycol

PEG/polysterene

grafts

Loading 0.1-1.5 mmol/g

Bead size

Swelling properties

Functionality: acid,

amide, amine, thiol

Lability

Mathias Junkers, SAFC

21

Solid phase synthesis - coupling agent


22

Solid phase synthesis

Advantage of capping


23

Monitoring synthesis process


Uv monitoring at 302nm

24

Solid phase synthesis: impact of capping


12/15/2016

Ac-Asp12 truncate

Ac-Ile14 truncate

Ac-Ser6 truncate

Full length peptide

Ac-Lys3 truncate

Ac-Val4 truncate

Ac-Ser11 truncate

Poor synthesis

Capping allows chromatographic

separation of truncates

25

Improving difficult syntheses – pseudoproline dipeptides


Oxazolidine cleavage through TFA treatment as part of standard deprotection conditions

26

Improving synthesis yield with pseudo-proline dipeptides

• Two pseudo-prolines incorporated at problem regions

• Increased crude purity from 15% to 70%


27

Overcoming Asp-Pro instability – acid mediated


Coupling agent23-mer

Crude purity

23-mer

Synthesis Yield

Oxyma 81% 47%

HOBt 81% 62%

28

On-resin dimerisation

Unusual dimer target required on-resin dimerisation


Peptide 2

Linker

Linker

Peptide 1

Peptide 1

• High-loading required to ensure effective dimerization

• Coupling agent crucial to effective dimerization

• Repeat PyBOP coupling the most effective

29

Problematic by-products


CO2H

CO2H

CO2X

CO2X

CO2

CO-PIPERIDINE

Piperidine

traces from

deprotection

Dimeric target

CO2

PIPERIDINE-CO

CO2HFmoc-NH

Standard amino acid coupling

CO2XFmoc-NH

Piperidine

trances from

deprotection

Fmoc-NH

CO-PIPERIDINEFmoc-NH

Stays in solution and

washed away

• Resolved by careful washing after preceding piperidine treatment step

30

Cleavage/deprotection


• Simultaneous removal of side-chain protecting groups and cleavage from the resin in a single step.

• Strong acid conditions.

• Cocktail of scavenger molecules to prevent the cleaved protecting groups recombining with the

peptide.

• Crude peptide isolated by precipitation

PGPGPGPG

31



90-95% TFA +

scavengers

Filter resin

Precipitate crude

peptide

Filter crude peptide

Strong acid and scavengers to mop up cations. Multiple simultaneous reactions.

Many potential by-products

• Irreversible addition of protecting groups

• Backbone hydrolysis

• Oxidations

Physical form crucial to enable good filtration

Isolated solid crude

peptide

32

Peptide degradation


33

Hydrophilic peptide – solution phase isolation


90-95% TFA +

scavengers

Filter resin

Precipitate crude

peptide

Filter crude peptide

Isolated solid crude

peptide

X

Very hydrophilic peptide

did not produce a

filterable solid.

90-95% TFA +

scavengers

Filter resin

Precipitate crude

peptide

Extract into water

Isolated crude peptide

solution

34

Solution phase telescoping – impact on purification


Smearing of

peak during

prep

chromatography

Sharper peak

and easier

chromatography

Evaporation to

remove excess

ether

35

Acid labile peptides


36

Cleavage without deprotection


PeptidePG

PG PG

PeptidePG

PG PG

NH2

Protected

peptide on acid-

labile resin

Crude-protected

peptide

PeptidePG

PG PG

Spacer X

Incorporation of

functionalised

spacer

Peptide Spacer X

Deprotected

peptide/spacer

couple

37

Impact of improving cleavage method



Cleavage

Purification/freeze-dry

Coupling with spacer


Deprotection


Cleavage step

was “10-20”

treatments with

1% TFA

3 purification

and freeze-dry

steps


Cleavage

Coupling with spacer

Deprotection


Cleavage

reduced to single

HFIP treatment

Single-pot

Single

purification /

freeze-dry

Overall yield increased from

15% to ~30%, and processing

time reduced by 1/3

38

Purification

• Standard approach for purification of pure

peptide from failure sequences and by-

products

• Non-polar stationary phase retains crude

peptide mixture, which is eluted with

acetonitrile / water gradient. Most polar

compounds elute first.

• Not perfect - more difficult for longer peptides

• Scalable – up to 1m diameter columns

Work-horse method: Reverse Phase HPLC


39

Purification fundamentals

• Usually includes a counterion exchange step

• Final product isolation by concentration and freeze-dry

• Development activities

– Crude peptide solubility

– Quantity per injection

– Packing media type

– Buffer composition

– Gradient

– Pooling criteria

– Pool stability

– Column re-use


40

Impact of column size

DiameterCrude peptide

loading (g)Flow rate

0.46cm 0.02 1 mL/min

1cm 0.10 4.7 mL/min

2cm 0.43 21 mL/min

5cm 2.4 118 mL/min

8cm 6.3 300 mL/min

15cm 22.0 1060 mL/min

30cm 88.0 4200 mL/min


Process development basics….

