© 2021 Protein BioSolutions, Inc. All rights reserved.

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Overview• Purify 1 to 24 samples in parallel• Eliminate known and unknown (!) sample degradation• mg+ protein production scale• Flexible sample volume scales from a few mL to multiple liters

24 x 1 ml

24 x 5 ml© 2021 Protein BioSolutions, Inc. All rights reserved.

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Overview• 24 independent flow paths• 9-port valve configuration• Compatible with common commercial columns© 2021 Protein BioSolutions, Inc. All rights reserved.

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Overview• 20 SBS deck positions (1 waste)• Walk-away automation• Perform parallel, 2-step purification of 12 samples© 2021 Protein BioSolutions, Inc. All rights reserved.

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Flexible column sizes and locations

24 x 1 ml

24 x 5 ml© 2021 Protein BioSolutions, Inc. All rights reserved.

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Now with optional 24 channel UV monitoring system!

6© 2021 Protein BioSolutions, Inc. All rights reserved.

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Individual UV Flow cell block for each of 24channels:© 2021 Protein BioSolutions, Inc. All rights reserved.

page 7© 2021 Protein BioSolutions, Inc. All rights reserved.

page 8© 2021 Protein BioSolutions, Inc. All rights reserved.

page 9Sizing column run:
Aggregate separation10© 2021 Protein BioSolutions, Inc. All rights reserved.

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Protein Maker : Purification Automation• High-throughput Protein Purification• Operation Modes:
1. Production Mode (up to 24 proteins, 1 resin)
2. Scouting Mode (up to 24 resin types, 1 protein)
3. Multi-column (automated 2 step purification)• Common Applications / Uses:
1. Antibody Production
2. Crystallography Prep
3. No Risk Scale-up

12 protein variants
purified in parallel© 2021 Protein BioSolutions, Inc. All rights reserved.

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Production ModeAntibody production/screening:
Purify mg+ amounts of up to 24 antibodies in parallel using up to or more than 1L

Case Study: Purification of Engineered Antibodies• Had: Fully humanized antibody against a viral antigen target, with high affinity for wild type target but noaffinity to a prevalent naturally occurring variant• Goal: improve affinity for variant without loss of affinity for wild type
Purified 50mL each for 24 mutations (expression in CHO cells) using 1 mL HiTrap Protein A column. Yield (2-10mg) provided sufficient material for biophysical characterization (analytical SEC/LS, SDS-PAGE, DSC) as well asaffinity measurement.

Example SDS-PAGE: Mutants 13-24

Example analytical SEC: Mutants 1, 22, and 24© 2021 Protein BioSolutions, Inc. All rights reserved.

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Production Mode
Production of multiple constructs:
Parallel processing enables scaled-up purification of multiple constructs (internaldeletion variants) to access those with high crystallizability

Case Study: Multi-construct design and purification
LysatesEluates

1 2 3 4 5 6 7 8 9 10 11 12

1 2 3 4 5 6 7 8 9 10 11 12Crystals

Genes optimized for E. coli expression
Expression yields: 0.1 to 15mg per liter cell culture© 2021 Protein BioSolutions, Inc. All rights reserved.

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Production Mode
Production of multiple constructs:
Parallel processing enables more efficient route to protein crystals

Case Study: Production of Kinase X – 17 constructs
Parallel nickel affinity purification(4-fraction elution)

9 constructs selected for his-tag
removal and crystal screening© 2021 Protein BioSolutions, Inc. All rights reserved.

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Production ModeParallel Lysis scouting:
Test 12 different lysis buffer conditions followed by small scale IMAC purification toscale up and move forward with the strong purifiers

Case Study: Parallel testing of Lysis Conditions

Acta Cryst. (2011). F67, 1015–1021© 2021 Protein BioSolutions, Inc. All rights reserved.

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Production Mode
Risk-free Scale-up via parallelization:
6 x 5 mL Column Bed Volume Parallel Purification
Ni-affinity
Chromatography

Fusion2nd Ni-affinity
Chromatography

ProteinCleavage

Situation• Increased purification from 20 mg to > 100 mgOptions• Develop protocol for 5 x scale up

3850 SEC 12182012 VA(1355882058)001:10_UV
3850 SEC 12182012 VA(1355882058)003:10_UV
3850 SEC 12182012 VA(1355882058)001:10_Conc
3850 SEC 12182012 VA(1355882058)001:10_Fractions

Size Exclusion
Chromatography

3850 SEC 12182012 VA(1355882058)002:10_UV
3850 SEC 12182012 VA(1355882058)001:10_Cond3850 SEC 12182012 VA(1355882058)004:10_UV
3850 SEC 12182012 VA(1355882058)005:10_UVmAU
2000%B

