Authored and Presented by: Cameron T. Phillips, Niket Bubna, Ph.D., David Chang and Sigma S. Mostafa, Ph.D

1. Primary Recovery & Harvest Processes for non-mAb Recombinant Proteins
Non-mAb proteins present challenges during primary recovery and harvest processes due to their
unique physicochemical properties which may be incompatible with platform harvest clarification
processes. From a CDMO perspective, the increasing number of non-mAb proteins entering
clinical trials has resulted in the need for a streamlined workflow to develop non-mAb harvest
processes to meet manufacturing timelines. Using two case studies, we demonstrate a workflow to
develop a primary recovery and harvest process for non-mAbs which presented challenges in
recovery and product quality. The process was then scaled to 2000-L scale for manufacturing.
Abstract
Cameron T. Phillips, Niket Bubna, David Chang and Sigma S. Mostafa
KBI Biopharma, Durham, NC
References
Conclusions
Acknowledgments
We thank Sigma Mostafa for support and guidance, and express our gratitude to Lynwel Cunanan
and James Hamlin for their help.
Primary recovery and harvest processes for two challenging non-mAb proteins were developed
for 2000 L scale. Depth filters from several different filter media types were tested, but cellulose
depth filters had the best performance for recovery and product quality. Final processes for both
molecules included single-use centrifugation step using kSep 400, then scaled to 2000 L with the
kSep 6000. Yield was increased from near 0% to > 90% for non-mAb 1, while bioactivity was
significantly improved for non-mAb 2 up to 200 L scale.
Scale-up to Manufacturing
Primary recovery and harvest of cell culture processes typically consist of centrifugation, depth
filtration, or a combination of both. Centrifugation of cell culture material removes large particles
and cellular debris whereas depth filters can be used for removal of cell debris and some non-
specific process impurities. Typical product recovery for mAbs are > 95%. During primary recovery
of two non-mAb molecules, significant product loss and loss of bioactivity was observed.
Manufacturer Material Filter Type Retention Range (μm)
Millipore HC Dual Layer Cellulose/DE D0HC
C0HC
B1HC
A1HC
F0HC
X0HC
0.5 – 9 μm
0.2 – 2 μm
0.1 – 0.7 μm
0.1 – 0.5 μm
0.1 – 0.4 μm
< 0.1 μm
Millipore HC Single Layer Cellulose CE15
CE20
10 – 15 μm
5 – 11 μm
Millipore Polypropylene Polygard (10 um)
Polygard (5 um)
Clarigard (3 um)
Clarigard (1 um)
10 μm
5 μm
3 μm
1 μm
Pall Profile II Polypropylene Profile II 200
Profile II 100
Profile II 50
20 μm
10 μm
5 μm
Pall Ultipor Glass Fiber GF Plus (10 um)
GF Plus (6 um)
10 μm
6 μm
Pall Supracap 100 Cellulose P900
PDK5
8 – 20 μm
1.5 – 20 μm
Sartorius Sartoclear Cellulose with inorganic filter
aids
PB1
C8HP
4 – 11 μm
4 μm
Sartorius Sartoclean Cellulose Acetate CA
GF
0.8 – 3 μm
0.8 – 3 μm
Sartorius Sartopure Glass Fiber
Glass Fiber
Polypropylene
GF plus
GF
PP3
1.2 μm
1.2 μm
20 μm
GE Healthcare Glass Fiber ULTA Capsule GF 5 μm
Initial depth filter screening was conducted at bench-scale with different filter media across
multiple vendors and pore sizes. Top filters were chosen based on product recovery, product
quality of the filtrate, and ease of manufacturing.
Introduction
Results from Harvest Process Development
Experimental Plan
Depth Filter Screening Plan
Initial harvest material was generated in 3 L glass reactors for initial depth filter screening studies.
After identifying top filters based on recovery and product quality analysis, the process was scaled
to pilot (50 and 200 L) using single-use vessels. Primary recovery process was evaluated with top
filters, with and without single-use centrifuge (kSep 400). Based on pilot-scale results, the process
was then scaled to 2000 L SUB for manufacturing.
Harvest
Process Lock
Recovery/Bioactivity
analysis
Choose top
filters
Molecule Problem
non-mAb 1 0% recovery with platform process
non-mAb 2 Low product bioactivity with platform process
+
Single-use centrifuge Depth filtration
Depth filtration
Depth filter screening
Jackalope
non-mAb 1
non-mAb 1
non-mAb 2
non-mAb 2
non-mAb 2
non-mAb 2
Bench-scale depth filter screening
3 L
50 L
cGMP
2000 L MFG
Pilot-scale (50 L / 200 L) harvest process development
Choose top filters for scale-up
non-mAb 1, platform process non-mAb 2, platform process
No product recovery
non-mAb 1 non-mAb 2
without kSep
with kSep
non-mAb 2
Pre-kSep turbidity
Post-kSep turbiditySartorius kSep single-use continuous centrifuge
Harvest Process Lock
Low product quality
non-mAb 1
Yield increased from ~0% to >90%
non-mAb 2kSep Scalability (non-mAb 1)
90% reduction in turbidity from 50 L to 2000 L Slight decrease in yield at 2000 L (>80%)
Bioactivity data unavailable
Top filters
Top filters
Top filters
Top filters
Top filtersTop filters
Millipore Millistak
Singh, Nripen. “Clarification technologies for mAb manufacturing processes: Current state and future perspectives.” Biotechnology and Bioengineering (2016).
Jungbauer, Alois, and Nikolaus Hammerschmidt. "Integrated continuous manufacturing of biopharmaceuticals." Kleinebudde, P., Khinast, J., Rantanen, J., Eds (2017).
Roush, David J., and Yuefeng Lu. "Advances in primary recovery: centrifugation and membrane technology." Biotechnology progress (2008).
http://www.emdmillipore.com/
https://www.sartorius.com/Depth filter schematic
kSep schematic