almac - the manufacture of peptides for clinical trials - 2nd irish peptide symposium june 2012

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Confidential © Almac Group 2012

The manufacture of peptides for clinical trials

Steven McIntyreTeam Leader, Peptide Operations

2nd Irish Peptide Symposium14th-15th June 2012

• Background• The Almac approach• Case Studies• New technologies• Summary


• Contract Research / Manufacturing Organisation– Fully integrated service provider

– Pharmaceutical & Clinical Development Services

• Founded in 2003, privately owned

• Total employees ~3000

- 2000 UK, 1000 USA

- Headquarters in Craigavon, Northern Ireland

- R&D and Custom Synthesis Peptide site in Elvingston, Scotland


GMP Peptide GMP Peptide ManufactureManufacture

Drug ProductDrug ProductManufactureManufacture

GMP Small GMP Small moleculemolecule

ManufactureManufacture

Form.Form.Develop.Develop.

PackagingPackagingand Labellingand Labelling

RadioRadioLabellingLabelling

SS andSS andAnalysisAnalysis

StabilityStability

Peptide and Protein Technology Profile

• 35 Staff spread across research, development, manufacturing & analytical

• >150 man years Peptide manufacturing experience• Manufactured and supplied in excess of 9000 Peptides• Proven track record of high quality supply• Reputation for success with very challenging chemistries and

sequences• Routine synthesis of sequences up to 200 AA • Peptide and Protein ligation expertise (pegylation, labelling,

conjugation)• Site specific Protein engineering and chemical Protein synthesis

Non-GMP custom synthesis

• Rapid throughput, high quality manufacture• Manufacture and supply of >100 Peptides per month from mg to

gram scale• >75% of custom manufactures delivered within 20 days of order• Ways of working:

– Order accepted by phone, e-enquiry, fax– Technical owner assigned from order to dispatch – Highly experienced Peptide scientists available for direct contact – Multiple assets for parallel processing of multiple products– Range of analytical characterisation approaches available

Catalogue products

Human Chemokines and site-specifically labelled derivatives

Almac Methodology

• Extend scope of SPPS into long peptide manufacture• Methods offer high degree of control over synthesis• Flexibility to introduce unnatural building blocks• (custom or commercial)• Flexibility to introduce labels• Learning from each synthesis captured and applied

through “best-process” tool

• Conventional methodology enables step-wise synthesis of peptides up to 40-50 a.a.

• Novel methodology developed for synthesis of peptides and proteins(50-150 a.a.)

– Coupling reagents– Novel solubilising protecting

groups– Linkers and solid supports– Online monitoring– Tagging based purification– Folding conditions

Linker ResinHNFmoc

Linker ResinH2N

Linker ResinHNFmoc

PGSC

COOHHNFmoc

PGSC

Linker ResinH2N

PGSC

Linker Resin

PGSCPGSCPGSC

PGSCPGSC

H2N

COOHH2N

COOHHNFmoc

PGSC

Deprotection

Coupling

i) Couplingii) Deprotection

n

Cleavage and Purification

Refold

Deprotection

Almac Methodology

190 sub unit operations described

8 Families defined

Almac

Confid

entia

l

New process roadmap

• Almac Experience captured in a Process Best View summary• Best View updated based upon evolving experience• Peptides categorised into 8 families• Best View Process broken down into 190 sub unit operations for each family

• Best View Process establishes a high quality start point for any new synthesis

GMPManufacture

ProcessDefinition

Developmentlimited and only

if absolutelynecessary

ScaleUp

Proof ofProcess Make

Fit for purposeProcess

right first time

Done on accuratesmall scale modelsfor representative

data

New process roadmap

• Process developmentAlmac philosophy and approach to early phase makes:

Experience based>150 man years>8000 peptides

What is GMP?

• Manufacturing and testing practise that helps to ensure a quality product

• Manufacturing processes are clearly defined and details of each process are well recorded

• Any deviations are documented and investigated

In summary a system to ensure patient safety

The phases of clinical development (and typical purities of products)

• Pre-clinical: Determine toxicity of product in animal models (80-90%)

• Phase 1: Confirm safety and tolerance of compound in healthy volunteers (95%+ with no new impurity >0.1%)

• Phase 2: Confirm proof of concept in patients (97-99%, with no new impurity >0.1%)

• Phase 3: Larger studies (validated process, all impurities well understood and controlled)

• In summary a system to ensure patient safety

• Four independent manufacturing trains:

