Technological Advances in Host Cell Protein Testing and Risk Management: Enhancing the Quality of Clinical ProductsNed MozierPfizer

12 September 2018

15TH SYMPOSIUM ON THE PRACTICAL APPLICATIONS OF MASS SPECTROMETRY IN THE BIOTECHNOLOGY INDUSTRY

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ABSTRACT

Technological Advances in Host Cell Protein Testing: Empowering Quality Decisions and Risk ManagementImprovements in databases and mass spectrometry (MS) technology have radically changed the landscape for HCP testing and impurity management. The often debated question as to whether MS has a function in the quality control testing laboratory is no longer hindered by its capabilities, although this remains a strategic decision for corporations. The power of MS for identification of HCPs is now common in development, especially in regards to process development and for risk management. The immunoassay remains the bedrock by which most quality decisions are made, but increasingly MS is being used as an orthogonal method. This has enabled more useful risk analysis and intelligent decision-making. Together, immunoassay and MS are the most important tools for managing the risk of impurities such as HCPs and have led to higher quality products. How various technologies are applied during the development paradigm has evolved, and been accelerated with the advent of biosimilars. This presentation will review the known and unknown risks of HCPs, and provide cases where individual proteins have been shown to affect clinical trials, portfolio decisions and how the technological innovations have influenced this direction.

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My Main Points

• Mass Spectrometry for HCPs is happening • It will not replace ELISA / Immunoassay for HCPs

The real POWER is using both• HCPs have damaged and/or destroyed clinical programs

– Total HCP content has been (and is) an issue– Clinical events due to HCPs have occurred

The Newer tools (e.g. MS) facilitate HCP issue resolution• Don’t be afraid of discrepant data.. seek to understand

– All test methods have limitations (cons)

– And advantages (pros) if properly applied

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..but you probably knew that

Content of My Talk

• Impurity Testing – The Fundamental problem–Type of impurity drives the need for sensitivity / orthogonality–Do we even need HCP control limits / product specifications?

• What LC-MS/MS has added to the HCP Toolbox• How Assays Relate to HCP Product Specifications• How to Conduct a Proper HCP Risk Assessment• HCP Cases That Mattered for Patients• Comparative Data Tells the Story• The Future is Bright

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The Problem with “Purity” as a Concept….• SDS-PAGE (1D)• SDS-PAGE (2D)• HPLC• Westerns – 1D• Westerns – 2D• ELISA (from null cell, most commonly)• Improved ELISA/Immunoassay (USP<1132>)• Peptide mapping / MS• Total proteome LC-MS/MS vs known genome• Single HCP ELISA or LC-MS/MS(if needed)• Biological / activity assays

JBC,263(10), 4527-4531, 1988, Mozier et al 5

The Analytical Problem is Great: Quantitation Typically Requires a True Standard and Validation

• Host Cell Proteins (HCPs)– Proteins produced or encoded by the host

organisms used to produce recombinant therapeutic proteins

– Constitute a major group of process-related impurities in a drug substance/product

• FDA Guidance

“Whenever possible, impurities introduced by the recovery and purification process should be below detectable levels

using a highly sensitive analytical method”

MW

pI

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What (really) is an HCP?

• One of potentially 40,000 proteins?– Each x 1000 isoforms/PTMs/degradants

= millions / billions / trillions / …… ? of possibilities

• What, really, is an HCP?– Heterogeneity varies– Single gene products rare– How should we measure?

• Immunoassay or LC-MS/MS?

YES!

