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The 3 P’s: Platform, Pre-analytical and Processing Considerations Using Unconventional Matrices

Alison JoyceAAPS NBC Conference, Boston, MA

May 17th, 2016

Outline

• What are unconventional matrices (UCMs)?• Platform/assay considerations

– Which platform to use?– Assay requirements

• Sensitivity, sample volume, Std/QC prep

• Pre-analytical/Processing considerations– Sample processing– Blood contamination– Normalization

• Tissue homogenate as UCM– Tissue Case Study for Mab PK assay

• Synovial fluid as UCM– Synovial Fluid Case Study for Mab PK assay

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• Bioanalysis of large molecules is typically carried out in blood-derived fluids (serum/plasma)– Easily accessible– Large quantities available– Often gives information about exposure, target levels, etc.– Deep knowledge in working with these matrices

• Unconventional matrices (UCMs) are non-serum or plasma biological fluids or tissues– Not easily obtained in many cases– May be very small quantities available– Matrix may be diluted/changed upon sample collection– Constitution may be very different from serum/plasma matrix

• Biophysical/biochemical properties different• Viscosity, homogeneity

• The default conditions that we know and rely on for serum/plasma bioanalysis may not be appropriate for these kinds of matrices!

What are Unconventional Matrices?

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Platform/Assay Considerations

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• Sensitivity/Sample volume requirements– Concentration of drug or biomarker in unconventional matrices may be

quite low• More sensitive platforms required

– Precious sample (volume, difficult to acquire)• Construction of Standards/QC’s

– Quantity may be limited for assay development, preparing standards/QC’s

– Use of synthetic or surrogate matrices to mimic the matrix• Can try buffer, serum/plasma, urine, other species tissue

• Tissue or cell lysis buffer may contain harsh detergents to extract analyte– LC/MS/LBA platforms may not be compatible – More contemporary LBA platforms may be more resistant to harsh

buffer conditions

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Bioanalytical Platform Considerations

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Assay Parameter LC/MS LBA

IP method non-IP method ELISA MSD Gyros Singulex Quanterix

Sensitivity √ √ √ √

Specificity √ √

Sample Volume √ √

Throughput √ √ √

Matrix interference √ √ √

Harsh buffer conditions √ √

Dynamic Range √ √ √ √ √ √Multiplexing √ √ √ √ √

• Bioanalytical platforms used for serum/plasma matrix may be used for UCMs– Unique matrix challenges should be considered when choosing the right

platform.

Pre-analytical Considerations

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• Sample processing– Pre-clarification steps may be needed

• Particulate matter, cells, tissue fragments, etc.• Can clog microfluidics, precision effects• Filtration/centrifugation impact on analyte measurement

– Extraction/processing procedure effect on analyte recovery in the matrix

• Homogenization, heating, addition of protease inhibitors, enzymes, etc.

• If lysing, is the buffer LBA compatible?• Stability of the analyte in the buffer, F/T or during the processing

procedure

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Pre-analytical Considerations (cont.)

• Blood contamination/sample contamination– Traumatic sampling procedure or disease (e.g. hemophilia)– Drug concentration may be very high relative to challenging matrix

concentration– A little bit of blood contamination could have a great impact on accuracy

of site of action measures– Perfuse organ and/or monitor for blood components

• Normalization – Total protein concentration – Tissue weight

• Assumes target concentration is uniform across tissue• Take into account size of tissue (e.g. biopsies can be very small)

– Tissue housekeeping proteins– Urine dilution normalization

• Creatinine, specific gravity7

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• Tissue: A collection of similar cells that perform a specialized function

• Drug/target interactions frequently occur in tissues. Quantitative PK or biomarker tissue measurements may enable:– PK/PD modeling for dose projections, exposure information, target

occupancy measures, patient stratification, normal vs disease biomarker levels

• Tissue must be disrupted for LBA or LC/MS analysis; disruption method depends on the tissue type– Homogenization: The breaking of tissue structure to form a suspension

of tissue solids, proteins and fluids; may be a challenging matrix for bioanalysis

• Extraction method determines recovery, accuracy, inter-sample variability• “Total” tissue measure- not compartment specific