Able to simultaneous progress multiple manufactures

Akta

Varian80/150

Bench top

Virtis

CS Bio536

Wet Chemistry (cleave, PEGylation, conjugation)

full coverage 10 – 630 litre

PURIFICATION FREEZE DRYSYNTHESIS

100s of grams

Small scale modelling

Akta Bench topCS Bio536

Small scale modelling

Novasep50/80 VirtisCS Bio

536Up to 100 grams

Dev

GMP

CS Bio936

GMP manufacturing

GMP manufacturing

GMP manufacturing

• Manufacturing rate ca 15-20 batches per year• Scales up to 500g per batch• Multiple products >70mer• World’s first >100mer made to GMP by solid phase

synthesis• Current customer base UK / Europe / USA,

Small/Medium Biotech / Big Pharma

h-MDC required for clinical trial

Case study 1: h-MDC

Request: Vials of injectible h-MDC for clinical use

1. cGMP API Manufacture and Release• Define Manufacturing Route• Analytical Development for API Release

2. cGMP Drug Product Manufacture and Release

Case study 1: h-MDC

Human-Macrophage Derived ChemokineMember of the CCL22 family, binds to CCR4 receptor

GPYGANMEDS VCCRDYVRYR LPLRVVKHFY WTSDSCPRPG VVLLTFRDKE ICADPRVPWV KMILNKLSQ

• 69 Amino acids• 2 Disulfide bridges

Case study 1: h-MDC

Technical challenges• h-MDC is a small protein and technically challenging to synthesize• Effective characterisation of the product is required

Quality• A high purity product is required (typically >95% for clinical trial

using a suitably validated analytical method) • The manufacture is according to ICH standards

Commercial• The customer’s timelines and budget must be respected• Rapid development and delivery essential

Case study 1: h-MDC

For h-MDC there are several options for synthesis- Recombinant - Fragment synthesis- Linear SPPS

Preferred option: Linear

Offers rapid and cost effective entry to clinical programme

Appropriate to scale and customer timeline

Technology developed in house

Deep expertise in house

Case study 1: h-MDC

Synthesis of Linear 69 mer

Cleavage from Resin

Purification of Linear 69 mer

Folding of 69 mer

Isolation of h-MDC. Acetate

Technical Challenges

Finished Product. Acetate

Case study 1: h-MDC


Cleavage from Resin


Folding of 69 mer




Achieve high coupling efficiency

Case study 1: h-MDC

Remove closely related impurities- Fold crude or isolate intermediate


Cleavage from Resin


Folding of 69 mer




Case study 1: h-MDC

Identify critical folding parameters- Avoid dimers and misfolds- Drive reaction to completion- Achieve correct activity- Achieve yield and throughput


Cleavage from Resin


Folding of 69 mer




Case study 1: h-MDC

Remove closely related impurities Achieve exchange to acetate


Cleavage from Resin


Folding of 69 mer




Case study 1: h-MDC

High Purity DP requiredHigh Purity DS requiredAchieve correct activity of molecule


Cleavage from Resin


Folding of 69 mer


Quality Challenges

High level of control neededduring manufacture


Case study 1: h-MDC


Cleavage from Resin


Folding of 69 mer


DevelopmentBest view process used as basisIncorporated multiple couplings at

appropriate points

Complex scavenger mixture requiredto minimise side reactions

Case study 1: h-MDC


Cleavage from Resin


Folding of 69 mer


DevelopmentComparison of two routes done• Direct fold of crude• Intermediate purification

Yield and quality similarIntermediate purification eliminates

variability

Case study 1: h-MDC

Purification developed to givecorrect selectivity and yieldTwo media compared


Cleavage from Resin


Folding of 69 mer


Development

Case study 1: h-MDC

Critical to peptide activityFocussed development on

achieving correct activityTwo sets of conditions tested

against ‘standard batch’Bioactivity tested and confirmed


Cleavage from Resin


Folding of 69 mer


Development

Case study 1: h-MDC

Best view applied directly to achieve isolated acetate salt


Cleavage from Resin


Folding of 69 mer


Development

Case study 1: h-MDC

Effective synthesis achieved- UV deprotection profile is used to monitor assembly- Average coupling efficiency > 99%

GMP Synthesis of Linear 69 mer

010

203040

506070

8090

x S L K N L I M M K V W P V R P D A C I E K D R F T L L V V V G G P R P C S D S T W Y F H K V V R L P L R Y R V Y D R C C V S D E M N A G Y P G

h-MDC

Case study 1: h-MDC


Folding of 69 mer

Isolation of h-MDC

Crude peptide purified to remove major impurities before folding- Major impurity is methionine oxidation product (+16)- Separable by RP – HPLC - Deletions and truncates also removed

Case study 1: h-MDC

Folding monitored by HPLC Final purification used to isolate product at > 95.0 % purity


