Continuous mAb Purification Process: Design Features and Practical Considerations

Joanna PezziniCASSS DC Area Discussion Group Meeting

December 7, 2017

Continuous Process Overview

Continuous Technologies

PAT Opportunities

Closed Processing

Outline

2

Continuous Process Overview

Continuous Technologies

PAT Opportunities

Closed Processing

3

Batch Purification Process Overview

Entire batch is pooled in product hold tank between each process step.

• pH adjustment in

stirred tank

• low pH hold time

in stirred tank for

viral inactivation

• Flow through

chromatography

or membrane

• Diafiltration

• Final

Concentration

• Excipient spike

• Final filtration

• Bottle fill

• Protein A bind &

elute

chromatography

• Cation exchange

bind & elute

chromatography

• Virus pre filter

and virus filter in

series

ProA

Capture

Viral

Inactivation

Anion

Exchange

CEX

CaptureViral

Filtration

UFDF &

Formulation

Continuous Purification Process Overview

5

Multi-Column Chromatography

(ProA)

Flow-Through Low pH Viral Inactivation

Filtration Train (Includes AEX

and Viral)

Multi-Column Chromatography

(CEX)

Single Pass UFDF

• Inline pH

adjustment

• Residence time

in SEC column

provides low pH

hold time for

viral inactivation

• Depth Filter

• Sterile Filter

• AEX Membrane

• Virus Pre-Filter

• Virus Filter

• Diafiltration

• Final

Concentration

• Inline excipient

spike

• Final filtration

• Bottle fill

• Protein A bind &

elute

chromatography

• Cation exchange

bind & elute

chromatography

Continuous flow through all steps for the duration of the process.Steps are separated by small product break tank.

Benefits of Continuous Processing

ProA

Capture

Viral

Inactivation

Anion

ExchangeCEX

CaptureUFDFVirus Filter

time

ProA Capture

Viral Inactivation

Anion Exchange

CEX Capture

UFDF

Virus Filter

Scale by Time

Sca

le b

y V

olu

me

➢ Improved productivity

➢ Flexible capacity management (scale by time in addition to volume)

➢ Decreased downstream cost of goods

➢ Amenable for labile proteins

➢ Facilitates adoption of single use technologies

time

Sca

le b

y V

olu

me

Scale Up of Batch Upstream Process

7

200 L Scale 2,000 L Scale 20,000L Scale

Column Diameter 4.4 cm 10 cm 30 cm

System Piping Inside

Diameter

1/32’’ 1/8’’ 1/4’’

Break Vessel Volume 200 ml 2 L 20 L

Eluate Tank Volume 5 L 50 L 500L

Proposed design applies to processes from 200 L to 20,000 L.

Small Volumes

Enable Single

Use Containers

Continuous Process Overview

Continuous Technologies

PAT Opportunities

Closed Processing

Outline

8

Multi-Column Chromatography- an Enabling Technology

9

Benefits

• Continuous feed

• Increased resin utilization (g/Lresin)

• Improved productivity (g/Lresin hr)

• > 50% resin savings

• > 20% buffer savings

Single Column Operation Multi-Column Chromatography

Column Size 60 cm diameter x 20 cm height 30 cm diameter x 10 cm height

Column Vol. 56 L 28 L total

Resin Cost $616,000 $308,000

Process Time 8.2 hours 6 hours

Productivity 20 g/Lresin hr 60 g/Lresin hr

Feed Wash Elution

Comparison of chromatography systems for batch process to demonstrate improved efficiency.

• Continuous flow design

• Easily scalable

• Inline titration leads to consistency

• Process range 30-60 min

Novel Flow-Through “Hold” Design for Viral Inactivation

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Product

AcidProduct

Acid

0

200

400

600

800

1000

0 10 20 30 40 50 60

MA

U

TIME (MINUTES)

SEC Column Transition

Traditional Titration/Hold Method Continuous Titration/ Residence Time Method

Base

Base

Next

Unit Op

Novel SPDF Achieves 99.75% Buffer Exchange

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• Single Pass Diafiltration (SPDF) designed to meet or exceed buffer exchange equivalent to a 6 diavolume

traditional process (99.75% buffer exchange).

