Bridging the Gap – Overcoming Bottlenecks in the Development of Therapeutics for Infectious Diseases

Bottlenecks in Biopharmaceutical Development: Chemistry, Manufacturing, and Control

Steven Giardina, Ph.D., RACSenior Scientist

SAIC

August 2012

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Topics

Brief history of cGMP for biopharmaceuticals CMC Issues – Perils of not planning backwards Building – Not testing – Quality into the Product Product Safety – How soon do I need to start worrying? Case Study – Ch14.18 – A monoclonal antibody for

treatment of late-stage neuroblastoma (or making your own biosimilar without intending to)

Lessons Learned

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Foundations of GMP

All drugs (small molecule and biologic) have to be shown to be safe and effective.

It was not always so. The history of drug law shows us that most of the laws

and regulations we have today were in response to tragic events.

A sampling…

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It all started with a horse named “Jim”

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Diphtheria Antitoxin

Made in old milk horses – like Jim.

In St. Louis, Missouri. The blood of a tetanus-infected retired milk-wagon horse was used for this purpose. Thirteen children who were given the antitoxin died from tetanus.

*Led to the Passage of the Biologics Control Act of 1902.

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Guidance Documents

Put meaning to the ambiguities in the statutes and regulations.• Points to consider• ICH (International Conference on Harmonisation)

Do not have the force of law but the FDA does expect investigators to adhere to them unless they have an equally sound approach.

Can be found on FDA web site

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GMP Rules of Thumb

If it is not written down – it never happened. Until you know it does not matter – it matters. GMPs really begin at the bench prior to what would normally be

perceived as the true beginning of GMP PD and manufacture. • Important to keep good records of anything that is needed for

inclusion in the Chemistry, Manufacturing, and Control (CTD Module 3).

• What would affect a decision on product safety? Can reach back into the origins of the discovery phase of PD.

You cannot test quality into the product – it must be built into all aspects of the drug’s manufacture (quality systems and risk management).• A product is adulterated – by definition – if it is not made in

conformance with cGMP requirements even if it tests to spec.

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Example from ICH-Q5D – Cell Substrate – Cell Origins

The cultivation history of the cells should be documented. The method originally used for the isolation of the cells should be described as well as the procedures used in the culturing of the cells in vitro and any procedures used to establish cell lines (for example, use of any physical, chemical, or biological procedure, or added nucleotide sequences). A description of any genetic manipulation or selection should be provided. All available information regarding the identification, characteristics, and results of testing of these cells for endogenous and adventitious agents should be provided.

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Common CMC Pitfalls – Biologics

No traceability on what was used to manufacture the drug.• Origin of DNA used to make cell substrate?• Chain of custody/identity of cell lines, vector, plasmids, etc. • Exposure to animal-derived raw materials

No scientifically valid assays for purity, potency, etc.• No agreement on what pure and potent even mean• Impurities/residuals

– LPS levels?– Genomic DNA?– Host Cell Proteins?– Aggregates? Clipped forms? Other product-related “impurities.”

No qualified reference standard for establishing “scientifically sound” assays before they are needed to track process development and product quality during manufacture, release, and stability period.

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Common CMC Pitfalls – Biologics (cont.)

What are the stability-indicating assays? Not just a list of what is easy to do – e.g., SDS-PAGE may be easy and needed but not necessarily stability indicating.

Drug does not meet obvious GMP requirements. Inadequate supply. Formulation does not ensure stability over time and during

shipment. Optimal formulation/fill-finish/concentration/amount per vial,

etc. for actual use over duration of the trial? No agreement on release criteria. Inadequate documentation of what was done and when. Poor quality systems (investigations, deviations, not

completed in a timely manner). Executive management and QA are “missing in action.” Hubris.

