PhyTip columns provide high throughput small scale automated purification and enrichment of antibodies from microliter to milliliter samples

1. Application Note PhyNexus
www.phynexus.com
Enabling High-Throughput Functional
Characterization of Therapeutic Antibodies
Introduction
The process of drug discovery over the last 50 years has seen momentous change1. In the 1950’s, lead molecules were
found largely by serendipity through in vivo screening of the chemical diversity available at the time, where
subsequently identified candidates were quickly moved through full development to market.
By the 1980’s, with the implementation of systems research and focused screening, discovery developed into a
lengthier process involving a deeper understanding of the molecular structures and the associated functional
consequences that contribute to the affinity and efficacy of a specific drug compound. In addition, advances in
molecular and cell biology allowed for direct study and screening against human receptor targets in more
representative and complex environments such as animal cells and tissues.
In today’s post-genomic era where development costs are increasingly significant drivers, this process has expanded
significantly to include target identification and validation, as well as lead generation and optimization. With the
greatest expense involved in a new drug being associated with clinical trials, pressure on the discovery process to identify
the lead candidate with the highest likelihood of success has increased considerably. This requires the ability to make
increasingly informed decisions about the individual leads at earlier stages.
Antibody therapeutics and their development
One class of drugs that first appeared on the market in the 1980’s is antibody-based therapeutics. Monoclonal
antibodies (“Mabs”) represent a significant growth opportunity as indicated by the present value of therapeutics
already available on the market (2002 global market of US $5.4B for 11 approved drugs), with hundreds of antibodies in
various phases of clinical trials. In addition, Mabs are known to have a higher likelihood of approval as compared to
other drug classes once they enter clinical trials, with approval rates for certain Mab types as high as 26%2. Despite this
clear path to future Mab successes, there is always a need to establish even higher approval rates in less time and with
lower costs.
1. E. Ratti and D. Trist, “Continuing evolution of the drug discovery process in the pharmaceutical industry”, Pure Appl. Chem., 2001, 73, 67-75.
2. J. Reichert and A. Pavlou, “Monoclonal antibodies market”, Nature Reviews – Drug Discovery, 2004, 3, 383-384.

2. Page 2 Enabling high-throughput functional characterization of therapeutic antibodies
In the last ten years of Mab lead discovery, high-throughput strategies have been implemented to initially screen antibodies
to yield sufficient numbers of high-quality leads that are passed on for optimization prior to preclinical development. Initial
screens typically utilize assays to determine the apparent affinity of the potential antibody lead for a given target antigen; it
is usually at lead optimization when numerous functional assays are performed to generate data of greater physiological
relevance, thus providing more decision-making power as to which lead compounds should proceed for preclinical
development.
By necessity, this strategy places a great deal of importance on the affinity of antibody-target interactions. While there
certainly must be sufficient affinity for the antibody to bind the target, it has been shown in various contexts that affinity and
drug potency are not well correlated.
For example, antibodies that possess too high of an affinity have been shown to achieve poor tumor penetration in vivo due
to the so-called binding site barrier effect3, and that antibodies which successfully penetrate tumors typically have affinities
hundreds to thousands of times lower than the highest affinity antibodies4,5,6,. Issues such as these are highly correlated to
drug potency and ultimately the efficacy determined within clinical trials. Therefore, the ability to make an earlier decision
as to successful leads on the basis of selection criteria more closely correlated to potency (i.e., criteria other than affinity)
is extremely valuable as it translates into less drug being required for desired efficacy, which consequently leads to
potentially lower toxicity due to lower drug loads (thus improving chances for approval), as well as lower cost-of-goods
per dose once the drug is approved. Beyond issues related to potency and lowered toxicity, there are also criteria against
which to select antibodies that involve the ability to efficiently and cost-effectively manufacture the therapeutic antibody –
namely, the stability of the antibody (both in vivo and ex vivo) as well as its efficiency of expression.
Selection across these different criteria of the most promising antibody leads requires the ability to have precise control
over the structural – and hence, functional – aspects of the therapeutic antibody under development. Therapeutic antibody
discovery benefits from today’s capabilities within molecular and cell biology to exert direct influence over these structure-
function relationships. The combination of automation and the ability to create vast numbers of different versions, or
“libraries” of a biomolecule provides the ability to fine-tune the structure of biotherapeutics in general (including
antibodies) to the level of individual amino acids as well as their modifications, and hence optimize the potential drug for
any number of the criteria described above7,8,9. Furthermore, so as to impose the various selection criteria in the most
biologically meaningful context, cell-based assays are increasingly utilized as an in vitro mimic of highly complex physiological
processes – thus providing deeper and more descriptive data for each lead, upon which more informed decisions can be
made earlier in the process.