Folding of 69 mer

Isolation of h-MDC

Case study 1: h-MDC

Proof of Identity Mass Spec Sequence by Mass Spec Sequence by AAA

CompositionPurity by HPLC (2 methods) Peptide Content Counterion Content Water Content Residual Solvents

Activity In vitro assay by client

Analytical Characterisation

Release - Sequence

Peptide digested and fragmentssequenced to build picture of peptide

GPYGANMEDSVCCR

GPYGANMEDSVCCR DYVR YR LPLR VVK HFYWTSDSCPR PGVVLLTFR DK EICADPR VPWVK MILNK LSQ




















GPYGANMEDSVCCR DYVR YR LPLR VVK


Phase appropriate validation during development programme

Selectivity/Specificity• Selectivity of h-MDC with Related Substances

Truncates Linear PeptideMisfolded peptide Oxidised product

• PrecisionRepeatability

• Linearityh-MDC Assay Range

• SensitivityLimit of Quantitation and Limit of Detection established

• Solution Stability

HPLC analysis

Method 2: 97.38%

Method 1: 97.73%

Orthogonal methods

HPLC analysis of h-MDC

Drug Product

• Drug Product formulation as lyophilisate in vials

• Sterile filtration of aqueous solution of drug substance through 0.2um filter

• Drug Product release testing• Minor amounts of oxidation (of methionine)

observed during formulation• Activity testing confirmed batch as suitable for

use in the clinic

Case study 2: Co-eluting impurity

• 49-mer peptide produced on solid phase• cGMP manufacture completed • Analysis of cGMP product indicated the presence of a

significant level (~5-10%) of an impurity -114 mass units from product as determined by mass spectrometry

• Product to be used in phase 2 studies – the -114 impurity was not present in any previous batch

• Principal of clinical development is that each phase of development should use material of higher purity

Case study 2: Co-eluting impurity – removal?

• Best purification conditions still failed to completely remove impurity

1716-143 TFA purification

0.0%

10.0%

20.0%

30.0%

40.0%

50.0%

60.0%

70.0%

80.0%

90.0%

100.0%

B8 B6 B4 B2 C1 C3 C5 C7 C9 C11

Fraction number

% p

urity HPLC purity

%impurity (TIC)

Case study 2: Co-eluting impurity – identification?

• Most likely impurity was thought to be capped truncate where UV profile had indicated coupling was not efficient

• Synthesis of this truncate had the correct mass but was not the impurity!!

• Reacted contaminated product with purification TAG (as activated ester) – both full length peptide and impurity reacted

• Trypsin digest confirmed that the impurity was a deletion product

• Still present in GMP batch however!

Case study 2: Co-eluting impurity – identification?

• Quantified level of deletion impurity by mass spec versus a standard of the impurity

• Initially reported level higher than actual level (actual level only ~1%)

• Deletion impurity tested in biological study – showed similar response to full-length peptide so material passed as fit for use in clinical study

New technology – tagging as a means of purification

• “Tagging” is a term we have coined for a process that facilitates more effective and efficient purification of crude peptides thanclassical methods.

• Analogous with common practices used in recombinant protein synthesis.

• It involves the temporary labelling of a peptide with a small molecule (the tag) that has been specially designed to aid purification.

• Due to the “capping” step commonly employed in peptide synthesis, the tag only attaches to the desired, full length peptide and not to truncated sequences.

The tag….

Cleavable spacer unit

Purification handle

Reactive group for attachment

to peptide

Tag-based purification – tag-peptide linkage

Tagged peptides on resin

Tag-based purification – resin cleavage

Crude tagged peptides in solution

Tag-based purification – binding

Immobilisation through Affinity Chromatography

Tag-based purification – cleavage

Elute recovered peptide

min5 7.5 10 12.5 15 17.5 20 22.5 25

mAU

0 100 200 300 400 500 600 700 800

min5 7.5 10 12.5 15 17.5 20 22.5 25

mAU

0 100 200 300 400 500 600

IMAC Purification of Tag2-12merCrude Tag-Peptide

Purified Peptide

Tag2-peptide

Peptide

Capped truncate

• Complete removal of co-eluting capped truncate through use of TAG

• Technology applied to range of peptides

Summary of services

• In-house services within PPT:• Process Development• Analytical Method Development• Phase-appropriate Method Validation• Manufacture (GMP & non-GMP) mg to kg• DS Release testing• DP Release testing• Stability Trials (DS and DP)• QA Support• Supply Chain Management

Summary of services

• Leveraged from wider Almac Group and Partners• Radiolabelling• Formulation development• Sterile formulation• Fill finish• Tox studies• QP release• CMC documentation / authorship of IMPD