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Drug Substance and Drug Product

Release vs. characterization (FIO) assays. Reference standard – why is it suitable for the purpose? Impurity profiles – product or process?? Stability in bulk or primary container – outsourcing to CMOs

for different aspects of the product manufacture? Shipping stability – cold chain? Lyophilization? Mechanical

stress? Drug product stability for toxicology studies? GLP

requirement. Drug product stability under proposed conditions of

administration – each center may be quite different despite what is in the Investigator’s Brochure.• Lost due to adsorption on the IV bag and infusion set tubing?• No toxicity = no drug administered? Proof?

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Purity

21 CFR 610.13• “Products shall be free of extraneous material except that

which is unavoidable in the manufacturing process described in the approved biologics license application.”

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Identity

21 CFR 610.14• “The contents of the final container of each filling of each lot

shall be tested for identity after all labeling operations shall have been completed. The identity test shall be specific for each product in a manner that will adequately identify it as the product designated on the final container and package labels and circulars, and distinguish it from other product being processed in the same laboratory. Identity may be established either through physical or chemical characteristics of the product, inspection by microscopic or macroscopic methods, specific cultural tests, or in vitro or in vivo immunological tests.”

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Certificate of Analysis

Product Name: XX Project Number: YY Part Number: ZZ Date of Manufacture: 08/10/12 Lot Number: L123456 Lot Size: 3000 vials Container Size/Fill Volume: 3ml/0.5 mL Storage Temperature: 2-8 °C Description: A chimeric monoclonal antibody (hIgG1) to the GD-2 disialoganlgioside antigen. Colorless liquid at

a concentration of 5mg/mL in a solution of 10 mM sodium phosphate, 150 mM sodium chloride, pH 7.4

TEST DESCRIPTION SPECIFICATION SOP OR QC RESULT

STUDY REPORT #

Identity

Appearance Colorless liquid 123 456 Colorless liquid Essentially-free of particulates Essentially-free of particulates

SDS-PAGE/R and NR/ Report Major Bands 444 777 166.8 kDa (NR)

Densitometry HC @ 51.29 kDa (R)

LC @ 28.58 kDa (R)

Human IgG Subclass human IgG1 – kappa subclass 333 888 human IgG1κ

Content

Protein Concentration E @ 1mg/mL = 1.0 222 111 5.14 (5.1)

by Absorbance @ 280nm

Potency

GD-2 ELISA Binding within 50-200% of 000 222 Binding is 104% of Standard

Standard 1

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Certificate of Analysis (con’t)

Product Name: XX Project Number: YY Part Number: ZZ Date of Manufacture: 08/10/12 Lot Number: L123456 Lot Size: 3000 vials Container Size/Fill Volume: 3ml/0.5 mL Storage Temperature: 2-8 °C Description: A chimeric monoclonal antibody (hIgG1) to the GD-2 disialoganlgioside antigen. Colorless liquid at

a concentration of 5mg/mL in a solution of 10 mM sodium phosphate, 150 mM sodium chloride, pH 7.4

TEST DESCRIPTION SPECIFICATION SOP OR QC RESULT

STUDY REPORT #

Purity

HPLC-SEC Monomer purity ≥ 95% 666 222 Monomer purity:97.2%

cIEF Report % and pI of major forms 222 666 Single peak at pH 5.5

Safety

Sterility No Growth USP 26 <71> 555 No Growth

Endotoxin < 5EU/mg 333 777 1EU/mL (0.19 EU/mg)

gDNA Content Report Results 111 999 0.5 pg/ML murine gDNA

pH 7.5 ± 0.4 XXX YYY 7.3

1. reference standard is lot L098765

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Certificate of Analysis (con’t)

For Information Only – Possible Additions – Comparability?• Silver Stain – impurity/degradation• Western Blot with Product Specific Antibody -Identity• N-terminal Sequencing – Identity• Biolayer Interferometry or Surface Plasmon Resonance

(kinetics/KD) w/ conformation sensitive ab or antigen or ?? – changes in structure = telegraphing a change in potency??

• Carbohydrate Composition – glycoform distribution and identity• Host Cell Proteins – Important and a problem when using

commercial kit s• Process Impurities – BSA?, Bovine IgG ( if using serum-

containing medium)• Other chemicals/reagents that should be removed –

antifoams? Selective agents?