Impurity vs Purity Further Explored: Single Protein Impurities

% Impurity (w/w) ppm (ng/mg) %Purity (w/w)100.00000 1,000,000 010.00000 100,000 901.00000 10,000 990.1000 1,000 99.90.0100 100 99.990.0010 10 99.9990.0001 1 99.9999

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Sensitivity for a single HCP

CGE/HPLCPeptide mapSDS-PAGE

Immunoassayor LC-MS/MS

“total” HCP is how specs are most often set, but if the HCP is known, the sensitivity can be “tuned” even lower than ppm (by immunoassay or MS)

Any method can be exquisitely sensitive if the

analyte is known

=1 μg/mg

Immunoassay vs LC-MS/MS are Orthogonal: Both are sensitive and state of the art for HCP Detection

Pro ConImmunoassay High throughput (>30 samples per

day per FTE) – can be increased by simply adding headcount

No HCP identification (only a value)

Low cost of equipment/facilities,easily automated

Non-immunogenic HCPs can be missed / under-estimated

LC-MS/MS Identification of HCPs allows risk analysis and goals for process development

Low throughput (2-3 samples per day), high instrument time

Not limited by HCP immunogenicity in rabbits or goats

High capital investment / maintenance

Experts required for development, troubleshooting and data analysis

Experts required for development, troubleshooting and data analysis

The Addition of LC-MS/MS to the Toolkit has been an Enormous Step Forward for our Industry

• Compensates for what the immunoassay may miss• HCP identification offers

– More intelligent process design• i.e. means for their removal by purification

– Better informed risk assessments• To patients (directly)• To patients (indirectly) through actions on the product itself

– E.g. proteolysis, degradation of polysorbate

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Using Information from both MS and ELISA

• Neither are necessarily quantitative– Both can underestimate the true levels of HCPs in samples

• Immunoassay: insufficient antibody, “super” antibody to some HCP

• LC-MS/MS: PTMs could be missed, genomic data accuracy requires vigilance

– Numbers can deceive if not understood

• ELISA and LC-MS/MS sensitivity are greatly enhanced if the identity of the HCP is known– ELISA standardized with single HCP – LC-MS/MS standardized with exact HCP concentrations

10 ng/mg sensitivity common

< 1 ng/mg sensitivity possible

Why do we need HCP specifications

• We may not – if the process and test methods are demonstrated to be highly effective, robust …..

• But often we do - To assure that production is performing as expected during routine manufacturing– Confirmatory that the controls in place are working as intended– At least one regulatory agency is likely to require it

• The HCP limit is part of the system that assures that each and every unit of medication is safe– Linking to the quality of the pivotal trial clinical outcome is critical

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HCP is a special case because the assay is so difficult to design and interpret and must be customized to the

process and product.

Spec Limits for Impurity Methods

• Process related– Host cell related

• Host cell protein• DNA (nmt 10 ng per dose), primers/cell line and standards commercially available

• Endotoxin (nmt 5 EU per kg in most cases), international standards

– Others• Residual protein A for mAb processes (often 10-20 ng/mg)

• Other residuals (e.g. insulin, process additives) – case by case

• Product related (impurities vs substances) – out of scope today

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Contaminants (e.g. endotoxin in CHO) ≠ impurities

Much uncertainty – that’s why we are here

Linkages: Remembering Our Analytical Role is Ultimately about our Patients

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Patient

Product

Process

Clinical outcome

Critical Quality Attributes

Material Attributes & Process

Parameters

Risk Factors for Total HCP (when none are identified)

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Wang et al 2009

Risk Factors for HCPs (when they are known)

• Amount in product• Dose quantity of product• Type of HCP in product• How product is dosed (intradermal vs subcutaneous vs

intravenous)• Dose frequency / duration of exposure (chronic vs short term)• HCP effects in combination with product effects• HCP effects when physically associated with product • Age and health status of humans (infant vs adult, disease)

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Vaccines are typically small quantities of low dosing frequency (1-3 lifetime), hence HCP is generally less of a concern

A Sampling of Emerging LC-MS/MS Publications

• Mass spectrometry-based methods are emerging as a routine method for HCP analysis where one can detect, identify, and quantitate residual HCP directly due to ever increasing instrument performance.