Keep bed height constant

Keep linear flow rate constant

41

Stability during purification - pyroglutamate


Peptide

Peptide

22.8

76

24.2

32

25.0

36

25.6

06

25.9

04

C22-A

5 -

26.5

67

27.0

83

27.4

22

27.7

01

27.9

56

28.6

42

30.1

43

30.2

83

31.3

19

31.4

68

34.4

69

34.5

04

34.6

05

34.8

29

Pry

o-G

lu -

35.5

44

38.1

77

39.4

65

AU

0.002

0.004

0.006

0.008

0.010

0.012

0.014

0.016

0.018

0.020

0.022

0.024

0.026

Minutes

20.00 21.00 22.00 23.00 24.00 25.00 26.00 27.00 28.00 29.00 30.00 31.00 32.00 33.00 34.00 35.00 36.00 37.00 38.00 39.00 40.00 41.00 42.00

• Usually well separated, but impossible to avoid formation

• Minimise by avoiding extremes of pH during purification

42

Long peptides – orthogonal purification


Peptide

• 104-mer peptide, 12kDa

• 2 Cys existing in reduced form

• Oligomers formed through oxidation of Cys to

disulfide bridges

• Crude peptide could not be purified by HPLC

alone

43

Size exclusion chromatography


Crude peptide

Peak 2

Peak 4

Peak 1

Reduced peptide

Peak 4

Peak 3

Peak 1

Peak 2

Peak 3Peak 1: high MW oligomer

Peak 2: product

Peak 3: major truncate

Peak 4: minor truncate

44

104mer final process




Reduction/SEC

purification

HPLC purification

Counterion exchange

Freeze-dry

45

Counterion exchange

• Purification usually results in undesirable TFA salt form – need to exchange

Why bother?


• Acetate usually chosen - weak salt has advantages in physiological conditions, but others also

used (chloride, sulfate)

• Can also use cations such as sodium or ammonium – care with stability!

46

Counterion exchange process


Product pool from

purification

Dilute and reinject on

column

Wash over with

counterion

Elute with acetonitrile

gradient

Hold on top of column

and wash

47

Control of ionic form of some peptides can be difficult


Split elution

48

Better control using ammonium acetate directly for purification


Purification using ammonium acetate Standard process using TFA

Also decomposition at low pH

(Asp-Pro) hydrolysis

49

Process intensification means win-win


Crude peptide

Portion 1

purification

(20 injections)

Portion 1 AEX

(4 injections)

Portion 1 freeze-

drying

Portion 2

purification

(20 injections)

Portion 2 AEX

(4 injections)

Portion 2 freeze-

drying

Reconstitution and

freeze-drying

Overall process time ~ 6 weeks

Crude peptide

Neutral purification

(32 injections)

Buffer removal and

concentration

(4 injections)

Freeze-drying

Overall process time ~ 3 weeks

50

Counterion exchange - dealing with instability

Maleimide containing peptide


AU

-0.10

-0.05

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0.50

0.55

0.60

Minutes

16.00 16.50 17.00 17.50 18.00 18.50 19.00 19.50 20.00 20.50 21.00 21.50 22.00 22.50 23.00 23.50 24.00 24.50 25.00 25.50 26.00 26.50 27.00 27.50 28.00 28.50 29.00 29.50 30.00

HPLC of acetate salt prior to

freeze-drying (pH = 4) HPLC of acetate salt after freeze-

drying (pH = 6-7)

In the end, sulfate gave the best mix of product stability and physical form

51

Peptides – final thoughts

• High level of interest remains in peptides as therapeutics

• Solid phase synthesis allows anyone to make a peptide, but making it well is more difficult

• Robust principles of process development should be applied to peptides as they would to

small molecules


52

Acknowledgements


David Anderson

Colin Dunsmore

Emma Duffy

Gillian Gray

Craig Johnston

Jennie Jamieson

Andy Kennedy

Beatrice Maltman

Kevin Shaw

Rachel Slater

Andrew Stewart

Brian Whigham

Kerry Woznica

Yannick Borguet

Anthony Clouet

Linda Devine

Lynda Henderson

David Lagnoux

Hazel Moncrieff

Gareth McConville

Steve McIntyre

Alex Saunders

Nhlanhla Sibanda

Alan Thompson

Ruth Bell

Nicole Burke

Osama Chahrour

Katarzyna Kalternberg-Ziolkowska

John Malone

Claire McCambley

Barbara O’Connell

Chaitali Patel

Thank you!

winter process chemistry conference - almacgroup.com · cold storage formulation development...

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