Results and Findings

Experiments and DesignDuring this project, phage display techniques were used to identify possible affinity peptide hits, whichwere then synthesized onto both Celluspot peptide array discs and agarose resin beads. After beingsynthesized, these peptide ligands are then analyzed using high throughput techniques. Resins are putthrough a batch screen process and analyzed. Afterwards, SDS-PAGE gels and RPLC chromatograms areused to visualize purification success of peptide resins.

Investigation of Affinity Peptide Chromatography Techniques for the Defense Advanced Research Projects Agency’s Biologically-derived Medicines On Demand (BioMOD) Program.

AbstractThe main objective of the research outlined is the purificationof certain molecules for the Defense Advanced ResearchProjects Agency (DARPA), which involves a mobile processdevelopment platform. The project claims to develop anentire upstream and downstream process developmentlaboratory that will be able to manufacture one dosetreatments while being geographically mobile. The entireprocess is contained within a very small area and only threecolumns are available for downstream process development.Underneath the guidance of the Downstream AffinityChromatography Resin team, the project will be to assist inthe development of novel separation technology usingspecialized chromatography resins. The project will focus onkey unit operations such as generating peptide libraries andsynthesizing peptides that will be used in high yield treatmentruns. The process begins with the generation of a peptidelibrary by looking through the known binding partner of thetarget antibody or molecule. Then synthesis of the peptidelibrary occurs and each of the peptides are printed onto a highdensity microarray slide. Then assays are used to determinethe affinity and selectivity of each of the unique printedpeptides on the microarray slide. After initial screening andtesting of each of the unique ligands, the new resins are thensynthesized again at larger scale, but this time synthesizeddirectly onto chromatographic resin. Afterwards, assessmentand development of the resin is done at a large batch scalethat screens peptide performance. The best performing resinsare then synthesized again, though this time for a largercolumn scale. The ligand selection and development processcontains feedback loops that allow for additional sequencesto be tested based on results that we obtain at any stage. Themain objective of the project is to develop chromatographicresin that is able to selectively bind certain target moleculesat a very high binding capacity in order to maximize targetpurity, yield, and throughput.

Ray Parker1, Chaz Goodwine1, Dr. Steve Cramer1, Dr. Pankaj Karande1

(1) Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy, NY 12180 (2) Biomanufacturing Research Program, Massachusetts Institute of Technology, Cambridge, MA 02139

References• "Chemical Engineers Collaborate To Advance Battlefield

Biomanufacturing Technology." Chemical Engineers at Rensselaer…Collaborate To Advance Battlefield… Technology. RensselaerPolytechnic Institute, 2 Dec. 2013. Web. 8 May 2015.

• Ling, Geoffrey. "Battlefield Medicine." Defense Advanced ResearchProjects Agency. DARPA. "Biologically-derived Medicines onDemand.“

• Balagadde, Frederick. "Bio-MOD Files." Microfluidic ScreeningPlatforms. Kampala Labs LLC & Stanford University. Web. 8 May 2015.

• Chantalat et al. Protein Pept.Lett. 2: 333-340 (1995)

Results and Findings (Cont.)

Conclusion• By investing in novel, flexible methodologies for

genetic engineering of eukaryotic strains, andflexible and portable device platforms formanufacturing multiple biologics with high purity,efficacy and potency, Bio-MOD will provide abattlefield medical supply for medics at the frontlines of support that is responsive to far-forwardemergency settings.

• Shown that various methods of phage displayscreening can be utilized to identify initial peptidesequences with various performance propertiesfrom which affinity optimization may be carriedout at a later time.

• Identified peptides that show good affinity for hGHusing the standard phage display method, and alsopeptides that show good selectivity for hGH in thepresence of P. pastoris CCF via subtractive panning.

• Future studies will assess subtractive washing as aphage display method in an effort to obtainselective peptides while not compromising ontheir affinity. Furthermore, lead candidateoptimization will be carried out on high-throughput microarrays and ligands for additionalhigh-impact biologics will be identified.

AcknowledgementsRensselaer’s CBIS’ Undergraduate Research Program,Massachusetts Institute of Technology, RensselaerPolytechnic Institute, Defense Advanced Research ProjectsAgency (DARPA), Northeastern University, Pall Corporation,Latham Biopharma, PerkinElmer and SSC Pacific.