– Considerations:• Structural heterogeneity of the organ; size, shape, weight, consistency (tough

vs soft)• Blood contamination• Disruption can cause heat, drug/target may be thermal labile (keep on ice)

Tissue Bioanalysis

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• Mechanical– Grinding/Shearing (mortar & pestle)– Blending (high-speed blades for chopping and mixing)– Sonication for soft tissues– Bead-beating for smashing; density of beads used can be specific

for tissue type (soft vs hard) • Enzymatic

– Tough connective tissue– Proteinase K or collagenase

• Analyte stability in tissue– Proteases/phosphatases in tissue; can add protease and/or

phosphatase inhibitor cocktails– F/T stability

Homogenization Techniques

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I like to homogenize used tissues….

Case Study: Optimization of MAb BiotherapeuticQuantitation in Various Tissues

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In this case study, we will examine:

• Pre-homogenization tissue prep effect – radiolabeled approach

• Lysis buffer effect on:– Analyte spike recovery in tissue homogenates– Analyte freeze/thaw and storage stability

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Mouse(n=3)

Case Study: Optimization of MAb BiotherapeuticQuantitation in Various Tissues

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(1) Pre-homogenization tissue prep effect:• Mincing, enzyme addition, different lysis buffers• Effect on analyte recovery in various tissue types (soft vs fibrous)• Use radiolabeled approach

Lysis Buffer Formulation

A20mM Hepes, 150mM NaCl, 0.1% Triton X-100, pH 7.4, PI

B20mM Tris, 150mM NaCl,

0.1% Triton X-100, pH 8.0, PIC Pierce T-PER, PID Invitrogen TER I, PI

*Intact vs Intact optimized:-Use of lower buffer volumes for homogenization-Use of upgraded Bullet Blender and UFO beads

Pre-homogenization steps:• Intact• Minced• Minced + collagenase• Intact, optimized*

I125 Mabdosed

Recovery:• Compare total counts

before and after tissue homogenization

Case Study: Optimization of MAbBiotherapeutic Quantitation in Various Tissues

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Results:

• Best recovery achieved with intact optimized tissue preparation procedure

• Similar results with all lysis buffers tested

IgG Recovery from Softer TissueBuffer C IgG Recovery from Fibrous Tissue

Buffer C

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Case Study: Optimization of MAb BiotherapeuticQuantitation in Various Tissues (cont.)

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(2) Homogenate spike recovery with different lysis buffers in two tissues

Spike performance ranking: Buffer D>Buffer A>Buffer C>Buffer B

Results: • There were definite

differences in recovery of analyte in the various lysis buffers tested, for both tissue types

• Buffer D showed the best spike recovery performance

• Due to poor QC recovery of some buffers, a F/T study was conducted using high and low QC’s

Target capture

MAb drug in tissue lysate or serum control matrix

Biotinylatedanti-huIgG kappa

Streptavidin-HRP

HRP substrate

ELISA format

*Spiked tissue lysates underwent 1 F/T prior to being assayed

Recovery of Spiked MAb in Lung Lysate

Recovery of Spiked MAb in Kidney Lysate

Case Study: Optimization of MAb BiotherapeuticQuantitation in Various Tissues (cont.)

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(3) Effect of multiple freeze/thaw cycles and storage stability on spike recovery in different lysis buffers

Results: • Buffers A-C showed a consistent trend in

recovery loss after each F/T cycle (only buffer C on 1st panel is shown)

• Buffer D showed good performance, even through 5 F/T cycles

• Buffer performance: D > A > C > B

• Buffer D also showed the best stability profile at -20oC for 21 days. All other buffers showed significant instability.