Consequence of Finding HCPs During Clinical Development: Drives Need for Orthogonality

• Majority of mAb Portfolio (CHO) – 2013, Genentech– Solved and published: Phospholipase B-like 2 (PLBL2)

• Product in development (CHO) – 2013, BMS – Method of analysis published

• rFactorIX (CHO) – 2012, Inspiration Biopharmaceuticals– Solved: Process improved over substantial time

• ETC216 (E. coli) – 2006, Pfizer – Solved: HCP was flagellin, published

• Omnitrope (E. coli) – 2004, Sandoz– Solved: presentations provided in conferences

A Partial List of HCP “bad actor” Case Studies

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HCP Reference ImpactCellulase (E. coli) Shahrokh 1995 Viscosity increase in Drug Product

Cathepsin D (CHO) Bierau 2009, Gao 2011 Proteolysis of product (quality concern)

Furin (CHO) Monograph 2013 Antibodies raised in 19% of patients

Flagellin (E. coli) Huang et al, 2009 ≤~1 ng/mg associated with adverse events

TGFβ1 ortholog (CHO) Beatson 2011 Active in vitro (no clnical exposure)

Ribose phosphate isomerase (E. coli) De Zafra 2015 ~1400 ng/mg adjuvant-like effect to product

Monocyte Chemotactic Factor (CHO) De Zafra 2015 Clinical trial suspended*

Phosholipase B-like 2 (CHO) Vanderlaan 2015 ≤300 ng/mg caused anti-HCP

Tweenase CHO Labrenz 2014, Dixit 2016 degraded PS20 compromises product stability

Unknown or undisclosed Human Growth Hormone (KabiVitrum), Factor IX (Inspiration Biopharmaceuticals)

*In another case (BEBPA 2017) for another product, this MCP was found in a commercial product that had been on the market for many years – and was not associated with product safety issues

April 2018, Biotechnol progress

0

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0.5 0.2 0.05 0.02 500 150 50 500 150 50 500 150 50

LPS (EU/mL) PBS ug/mL DP ug/mL DS ug/mL new DS

IL-6

, (pg

/mL)

donor 1 donor 2 donor 3 donor 4

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0.5 0.2 0.05 0.02 500 150 50 500 150 50

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/mL)

donor 1 donor 2 donor 3 donor 4

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0.5 0.2 0.05 0.02 500 150 50

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IL-6

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/mL)

donor 1 donor 2 donor 3

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LPS (EU/mL)

IL-6

, (pg

/mL)

Process-Related Impurity (host cell protein)

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0.5 0.2 0.05 0.02

LPS (EU/mL) PBS

IL-6

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donor 1

A Comprehensive Portfolio Strategy

• Risk-based (proteins, mAbs, enzymes > vaccines)– Some categories have more historical regulatory interest

• HCP by Immunoassay from cradle to grave• Add LC-MS/MS analysis at key triggers in development, e.g.

– when HCP is present in DS or product reaches a particular stage– in response to particular regulatory requests

RegTox Ph. 1 Ph. 2 Ph. 3 Commercial

Initiation of clinical program

Commercial process lock

Stage 1 Stage 2

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Early Stage (non-clinical, tox batches) Testing Results

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Program No. BatchesTested

HCP ELISA Result (ng/mg)

LC-MS/MSResult (ng/mg)

mAb1 2 ≤10 ≤10mAb2 2 ≤10 ≤10mAb3 3 ≤10 ≤10mAb4 2 ≤10 ≤10mAb5 2 ≤10 ≤10mAb6 2 ≤10 ≤10mAb7 2 ≤10 ≤10mAb8 1 ≤10 ≤10Protein 1 1 ≤10 16Protein reagent 1 ≤67 ≤10mAb9 2 ≤10 ≤10Fusion Protein 1 28 ≤10mAb10 1 ≤10 ≤10

Conclusions and Suggestions

• HCP Risk Assessments add value– To assure quality of clinical supplies– May avoid late-stage re-work and program delays

• No single method is sufficient for HCP control– All the tools should be applied based on risk assessment above

• Further investment in the LC-MS/MS tool will add value– Database accuracy and integrity– Reach consensus on your analytical capabilities before collecting data!

• Regardless of analytical technology, err on the side of avoiding type 2 (consumer) risks vs type 1 (producer) errors

• First assume a signal (HCP) is not an artifact, then prove it

• Don’t’ fight about which method is best - use all the knowledge23

It’s about the Patient!

