Implementation of an Online UV monitoring system in Tinzaparin Sodium Manufacture

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For decades LEO Pharmawas a very diversified business

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AntiT

H

THE CLOTTING CASCADE

THROMBIN (IIA)

ANTI-THROMBIN

XA

CLOTTING FACTORS

ANTI CLOTTING FACTORS

EH P A R I N

ANTI-THROMBIN

EH P A R I N

ANTI-THROMBIN

EH P A R I N

ANTI-THROMBIN THROMBIN (IIA)

XA

EH P A R I N

ANTI-THROMBIN

HEPARIN –MODE OF ACTION

ML W H

ANTI-THROMBIN

ML W H

ANTI-THROMBIN

ML W H

ANTI-THROMBINTHROMBIN

(IIA)

XA

ML W H

ANTI-THROMBIN

TINZAPARIN (LMWH) –MODE OF ACTION

Assay of Heparin Ph.Eur 2.7.5

EH P A R I N

THROMBIN (IIA)

ANTI-THROMBIN

THROMBIN (IIA)THROMBIN (IIA)

Substrate PnaPeptide

405 nm

MAST CELL

MUCOSA IS REMOVED FROM INTESTINAL CASING THROUGH ROLLERS

MUCOSA

There is selective binding to the resin.

This is a the key impurity removal step

LEO Pharma Cork

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Heparin is a complex molecule

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Tinzaparin sodium (LMWH)

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HeparinaseSpecific MWD specifications

Tinzaparin Manufacture

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The degree of depolymerisation directly affects the potency and yield of the product

It is important to have a reliable method of monitoring the reaction in order to ensure a consistent and optimised process.

The reaction is currently monitored by taking advantage of the formation of an unsaturated iduronic acid derivative during the depolymerisation which absorbs ultraviolet light.

The change in ultraviolet light at 235 nm (delta A) is used as a measure of the number of bonds cleaved and therefore a measure of the degree of depolymerisation of Heparin Sodium.

Tinzaparin Manufacture

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Current method of Offline sampling

Advantages Disadvantages

Simple (Analytically) Laborious

Reproducible Not real time

Potential yield implications

Typically 10-12 offline readings obtained over the course of the reaction.

Aim is to get the Delta A within 1 AU

Tinzaparin Online Monitoring System

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The ideal situation is to implement an online method of monitoring the reaction, which would eliminate the

requirement for sampling and the delay in obtaining data used to terminate the reaction.

Tinzaparin Online Monitoring System

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Options considered; Lab scale experiments

• Univariate Online UV method

• Multivariate Online UV method

• Raman Spectroscopy

• NIR

• FIPA (Flow Injection Polymer Analysis)

Tinzaparin Online Monitoring System

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• Raman and multi-spectrum UV gave the best response

• Multi-spectrum UV was chosen as the most suitable technology for an onsite trial.

Development Challenges

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It can be seen that as the peak moves the absorbance at 235nm is at a different point on the curve and so the correlation with reaction progression is not linear.

The advantage of collecting data for a range of spectra means that issues like this can be dealt with, but it also means that the mathematical methods used to analyse the data are nearly as important as the raw data.

Tinzaparin Online Monitoring System

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Flowcell

Tinzaparin Reaction vessel

Recirculation Pump

UV Spectrophotometer

Pilot plant (10L) trials performed

Computer

Tinzaparin Online Monitoring System

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Optimisation of the system; Pilot Scale

• Concentration Effects – to determine if the product concentration in the solution has an effect on the UV signal.

• Temperature Effects – a reaction was ran at a different temperatures to determine the effects of temperature on the UV signal.

• pH Effects- Reaction 2012-UV-013 This trial was to determine if the UV signal was affected by varying pH levels.

• Flow cell width

Online Tinzaparin Reaction: Full Batch Scale

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Tinzaparin Online Monitoring System

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Further Optimization of the data: Batch scale (8 batches)

• Peak Analysis

• Peak Height Analysis

• Peak Centre Analysis

• Peak Area Analysis

• PLS (Partial least squares) Model

Tinzaparin Online Monitoring System

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PLS is a multivariate technique which assesses all the data available and then assigns weightings (or loadings) to each variable (wavelengths in our case) according to how much each variable changes with regard to the response variable in question.

In effect the first factor calculated (or ‘principal component’) is simply a weighted average of the raw absorbance values. This component is then analysed to determine how much of the variation this new composite variable explains another component calculated.

More components can be calculated until all the variation in the data is explained, ideally with as few components as possible. Therefore it is possible to reduce the information collected by dozens of variables down to two or three ‘principal components’.

Tinzaparin Online Monitoring System

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Predicted A235 measurements compared to reported offline measurements

Regulatory strategy

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Change type 1b other

Introduction on Online UV as an alternative option for monitoring reaction progression.

UV analysis already used in production (Offline)

No quality attributes were potentially impacted by change e.g. Assay and MWD not impacted by introduction of the online method.

Current controls for the reaction (Offline Measurement) remain in place

Key Learnings

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Selection of Data and Data processing• Identification of data that provide the best results

Engineering Challenges• Operation of UV instrument in production rooms• Recirculation loop cleaning and maintenance

Validation Challenges• Moving UV instrument into ATEX area

Regulatory Strategy• Meeting commitments.

Special Thanks

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Alison Winger PhD - MSAT Development ScientistJason Murtagh - Development Process EngineerLiam Ryan - MSAT Development ScientistKaren Colgan – Regulatory/Quality SpecialistSandra Patricia Auguste-Bowler – Principal Scientist

Clariet Scientific

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Thank You