– Achieved with three sequential dilutions ≥7.5X

– Product concentrated ≥ 7.5X with SPTFF membrane prior to each dilution to minimize buffer use

– 𝐹𝑖𝑛𝑎𝑙 𝐶𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 =Initial 𝐶𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛

𝐷𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝐹𝑎𝑐𝑡𝑜𝑟# 𝑠𝑡𝑎𝑔𝑒𝑠

Feed Retentate1

Diluent 1 Retentate2

Diluent 2 Retentate3

Diluent 3(DF

Product)

Co

ncen

trati

on

Continuous Process Overview

Continuous Technologies

PAT Opportunities

Closed Processing

Outline

12

Process Flow Diagram

13

pH Control

Pressure and Volume Control

Pressure

Control

SPTFF: Single-Pass Tangential Flow Filter

14

Control Plan

Arduino

Control Plan

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6 Manipulated Variables (MV):

Pump 1

Pump 2

Pump 3

14 Controlled Variables (CV):pH 1

pH 2

P1

M2

P2

P3

P4

P5

P6

M3

P7

P8

P9

P10

Pump 4

Pump 5

Pump 6

Arduino 1

Viral

Inactivation

Arduino 2

Filter

Train

Arduino 3

Single-Pass

Diafiltration

PAT Opportunities for Continuous

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Can PAT and automation improve product quality and/or process efficiency?

pH feedback to

control low pH

viral inactivation

acid and base

addition rates

Pressure

feedback to

control flow rate

during

chromatography

or membrane

filtration

Volume

feedback to

control break

tank volume in-

between

continuous

steps

HCP, DNA

concentration

for AEX

membrane

lifetime

Product

concentration to

monitor UFDF

step

Yield to evaluate

chromatography

resin lifetime

Current

ProposedAggregate

concentration

for wash/elution

buffer

composition of

CEX step

Charge variants

to determine

bioreactor

conditions

Steady state operation achieved with continuous manufacturing provides

time to measure and respond to changes in product quality attributes.

PAT Decision Tree

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How fast is feedback

needed?No need for PAT

yes no

No PAT Available to

Implement

<5 min

On-Line

5-10 min

At-Line

> 10 min

Is inline

instrumentation

available?

yes no

Inline PAT

Focus on automated on-line

sample collection and in-

time analysis for batch

release

Bring to Industry

Attention (BPOG,

Suppliers, etc.)

Focus on automated on-line

sample collection for at-line

analysis

Can PAT and automation improve product quality and/or process efficiency?

What can be achieved?

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On-line

Chromatography based

Methods:

SEC for aggregate,

fragments

IEX for charge variants

Mass Spectrometry based

methods for:

Oxidation

Deamidation

Glycosylation

Di-sulfide bond

modifications

At-Line

Automated/Robotic sample

collection, handling,

preparation, and injection

into the following

instrumentation:

Enzyme-linked

immunosorbent assay

(ELISA) for host cell

protein (HCP)

Polymerase Chain

Reaction (PCR) for

deoxyribonucleic acid

(DNA)

In-line

✓ Flow

✓ Pressure

✓ pH

✓ Conductivity

✓ UV

Concentration

(Density, IOR, A280)

Spectroscopy (Raman,

NIR, DLS, MALS, IOR)

✓ Routinely used in GMP biologics manufacturing facilities

Not routinely used in GMP biologics manufacturing facilities

Automated Sampling Need for Continuous

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• High sampling frequency, number of measurements, and number of sample points requires

automated on-line sample collection and stream-lined off-line sample analysis

– 1 per day x 5 sample points x 10 measurements = 50 samples per day

– High sampling frequency to enable feedback to control process, monitor for drift in process over time

– Close location of sample points enables automated sample collection with single piece of equipment

(sample lines feed to different sample points)

Continuous Process Overview

Continuous Technologies

PAT Opportunities

Closed Processing

Outline

20

2121

250 ml

sterile

bottle

0.2μm filter

250 ml

sterile

bottle

0.2μm filter

Chromatography/Filter

System

Sanitization

Solution

Our approach for bioburden control:

Gamma irradiated vessels and sanitization solution through chromatography/filter

systems upon startup and as needed.

Bioburden control

Continuous process operated over several days or weeks must maintain low

bioburden levels, similar to batch process requirements.

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• Vessels gamma irradiated.

• Vessels include 3-way valve on each of three

tubing inlets.

1. Upstream inlet

2. Downstream outlet

3. Buffer addition line

• Vessels also include a vent filter and a stir

bar.

• Vessels placed on stir plate that rests on

scale.

Bioburden Control- Vessels

Questions???

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30 L Brx + ATF

2.5 g/hr

ProA Multicolumn

Chromatography

1.7 g/hr

POD – AEX – VF

11 g/hr

Viral Inactivation

11 g/hr

Diafiltration and Final

Concentration

11 g/hr

MedImmune’s First Fully Integrated Continuous Pilot Scale Demo

Bioprocess Engineering

Purification Process Sciences

Cell Culture & Fermentation Sciences

David H. Koch School of Engineering Practice, MIT

Significant contributions by Lindsay Arnold

Acknowledgements

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