1500

0.10

0.15

0.05

1000

Yield: 176 mg• Repeat 5-6 x of the original purifications> 95% purity• Run multiple small-scale purifications inparallel – Protein Maker
No need for traditional “scale-up” if you use Protein Maker
0.00

500-0.05

0A1 A2 A3 A4 A5 A6 A7 A8 A9 A11 B12 B10 B8 B7 B6 B5 B4 B3 B2 B1 C1 C2020

406080100

Waste-0.10ml© 2021 Protein BioSolutions, Inc. All rights reserved.

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Scouting ModeParallel resin scouting:
Scouting a variety of resins (up to 24) for optimization of 1 protein

Case Study: Scouting Mode with a Step-Gradient Protocol
Glu-PGS – Antibody affinity column
A. Equilibration Buffer: 20 mM Tris pH 8, 100mM NaCl, 0.5% NP40
B. Elution Buffer: Equilibration buffer 1 plus 50 M EYMPTD peptide
HiTrap SP Sepharose – Cation exchange resin
A. Equilibration Buffer: 20 mM MES pH 6.0
B. Elution Buffer: Equilibration buffer plus 1 M NaCl
HiTrap Q Sepharose – Anion exchange resin
A. Equilibration Buffer: 20 mM Tris pH 8
B. Elution Buffer: Equilibration buffer 1 plus 1 M NaClHeparin Sepharose – Cation exchange resin
A. Equilibration Buffer: 20 mM MES pH 6.0
B. Elution Buffer: Equilibration buffer plus 1 M NaCl
HiTrap Phenyl Sepharose – hydrophobic interaction
A. Equilibration Buffer: 20 mM Tris pH 8, 1M NH4SO4
B. Elution Buffer: 5 mM Tris pH 8

HiTrap Blue Sepharose - affinity column
A. Equilibration Buffer: 20 mM Tris pH 8
B. Elution Buffer: Equilibration buffer 1 plus 1 M NaCl
© 2021 Protein BioSolutions, Inc. All rights reserved.

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Multi-Column purification
2-column Purification Schematic:

12 sample rack•••••

12 Protein Acolumns (1 ml)
12 Desalting
columns (5 ml)
Load volume: 50 ml(from sample rack)
Run time: 2.5 hunattended
operation
Run includes
columnregeneration

De-salting
column
20% EtOH

Strip
Load from deckWaste

Protein Acolumns

4321

56789Wash
EluteBuffer

De-salting
columns© 2021 Protein BioSolutions, Inc. All rights reserved.

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Complex Purification Mode
2-step Purification:
Ni and Protein A 2-step Fab purification

Case Study: Need medium scale throughput of 2-step protocol• Ni purification (GE His-Trap FF) of His-tagged Fabs alone requires additional purification, particularly for lowproducing proteins, but generally results in high yield• Ni requires buffer exchange post purification• Protein A (GE Hi-Trap FF) is added in series to increase purity and remove requirement for buffer exchange
Customized Potential Protocol:• Syringes 1-12: Load Ni, wash, and elute with 200 mM imidazole-containing buffer• Syinges 13-24: Load Ni eluate, wash, elute with pH 2.8 buffer with immediate post-column neutralization

Syringes 1-4 His-Trap FF (right); syringes
13-16 Hi-Trap Protein A FF (left)© 2021 Protein BioSolutions, Inc. All rights reserved.

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Customer Feedback
OPPORTUNITY COSTS
Make the best use of your time, money and equipment: “The Protein Maker
enables us to make the best use of our time and purification equipment by saving oursingle channel systems for complex purification experiments while the Protein Maker
quickly works through our routine and/or high-throughput purifications.”

Shrink your project timelines: “The parallelization of the Protein Maker enables
one technician to purify more protein constructs in one day than could be done in aweek by that same person. The practical result is that we can isolate the desired
protein construct in four weeks faster on average for every project. That is extremelyvaluable!”

Enable new strategies: “Parallel purification of 12-24 protein samples enables newand otherwise impractical screening and scouting techniques that we wouldn’t attempt
without the Protein Maker and have lead to key successes.”

INTANGIBLES
Pleasing your colleagues, clients and project leaders: “Just knowing that wehave this capability in house gives me the confidence to tell the end users of theproteins we produce that I can get them their pure protein in two weeks…when theywould expect it to take six or more.”© 2021 Protein BioSolutions, Inc. All rights reserved.

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