Acknowledgments

• Phoebe Baldus• Justin Sperry• Jessea Campbell• Ying Zhang• Jason Rouse• Ranga Godavarti• Joann Parker• Marta Czupryn• Meg Ruesch

• FDA collaborators24

Close partnership between our analytical groups and our process, regulatory and drug safety groups was a key to our success

ARD: Bioassay and Impurity Testing group (BIT)

ARD: Mass Spectrometry and Biophysical Characterization (MSBC)

3

QUESTIONS?

Manual of Policies and Procedures (MAPP) from FDA was effective 18-Jan-2018

• US, FDA-CDER-specific, small-molecule orientation• “Clinically relevant acceptance criteria are defined as a

set of acceptances ranges to which an impurity should conform in order for the product to be safe and effective when used as labeled.”

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MAPP (continued)

• Non-clinical studies usually not informative for HCP risk• Comparison to an FDA-approved product may be useful!

– For total HCP in one mAb vs another within a company– Identification (e.g. by MS) of single HCPs may be leveraged

• Method Precision is a factor (discussed later in validation)27

MAPP (continued)

The concept of a specified / unspecified impurities has traditionally been considered irrelevant for biologics (vs small molecule drugs)

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e.g. an HCP that is present and has its own spec limit?

For unidentified peaks/bands – never the case for HCPs

For peaks/bands below DL – never the case for HCPs

Most relevant for HCPs: Analogous to total HCP (ng/mg)

Similarly, “qualification” of impurity levels in animal tox is not relevant to HCPs in biologics

MAPP (continued)

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While limited, this body of data about individual HCPs is growing

LC-MS/MS has changed this

This has always the case for total HCP –it is sometimes viewed as a canary in the coal mine for process performance – i.e. it is why HCP is typically a CQA

Specification Concept / Relevance to HCP Assays

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General Concept Relevance to HCP specsLinked to a manufacturing process High: CHO≠NS0≠E≠coli≠SP/2≠ …..most

products have unique recovery/ isolation stepsLinked to product Med: Each can interact differently with HCP (e.g.

hitchhiker) during DS processingLinked to product stability Low: HCPs can’t increase, assumed constantLinked to preclinical / clinical safety studies (especially pivotal)

Low for pre-clinical; High for clinical

Based on commercial process High: data for DS made at manufacturing scale demonstrates capabilities

Method capabilities High: HCP assays are unique / challengingReliant on Reference/AssayStandard

High: External standards not available, a proper HCP assay standard is essential

Method Sensitivities for HCP Characterization• HCP ELISA is the industry “gold

standard”– Requires custom reagents

– Detects HCP as a population

– Pro: Good sensitivity (≤10 ng/mg)

– Con: may not detect every HCP

• Emerging, orthogonal mass spectrometry-based method– Sensitivity should match ELISA

– Detection of HCPs in the presence of mAb

– Pro: identifies the impurity

– Con: may not detect every HCP

5000ng/mg

Western Blot

CGE Non-reducedCGE Reduced

Peptide Map

1D SDS-PAGE

ELISA

500ng/mg

5ng/mg

50ng/mg

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Sperry, BEBPA-HCP, 2014-00470

Total HCP by Immunoassay

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Total HCP by Immunoassay

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Pro ConEstablished, accepted by health authorities Blind to certain HCPs*Validateable, QC-friendly, transferable Non-linear behavior**Cost effective (instrumentation, lab footprint) No identification***High throughputSuitable for process clearance and final drug substanceNumerical limits / specs allow simple batch dispositionPlatform-friendly

*Mitigation= orthogonal methods, e.g. gels, HPLC, **Mitigation= process improvement, antiserum treatment, dilutional approach (USP<1132>)***Mitigation= isolation / sequencing or LC-MS/MS

LC-MS/MS

How Low Must We Go?

• When HCPs are not detected100?20?10?1?

• If they are detected?>1000?1000?100?10?1?

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It depends – is the method acceptable? Is it missing anything?

Is this the best the process can deliver? Do we have clinical safety

data with these batches?

