Biotechflow expanded bed columns + streamline 2018

The cGMP commercial purification of a Monoclonal Antibody
direct from unclarified broth using
expanded bed chromatography with
PAT Control using Ultrasound.
Martin Hofmann
Managing Director
Biotechflow Ltd
Mobile +44 (0) 7980 74 77 30
Website biotechflow.com
Email martin@biotechflow.com
Copyright ©Biotechflow 2018

2Martin Hofmann. Copyright ©Biotechflow 2018
Expanded Bed
Adsorption
Crude, unclarified high cell density, highly-viscous feed
Whole cell mixture; homogenized cell broth; foods; plasma
Streamline Media
Or Rhobust®
MabDirect
Protein A
Captures MAb
Velocity
450-500 cm/hr
600 mm column volumetric flow:
24 L/min
1414 L/hr
Cells, cell debris, host cell proteins (HCP’s) and DNA

3Martin Hofmann Copyright ©Biotechflow 2018
1). Here we see the harvest
from the bioreactor fed
directly into the
bottom (upflow) of the
Biotechflow EBA 300.
The yellow supernatant being
unretained cells (viable) and
cell debris.
The target Mab was
extracellular (secreted).
2). Next is the first wash, flow is always
upflow. This is to remove unretained
species, cell debris particles still in the void
volume of the bed and to obtain a lower
stable pH.
A complete Harvest to
Elution of Mab cycle.

3). Wash Two (see chromatogram next
slide).
Here we see a clear supernatant now that
all cell debris has left the column.
4). Now, on dropping the pH to 4.3 the Mab is
eluted.
The concentration of the Mab is clearly high
as evidenced by the appearance of the
supernatant.
A complete Harvest to Elution of Mab cycle, part 2.

Chromatogram of the complete Harvest to Elution of Mab cycle

6Martin Hofmann Copyright © Biotechflow 2018
PISTON DESIGN

Martin Hofmann. Copyright ©Biotechflow 2018 7
Piston Design Criteria
There are no meshes, and the process is always upflow
Design has to prevent of the formation of vortices in the expanded
bed
Air must freely leave freely
Move at ca. 2.5 mm sec-1 1000 mm immediately in response to
control
Fail-safe

Piston Seal Functions
Piston seal to perform 4 main functions:
Provide efficient sealing
Hold the piston securely in place at 4.5 bar test pressure
Provide a mechanism to form a gap for cleaning between
seal and tube wall
Afford zero dead space between piston and tube wall
during process

Piston Design
Slope angle critical –
For air removal
and resin retention

Piston seals - final

Piston seal mechanisms
When deflated seal, reduces in diameter by 2mm
revealing a 1mm circumferential gap which can be
flushed free of medium and cell debris etc.
Access to this gap is via an access hatch in the topplate.
Two steel rings are incorporated inside the inflatable seal
at the top and bottom of the seal
Inflated, the seal contacts fully around the top and
bottom edge of the seal face, thereby leaving no unswept
areas.
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1
Martin Hofmann. Copyright ©Biotechflow 2018

ANTIVORTEX DESIGN

Anti-jet Top MP outlet
PROBLEM: Resin leaving the column via a vortex
The critical parameters were:-
Veins
(Kiviniemi, 2009 and Rindels, 1983)
Outlet holes
Anti-jet rod
Cone

Anti-vortex device type A
These designs failed to prevent a vortex
Resin beads swirled up and left the column

Piston seal mechanisms
It was found, by experiment that the critical parameters were:-
The number and angle of ‘veins’
(Kiviniemi, 2009 and Rindels, 1983)
The dimensions of the outlet holes
The length of the anti-jet rod
The angle of the cone of the piston
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Martin Hofmann. Copyright ©Biotechflow 2018

Anti-vortex device final design
120-degree angle
Three veins
Anti-jet rod extends beyond cone
Sacrificial and disposable

Successful anti-vortex device B

PERPENDICULARITY

Inclinometer within 0.01-degrees.
Ref and thanks to:
Piet den Boer
Sr. Consultant
Xendo B.V.

20Martin Hofmann Copyright © Biotechflow2018
MANUFACTURING TIME

Martin Hofmann Copyright © Biotechflow 2018 21
Progress in 3 Months
Time from PO to Delivery 6 months

6 Months Max Manufacturing and FAT

FLOW RATE CONTROL
USING ULTRASOUND

Use of Ultrasound
6 or 8 ultrasound transceivers monitor:
Monitor the position of piston
PRIMARY FUNCTION:
Feedback to the skid flow meter and pump
Flow automatically changed by PID loop as viscosity changes
tokeep piston at 90 mm of supernatantabove
the top of expanded bed
New Functions on latest column REAL TIME PAT by
monitoring ultrasound at various bed heights.