F/T Stability of QC Spikes in Buffer C

F/T Stability of QC Spikes in Buffer D

Storage Stability of in vivo study tissue lysates after 21 days (-20oC)

5 F/T cycles prepared vs. fresh spikes (FS)

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Optimization of several pre-analytical and processing steps for tissue samples is critical for accurate bioanalysis, including:

• Pre-homogenization techniques• Homogenization/extraction techniques• Lysis buffer choice for recovery and stability of analyte, and LBA

compatibility• QC’s should be treated as samples to ensure proper performance

of the assay for study support

Tissue Case Study Conclusions

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• Synovial Fluid– Syn (like) + ova (egg); joint fluid – Role: reduce friction between cartilage of synovial joints

during movement• Similar protein content to serum, except missing some HMW

species (10-30 mg/ml protein content)

– Viscosity• Very viscous nature attributed to high concentration of

polymerized hyaluronate• In inflamed, disease joint, often less viscous • Diseased synovial fluid is different from healthy synovial fluid

Synovial Fluid as a Challenging Matrix

1 2 3 4 5

1. Normal synovial fluid2. Non-inflammatory (e.g. OA)3. Inflammatory (e.g. RA)4. Septic (infection)5. Hemorrhage (e.g. hemophilia)

– Normalization• Total protein• Normalize using something that diffuses

freely between blood and intra-articular joint space

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Clinical PK Assay in Synovial Fluid

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• Goal: Develop a sensitive and selective clinical PK assay for MAb therapeutic in synovial fluid with limited reagents

• Why measure drug in synovial fluid? – Site of action for different diseases that

affect the joint (OA, RA, hemophilia, etc.)- need levels for PK/PD modeling

Microplate

Y

Anti-huIgG Fc-HRP

Mouse anti-Drug MAb

MAb drug

Assay Range:60 – 1448 ng/mL in

100% synovial fluid

Colorimetric substrate

• Challenge: Selectivity in individual donors was variable, inconsistent.– Attacked this issue on multiple fronts

Assay Buffer Optimization• DoE• Add synovial fluid pool to assay

diluent

Assay Format• Target vs anti-ID capture• Simultaneous vs sequential

Pre-analytical Sample Treatment/Processing• Addition of enzymes• Centrifugation• Dilution of sample

Pipetting• Technique very important• Positive Displacement pipettes

Troubleshooting Selectivity in Synovial Fluid

Go Forward Assay Conditions

Spike [c]

ng/mL

Buffer1

%Rec

Buffer2

%Rec

Buffer3

%Rec

Buffer4

%Rec

Buffer5

%Rec

Buffer6

%Rec

1448 ng/mL

182% 107% 107% 121% 117% 104%

295ng/mL

118% 116% 103% 92.4% 119% 104%

180 ng/mL

120% 110% 109% 97.1% 96.9% 101%

60.0 ng/mL

150% 119% 113% 223% 77.1% 316%

Original OptimizedFormat Target capture anti-ID captureBuffer Default DoE optimized

Centrifugation short, RT spin longer, 4°C spin, remove fat layer

Some DoE factors tested:• [NaCl]• Base buffers• IgG/serum

additives

• Assay Buffer Optimization • Pre-analytical Sample ProcessingSome sample processing tested:• Adding enzymes (chondriotinase, hyaluronidase)• Diluting sample before freezing• Filtration• Centrifugation and separation of fat layer

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Pipetting Viscous Body Fluids: Air Displacement vs Positive Displacement Pipettes

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Issue: Difficult to pipette with accuracy and precision when working with viscous fluids

• With Air Displacement pipettes:– Slow down pipetting speed; some fluid can

remain in the tip after dispensing– Don’t cut the tip point! Lack of accuracy– Can use reverse pipetting with pre-wetting step– Good technique is critical

• With Positive Displacement pipettes– Aspiration force remains constant regardless of

viscosity. A disposable piston in the tip ensures complete dispensing occurs (good accuracy)

Synovial Case Study Conclusions

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When working with synovial fluid as UCM:

• Optimization of assay format/conditions may be challenging for synovial fluid

• Pre-analytical processing needs to be evaluated for efficient recovery of analyte

• The viscous nature of the matrix makes accurate pipetting difficult– Consider the use of positive displacement pipettes

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Acknowledgements

• Joe Palandra• Ming Kuo• Rosemary Lawrence-Henderson• Mania Kavosi• Jeff Kurz• Denise O’Hara• Alok Rathi• Hendrik Neubert• Mauricio Leal• Sheldon Leung• Jim McNally• Boris Gorovits

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Andover, MA

Pfizer, Inc. Andover, MA