Finding out late that

HCPs are high can be traumatic

Early method qualification and

validation are good investments

HCP Specifications Early in Development

• When HCPs are Present– If <100 ng/mg, regulatory comments unlikely

• Dilutional linearity?

– If >100 ng/mg, more likely to get regulatory comments• Is the method commercial or process-specific?

• Is the process optimized?

• When HCPs are not detected– Is the method properly designed?

• Does it show clearance?

• Is it sensitive enough?– 1, 10 vs 100 ng/mg

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← sample dilution

← sample dilution

USP<1132>

Considerations LATE in Development

• What is in the DS going into your Pivotal Clinical Trial? This is what your safety and efficacy clinical data will be base on.– Orthogonal methods important to assure nothing is missed

• Anything identified needs a risk assessment

• In some case, upper limits may be required for individual HCPs

– Total HCP immunoassay values critical• These numbers will provide the argument for a numerical upper limit

• Consistency will be evidence of the quality of your process

• Limited clinical batches always bring risk for future commercial production– E.g. if process is changed later, the method capabilities are critical

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Is an HCP limit even needed on the DS spec?

• Some products have been approved without a DS specification• Success Factors

– A well characterized and controlled process • Especially those where HCP reduction is understood and part of the design

– Demonstration that HCPs are (1) below QL or (2) at consistently low levels• Assuming a properly developed HCP assay

• Dilutional linearity issues are solved

– A large quantity of data• HCP clearance data from scaled down studies where key steps identified

• Batches at intermediate and full scale showing HCP clearance

• HCP levels at the final step are well defined– Approval of a limit at an intermediate step is a fallback position

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A Variety of HCP Specification Upper Limits (≤ ng/mg)

Total HCP Single HCPIPC DS IPC DS

mAb1 100mAb2 10mAb3 60mAb4 20mAb5 10mAb6 10 10mAb7 1

rProtein1 100 ≤5000rProtein2 200rVaccine1 1,000rVaccine2 20,000

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All of these cases have

been successful – how were

these justified?

Pentamer

MonomerHexamer

5

6

12

20

Qβ VLP

Qβ Virus-like Particle

• Bacteriophage Qβ is recombinantly expressed in E. coli

• 180 repeats of the monomeric unit – 132 amino acids (14.1 kDa)• Composition: capsid (2.54 MDa), RNA (~25%) and HCPs (~1-2%)• Immunogenic carrier protein for vaccine conjugates

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Cryo-EM image

2%= 20,000 ng/mg

HCP Limits Require Sample / Method Interpretation First

• Multiple dilutions needed for “Final Pool” sample

• Which is the “true” value? 300 or <1?

• If this issue persists (HCP remains in DS), this testing approach needs to be solved before method validation– Can be very challenging if a single HCP exists at high levels – may require a separate assay

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USP<1132> suggests causes when a lack of

dilution linearity is observed, and

resolution

Specification Considerations when HCPs are not detected

1. Is the method properly designed?– Guidance in USP<1132> and other publications

2. Does it have the necessary sensitivity?– Typically 1-10 ng/mg (supports 10 or 100 ng/mg)– Caution: Method Validation (late in program) may increase the QL

depending on how the validation is done

3. Does it have the necessary selectivity?– Fails to recognize non-HCPs (especially the product)– Is not failing to miss HCPs in the product (orthogonal method needed)

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Thoughts on Coverage:

1. US regulators may ask for coverage by 2D gels2. Antibody-Affinity Extraction under “native” conditions3. Showing clearance of HCPs through your process is simple and effective

Rationale when no HCPs are detected

• Early: 100 ng/mg common• Late / after process validation

– Not more than 10 or 20 ng/mg is common– Not more than 50 or 100 ng/mg might get regulatory pushback

• Twice the QL is a reasonable approach– If QL is below 10: Make limit not more than 10 ng/mg– If QL is >10 and <100, set limit at QL*2

• E.g. if QL= 10, set limit at 20 ng/mg

• E.g. if QL= 25, set limit at 50 ng/mg

• E.g. if QL= 50, set limit at 100 ng/mg

– If QL is >100 ng/mg, may get questions

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Another approach is use method QL + 3X precision (intermediate): e.g. if