3. K. Fujimori, D.G. Covell, J.E. Fletcher and J.N. Weinstein, “Modeling analysis of the global and microscopic distribution of immunoglobulin G, F(ab’)2,
and Fab in tumors”, Cancer Res., 1989, 49, 5656-5663.
4. G.P. Adams, R. Schier, A.M. McCall, H.H. Simmons, E.M. Horak, R.K. Alpaugh, J.D. Marks and L.M. Weiner, “High affinity restricts the localization and
tumor penetration of single-chain Fv antibody molecules”, Cancer Res., 2001, 61, 4750-4755.
5. C.P. Graff, K. Chester, R. Begent and K.D. Wittrup, “Directed evolution of an anti-carcinoembryonic antigen scFv with a 4-day monovalent dissociation-
half-time at 37oC”, Protein Eng. Des. Sel., 2004, 17, 293-304.
6. L.M. Weiner and P. Carter, “Tunable antibodies”, Nature Biotechnol., 2005, 23, 556-557.
7. A.L. Kurtzman, S. Govindarajan, K. Vahle, J.T. Jones, V. Heinrichs and P.A. Patten, “Advances in directed protein evolution by recursive genetic recombi-
nation: applications to therapeutic proteins”, Curr. Opin. Biotechnol., 2001, 12, 361-370.

3. Enabling high-throughput functional characterization of therapeutic antibodies Page 3
However, cell-based assays require that antibodies screened within it are well purified and enriched once they have been
expressed, since typical antibody concentrations and contaminants inherent to cell culture completely confound direct
analyses. The process for adequately preparing antibodies requires that sufficient quantities of material be scaled up to at
least tens of milliliters, and then processed in a time-consuming and serial manner using expensive chromatography
equipment – all of which precludes taking a cell-based screening approach in the earliest stages. Consequently, cell-based
assays continue to be performed considerably downstream from the initial selection screens, thus relegating the high-value
information they provide to the latter stages of drug development and depriving therapeutic antibody developers of the
critical information they need to make increasingly informed decisions earlier in their processes.
Obtaining high-value biofunctional information at the earliest stages of
antibody discovery
Recent advances by PhyNexus in the area of miniaturized high-throughput tools for purification, enrichment and desalting of
antibodies and recombinant proteins now enable the implementation of cell-based assays at the earliest stages of lead
screening. By performing high-performance functional protein separations on samples as small as a few hundred
microliters, it is now possible to obtain more physiologically relevant data from thousands of distinct antibody variants and to
do so with complete automation – thus making substantial improvements in return-on-investment by dramatically
increasing the decision-making power available at the earliest stages in the antibody discovery process.
PhyNexus’ PhyTip® column technology has been developed for high-throughput preparation of antibodies and
recombinant proteins in order to facilitate the process of preparing hundreds to thousands of potential antibody leads that
are ready for cell-based assays without the need for scale-up. These unique columns are designed to operate either on
96-at-a-time platforms (such as those from Caliper, Tecan, Perkin-Elmer and Beckman) or on PhyNexus’ automated MEA
Personal Purification System. In either case, the PhyTip columns utilize a unique combination of design and process to
maximize the potential of the enclosed affinity resin to capture the protein of interest. The figures below show the MEA
Personal Purification System and the PhyTip columns.
The operational process utilized by the PhyTip columns requires the robotics platform to move to the sample and pass a
given volume over the resin bed at a specific flow rate to obtain the highest performance from the individual steps of
capture, purification and enrichment. Typical results from a 96-at-a-time platform indicate that >95% purity of fully
functional scFvs, Fabs or IgGs can be obtained with exceptionally high yields and enrichment factors in as little as 15
minutes for 96x200 µL samples10 or 60 minutes for 96x5 mL samples11.
10. J. Lambert, M. Anderson, C. Hanna, L. Jordan, A. Esterman and S. Cohen, “Improved process efficiency with 96-well protein purification and
characterization”, 15th Annual International Conference on Antibody Engineering, San Diego, CA, 1-3 Dec, 2004.
11. K. Kopacz, C. Pazmany, Q. Wu, J. Cosic, A. Nixon and D. Sexton, “Automated purification of phage display-derived antibody sFab fragments”, 10th
Society of Biomolecular Screening Annual Conference, Orlando, FL, 11-15 Sept, 2004.