Ultrasound on 300 mm EBA Column

Ultrasound on 600 mm EBA Column

REAL TIME MONITORING
BY USING ULTRASOUND

Use of Ultrasound – ‘SonoSensor’
This novel IP67 unit
was built for a crude
separation of
lactoperoxidase (as a
possible anti-tumour
agent) from milk;
the sensors being
attached to a steel
tank.
This portable device
can be retro-fitted
onto any column,
pipework or vessel.
The output is 4-20
mV. This unit has 6
sensors but more, or
less are easily
possible.

PAT During Process by Ultrasound
Monitoring Sepharose Compression
In 2m Steel Column (model by 2 m tank)

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58
60
62
64
15:50:24 16:04:48 16:19:12 16:33:36 16:48:00 17:02:24 17:16:48 17:31:12 17:45:36 18:00:00
Time, hr:min:sec
UltrasoundResp.
10 ml / kg
8 ml / kg
6 ml / kg
4 ml / kg 2 ml / kg

0
1
2
3
4
5
6
7
8
9
10
0 2 4 6 8 10 12
Acetone Conc., millilitre acetone / kg water
UltrasoundResp

0
10
20
30
40
50
60
70
80
90
100
8000 8500 9000 9500 10000 10500 11000 11500 12000
Time (secs)
-0.5
0
0.5
1
1.5
2
2.5
3
3.50
10
20
30
40
50
60
70
80
90
100
8000 8500 9000 9500 10000 10500 11000 11500 12000
Time (secs)
-0.5
0
0.5
1
1.5
2
2.5
3
3.5
Bed Relax after
off-line buffer
change step.
Bed Compression on
going back on-line
Buffer driving
IgG off
IgG peak
with shoulderBuffer Exchange from
pH 8.4 to pH 3.5
Blank ultrasnd
UV
Product ultrasnd

cGMP Commercial EBA

Final conditions cGMP process
High cell-density harvest material was used under the
following conditions:
Cell density: 60-90 million cells/mL (viability >80%)
Antibody titre: 1.3 g/L (continuous XD®(4) cell culture),
cumulative titre 5.8 g/L over 21 days
Load ratio: 22 mg IgG/mL settled bed
(both Protein A resins)

600 mm EBA
column with
Rhobust Media -
tungsten carbide,
agarose and
Protein.
Feedstock injected
directly from 2000
litre Bioreactor for
MAb.

The Two EBA 600’s in-situ

Process
Overall
Yield
Purity
HP-SEC
Buffer Use
Process
Time
Laboratory
Scale
Process Time
Manufacturing
Scale
(%) (%)
(Column
Volumes -
CV)
(hours) (hours)
Expanded Bed
Adsorption.
82 99.6 67 6.8 6.8
‘Traditional’;
clarification
followed by
packed Protein A
column.
70 99.4 118 12.5 18.5
Yield Purity Time

DNA and HCP removal
DNA (pg/mL) HCP (µg/mL)
PCR ELISA
EBA
Clarification
+ Packed Bed
EBA
Clarification +
Packed Bed
Crude Harvest 26,000,000 NA 2,800 NA
Clarified Harvest NA 23,000 NA 96
Neutralized Eluate 133 <10 2 5
DNA log10 reduction
factor
5.3 3.4 3.1 1
RF qty (log10) 5.8 5.1 3.6 3

IgG
concentration
in harvest
Harvest
Volume
(mg/ml) (ml)
EBA 1.34 348
Conventional Clarification
followed by
Protein A column.
0.35 1070
Final results from cGMP production
1.34 g/L IgG
384 ml

Conclusions
EBA column for Mab’s
43% decrease in buffer volume
Production-scale runs took 1/3 of the time necessary for traditional
Product purity was directly comparable
Product concentration was significantly increased,
from 0.35 (traditional) to 1.34 mg/ml (EBA)
EBA enabled higher throughputs and flow rates (450cm/hr)
EBA process more-effectively removed cell debris than traditional
600 mm ID x800 mm L 8x throughput, 2nd next year, 2019, 500 kg/year

Biotechflow expanded bed columns + streamline 2018

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