QL= 10 and CV= 20%:

10+(3*0.20*10)= 16 ng/mg

Mozier 2018-00743

A Variety of HCP Specification Upper Limits (≤ ng/mg)

Total HCP Single HCPIPC DS IPC DS

mAb1 100mAb2 10mAb3 60mAb4 20mAb5 10mAb6 10 10mAb7 1?

rProtein1 100 5000rProtein2 200rVaccine1 1,000rVaccine2 20,000

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In rare cases 2 limits are required

Mozier 2018-00743

Human Exposure to Small Doses of Protein A

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What about single HCP specs when the identity is known?

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Calculating Exposure Levels

kg weight5

205070100

46

mg drug0.510100250

ng/mg HCP1

10300

140010,000

(300 mg / 50 kg) (300 ng/mg)= 1800 ng/kg = 1.8 μg/kg

Ballows et al, 2013: Protein A as a drug

0.45 μg/kg= 7 of 8 patients raised Ab to drug0.3 μg/kg= 3 of 8 patients raised Ab to drug

0.06 μg/kg

Specs for Single HCPs

• Limit may be set by safety argument (direct clinical exposure with our without multipliers) based on risk assessment– Limits based on maximum content in clinical trial material is more

likely (vs statistical argument)

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PIVOTAL lots / Commercial Scale

Commercial ELISA suggested steady process improvements throughout development and process was in control post approval

Levels of HCP in new assay (post marketing commitment) at first caused concern that process changes may have altered quality

Retrospective analysis showed no change in HCP levels (and even improvements) leading to new (higher specification) since long patient history / monitoring– Sponsor would have avoided this stress by having improved assay earlier

• But this can also happen when new reagents are replaced, regardless of commercial or process-specific / platform

Case Study 1: Process change post approval simultaneously with anti-HCP reagent replacement

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Results in ng/mg

Mozier CHI 2015

Mass Spectrometry-Based Workflow for HCPs

SampleFinal DS/DSI or

Process pool/fraction

LC-MS/MSPeptides are analyzed

by Orbitrap MS technologyStrategic Capital Investments

for St. Louis (’14) and Andover (’15)

SeparationRP-HPLC

Formic acid-basedWaters CSH Column

Data AnalysisPeptides are identified

and quantified

Generation of PeptidesProteins are denatured, reduced and alkylated

followed by tryptic digestion

Abundances of Common CHO HCPs

• LC-MS “DIGE” – database MW/pI and relative abundance

0

20

40

60

80

100

120

140

160

180

200

3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0

Dat

abas

e M

W (k

Da)

Database pI

High ViabilityLow Viability

thrombospondin-1isoform X2

glucose-regulatedprotein precursor

calreticulinpeptidyl-prolyl cis-trans

isomerase B-like

glypican-1-like protein

Complement C1r-Asubcomponent

0.0010%

0.0100%

0.1000%

1.0000%

10.0000%

100.0000%

HCP-1 HCP-2 mAb

Basics of HCP Process Monitoring by LC-MS/MS

mAb Select AEX CEX DS

356Proteins

94Proteins

9Proteins

2Proteins

220 ng/mg

15 ng/mg

1392 ng/mg

20 ng/mg

ELISA Result~ 8 ng/mg

Sample Dilution for Immunoassay Testing for HCP Clearance

• Standard curve range is narrow vs range of HCP concentrations in samples

• Pipetting / Sample Handling can be a factor in the result

• A lack of dilutional linearity further complicates the analysis

USP<1132>

Standard range≤200 ng/mL

~10,

000X

~1,0

00X

~100

X

~2-10X

USP<1132>

Sample Dilution Provides Myriad Options

• Understand liquid handling / non-specific adsorption through careful evaluation of sample preparation

10X 10X 10X 10X

10X

1 µL in 10 mL?10 µL in 1 mL?20 L in 5 mL?

100 µL in 10 mL?