4. 8. M.A. Poul, B. Becerril, U.B. Nielsen, P. Morisson and J.D. Marks, “Selection of tumor-specific internalizing human
Page 4 antibodies from phage libraries”, J. Mol.. Biol., 2000, 301, 1149-1161.
Additionally, PhyTip 5K desalting columns used in conjunction with the MEA Personal Purification System allow for
completely automated desalting of 96 functional antibodies in less than 45 minutes. These capabilities have allowed various
groups’ to successfully enable high-throughput biofunctional screening data to be obtained for a wide range of antibody types
and assay formats.
For example:
• High-throughput cell-based assays of growth factor receptor-mediated Stat5 phosphorylation have been used at Amgen
to screen for potent antagonist antibodies derived from phage display12. Individual clones of human scFv recombinant
antibodies were expressed in E.coli overnight growths in 96x2 mL deep well plates. 300 µL periplasmic preps were pre-
pared for each clone and were individually processed with PhyTip columns containing Ni-NTA resin.
• High-throughput flow cytometry has been used at Dyax to perform assays for whole cell-binding IgGs13. Individual
clones of human Fab recombinant antibodies were initially selected by standard panning procedures, individual clones
were picked, reformatted into IgG structures and expressed as HEK293 transient transfections. 96x6 mL cultures were
incubated in 4 separate 24x10 mL deep well plates and were redistributed into 96x2 mL deep well plates, which were
then processed with PhyTip columns containing Protein A resin. This same group also uses PhyTip columns to perform
high-throughput surface plasmon resonance (SPR) array screening of recombinant antibody kinetics14.
• High-throughput target identification, target validation and lead screening have been streamlined and automated at
Raven by immobilizing lead antibodies on PhyTip columns and performing a range of assays with them – from IP and
CoIP to identify targets, ELISAs of whole cells and cell lysates to confirm targets, and immunohistochemistry (IHC) of
cell and tissue arrays as part of lead screening and target validation. This process ensures that only the most
well-qualified leads are pursued – thus leading to substantial improvements in process efficiency, more focused lead
development efforts and overall cost savings15.
Conclusions
Antibody-based therapeutics have begun to prove themselves as a powerful class of drugs. The intrinsic ability to fine-tune
antibody function with respect to the disease target, the proven track record of antibody therapeutics within clinical trials,
and the already burgeoning value of the antibody therapeutic market indicate a bright future for this class of therapeutics.
Nevertheless, a constant push will always exist to ensure even greater efficacy and improved approval rates so as to
maximize return-on-investment for those organizations involved in antibody therapeutics. An essential part of meeting these
goals is to insure that resources are directed towards implementing the most efficient processes to developing those
antibody leads that are the most relevant and have the highest likelihood of success; to do so requires obtaining as much
physiologically relevant data as early as possible so that key decisions are made before significant resources are committed.
PhyNexus’ PhyTip column technology for purification and desalting represents a critical component in the process of
obtaining this decision-making power at the earliest stages of antibody discovery and development.
12. C. Hanna, D. Gjerde, L. Nguyen, D. Burge, J. Lambert and T. Arvedson, “Novel strategies for assaying recombinant antibody function with high-
throughput cell-based assays”, 7th Annual Phage Display and Recombinant Antibodies Technology, Cambridge, MA, 16-20 May, 2005.
13. K. Kopacz, C. Pazmany, L. Huang, R. van Hegelsom, H. Pieters, N. Frans, M. Nguyen, A. Gorman, I. Roy, Q. Chang and A. Nixon, “Automated
purification of phage display-derived antibodies for downstream analysis”, 7th Annual Phage Display and Recombinant Antibodies Technology, Cambridge, MA,
16-20 May, 2005.
14. J. Lambert, C. Hanna, U. Banik, D. Sexton, K. Kopacz and S. Wiltshire, “A novel approach to high-throughput monoclonal and recombinant antibody
enrichment and characterization”, 6th Annual Phage Display and Recombinant Antibodies Technology, Cambridge, MA, 26-29 April, 2004.
15. C.Hanna, D. Gjerde, J. Lambert, T. Liang, J. Whelan, C. Fieger and I. Ni, “Automated enhancement and streamlining of therapeutic antibody discovery
through the application of micro-scale high-performance protein separation technology”, 20th Annual Drug Discovery Technology, Boston, MA, 8-11 August,
2005.
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