Figure 1. Universal schematic for the Phase 1 Bio-MOD system that

incorporates a novel, flexible engineering approach to genetically

manipulate single strains of prokaryotic cells or eukaryotic cells to

produce multiple protein therapeutics.

DARPA's Biologically-derived Medicines on Demand (Bio-MOD) program seeks to develop devices and techniques toproduce multiple protein biologics in response to specificbattlefield threats and medical needs. Bio-MOD iscomplementary to the DARPA Pharmacy on Demand (PoD)program that is developing capabilities for manufacturingmultiple small molecule drugs using shelf-stable precursors.Together, Bio-MOD will provide a battlefield medical supplyfor medics at the front lines of support that is responsive tofar-forward emergency settings & emergent needs.

Background Information

1. Phage Display to Identify Affinity Peptides

• Phage Display is used to identify protein-peptideinteractions using bacteriophages to connect peptideswith the genetic information that encodes them.These phages are screened against proteins and candetect affinity interactions, screening large libraries(109 unique clones in a library) of peptides forpotential interactions with a target protein.

2. Celluspot and Resin Peptide Synthesis

• Once protein-peptide interactions are screened, allobtained sequences of possible affinity ligands arethen synthesized on base matrixes of either agaroseresin beads or compressed cellulose discs. Cellulosedisc synthesized ligands are printed and assessed onmicroarray.

3. Batch Screens of Peptide Resins

• Batch screenings of the resins narrow down thelibrary that was synthesized onto the resin orCelluspot disks. This is done in a high throughputscreening fashion and multiple parallel analyses areused to improve ligand identification success.

Figure 2. Sequence of events that are followed in a phage

display screening to identify binding peptides.

0

1

2

3

4

5

6

7

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9

Q (

mg

/ml r

esi

n)

Ceq (mg/ml)

1. Phage Display Generation 2 Results: CCF Isotherms P2.4 P2.6 P2.8 P2.10 P2.12 P2.17 P2.23 P2.27 P1.7.2 P1.13.2 P1.10.2

Figure 3. Outline of the process for the selection and

evaluation of peptides ligands from phage display for the

purification of target biologics in host cell culture fluid.

Figure 6: Reduced SDS-

PAGE gel visualized

with Coomassie Blue.

The gel shows the CCF

load and elution from

peptides identified by

conventional phage

display and subtractive

panning.

Lane 1: ladder, 2 : CCF

load, 3: Flow through of

P1.13.2, 4: P1.13.2

elution, 5: P1.7.2

elution, 6: P2.12

elution, 7: P1.10.2

elution, 8: P2.23

elution, 9) P2.4 elution,

10) P2.27 elution

WashElute

Strip

Load IFN in Cell Culture Fluid (CCF)

3. Batch Screens of Peptide Resins

Figure 4: The performance of peptides identified by

standard phage display in capturing pure hGH in

100 mM Phosphate Buffer, pH 6.5. Two varieties of

the lead peptides were synthesized and assessed. The

performance of P1.13.2 and P1.13 differ greatly,

with better performance being attained upon having

a free N-terminus and shorter linker.

2. Celluspot and Resin Peptide Synthesis

hGH: 100 ug/ml, Anti-hGH mAb-HRP (1:1000) in 100 mM Phosphate, pH 5.8

No hGH, Only Anti-hGH mAb-HRP (1:1000) in 100 mM Phosphate, pH 5.8

Figure 5: Phage Generation

Two experimental procedure

consists of preparing the

resin, then loading on the

hGH Isotherms, for two

hours, rinsing with buffer,

eluting with citrate, striping

with guanidine and re-

equilibrating with run buffer.

Figure 7: Batch screens

of peptide resins were

further analyzed by

reverse phase liquid

chromatography (RPLC).

The ATKA chromatogram

shows in yellow the

signal of cell culture fluid

doped with 1 mg/ml hGH

(where hGH is eluting

just after 2 minutes). In

blue, the elution fraction

of P1.13.2 after being eluted with 100 mM Citrate, pH 3.0. It can be

seen that a considerable reduction in contaminants is achieved at the

conditions screened at batch scale. Further wash and elutions screens

will be conducted to improve performance of the resins.