~10,000X Dilution

Enormous Range of HCP Levels in Samples vs. Relatively Narrow HCP Calibration Curve

Each sample is diluted within range of the Working Range of the HCP calibration curve and each result is +/-30%

(therefore, normal distribution can range 2X for samples)

USP<1132>

• 1 vs 2 ng/mg?• 10 vs 20 ng/mg?• 100,000 vs 200,000 ng/mg?

Monoclonal Antibody#1 HCP Process Clearance

Step 1 Step 2 Step 3 Step 4

0Proteins

1 (5% FDR)1 (1% FDR)

Protein

4 (5% FDR)4 (1% FDR)

Proteins

301 (5% FDR)237 (1% FDR)

Proteins

Process Step ELISA(ng/mg) LRV LC-MS/MS

(ng/mg) LRV

Step 1 45211 N/A 104374 N/A

Step 2 38 3.1 967 2.0

Step 3 < 1 N/A 56 1.2

Step 4 (DS) < 2 N/A ND N/A

Monoclonal Antibody#2: HCP Clearance

• Process clearance studies conducted during process validation

• Assurance of commercial-ready process in clearing HCPs

Step 1 Step 2 Step 3 Bulk DSNot

DetectedNot

DetectedELISA

~6700 ng/mg

LC-MS/MS~15,400 ng/mg

ELISA~237,000 ng/mg

LC-MS/MS~163,000 ng/mg

NotDetected

NotDetected

What to do when finding HCP

• Key elements of a risk assessment– Is it known to be associated with reactions in non-

clinical species?– Is it known to cause adverse events in human beings?– Is it predicted to be biologically active in humans?– Has it been shown to be active in in vitro studies?– What is the risk of (direct) immunogenicity?

• Probability of inducing antibodies to HCP or product• Is there a human homolog?

– What is the risk of (indirect) immunogenicity?• HCP is active and degrades product leading to product

immunogenicity Our practice is similar to that of De Zafra et al, Biotechnology and Bioengineering 2015

The Special challenge for Impurity Tests: We see what we look for and are limited by imagination

From the other Donald

“There are known knowns. These are things we know that we know. There are known unknowns. That is to say,

there are things that we know we don't know. But there are also

unknown unknowns. There are things we don't know we don't know.”

HCP Assays: we know some

remain undetected!

What other impurities might be present ?Which methods are most likely to “see” something new?

Summary: Assuring the Absence of HCPs

• A properly developed, state of the art immunoassay remains a workhorse and is ideal for routine use and process characterization– Much to learn from the new Pharmacopoeial guidelines and recent publications

• Addition of LC-MS/MS as an orthogonal method is a powerful tool for confirming immunoassay and to identify HCPs (if present at 10 ng/mg) – Enables risk assessment of found HCPs and more intelligent process

development for their removal – and better quantitation in immunoassay by proper standardization

• A lack of 100% concordance between Immunoassay & MS is expected!– Both add unique value, compensate for each other, and are a powerful combo

– When discrepant, usually the method giving signal is the true one• False negatives more likely than false positive

• Publish what you can on HCP risk assessments and finding so all in our industry can increase the quality of our drug supplies for patients!

Practical Application of Orthogonal HCP Testing: How to interpret and Use the Data

Many companies are now using, or beginning to apply, LC-MS/MS testing in a new application for detection and monitoring of residual host cell proteins (HCP). These impurities are also tested by the more established immunoassay that was received reviewed in the USP<1132> guideline. Both approaches have value, and each has unique benefits and drawbacks. What appear to be contradictory results numerically can be understood if one considers the manner in which these methods detect and report results. Used properly in combination they are orthogonal and increase the power of the control strategy in our industry. For example, ELISA is simple, robust, QC-friendly, and useful for both drug substance release and for demonstrating clearance of HCP throughout processes. LC-MS/MS, on the other hand, though requiring more time and cost, has the benefit of identifying individual HCP species. Identification affords a more intelligent risk assessment and a tool for bioprocessing and removal of HCP where necessary. Case studies are provided where both data sets are shown to be complimentary. Discussed will be how this tandem approach enables a more powerful control strategy and means to characterize processes within and across a portfolio.