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COA – Last Page

Signatures and More Signatures – • QC – attests that data are true and accurate• Project Scientist• Program Director (Go Directly to Jail)• QA• Customer

Caution Statement – proscibed based on intended use• Not for use in humans? ( as opposed to not for human use)• For investigational use…

Name and Address of the Manufacturer of Record – not the subs

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The Challenges of Making a Biologic

Biological molecules – like immunoglobulins – are complex and large.

Complexity can be increased due to processing such as glycolsylation.

How do we define “pure” if there are multiple forms of the product?

How do we define its biological activity/potency?• Which is not the same as efficacy!

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Case Study – When is a product difference = a different product?

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New Therapy for Pediatric Neuroblastoma

Most common extracranial solid tumor in infancy. One of the most common malignancies of childhood. About 600–700 cases per year in the United States. Patients with low/intermediate risk neuroblastoma have

excellent prognosis and outcome. High risk = poor outcome despite intensive therapy. 70%–80% of patients older than 18 months present with

metastatic disease. Less than half are cured even with the use of high-dose therapy followed by autologous bone marrow or stem cell rescue.

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Ch14.18 Milestones

• MAb against GD-2 disialoganglioside is shown to be safe and effective in refractory neuroblastoma.

• Treatment includes the use of IL-2 and GM-CSF to increase efficacy via enhanced ADCC in addition to 13-cis-retinoic acid. Not a benign therapy (pain, allergic reactions)

• Multiple lots of ch14.18 manufactured in support of nonpivotal Phase III trial sponsored by the NCI over a period of ~7 years.

• All product lots manufactured by SAIC-Frederick’s Biopharmaceutical Development Program (BDP).

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Ch14.18 Milestones

In 2009 results of Phase III randomized trial showed event-free survival at 2.1 years was significantly higher (66%±5% vs 46% %±5%).• Overall survival also significantly higher (~86% vs. ~75%).• Based on these data ch14.18 randomization of the trial was

stopped and ch14.18 + 13 CRA + CM-CSF + IL-2 became the standard of care.

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New Challenges

Methods Legacy: The process used by the BDP in support of the NCI trials remains the only manufacturing process.

Undemonstrated Technology Transferability: The BDP was the only manufacturer of the product being used in the NCI-sponsored current trials.

System Capacity: Increased anticipated demand – still have existing process using hollow fiber production system.

Manufacturing Reproducibility: The challenge was to meet the demand focusing on lot-to-lot consistency using a process that was not originally designed to meet the new material requirements.

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Master Cell Bank Lot 32031

Working Cell Bank Lot 32032

Bioreactor Scale-up

Bioreactor Harvest

Thawing, Pooling, and Filtration ofHarvests

Run #1rProtein A Column

Run #2 rProtein A Column

Run #1 Viral Inactivation by LowpH Treatment followed by

Solvent-Detergent Treatment

Run #1 SP-Sepharose FFColumn Chromatography

Pooling of ch14.18 SP-Sepharose FFRuns #1 and #2

PLANOVA 15N Virus Filtration

Run #2 Viral Inactivation by LowpH Treatment followed by

Solvent-Detergent Treatment

Run #2 SP-Sepharose FFColumn Chromatography

Concentration and Diafiltration ofch14.18 for the Superdex Load

Chromatography on Superdex 200SEC Column and Pooling of Superdex

Main Peaks

Q-Sepharose FF ColumnChromatography

Concentration to 5.0 mg/mL byTangential Flow Filtration,Final 0.2 micron Filtration

Purified Bulk ch14.18Storage at 2 - 8°C

Purified Bulk ch14.18 Vialed toProduce ch14.18 Final Vialed Product

Storage at 2 - 8°C

DNA Content LALRetrovirus (TEM) IsotypeGD2 Binding (ELISA)

Testing before and after filtration:Pierce Coomassie Sterility TEM IEFSDS-PAGE Mycoplasma DNA MVMHPLC-SEC Western Blot BSA A280Human IgG ELISA Bovine IgG LALActivity ELISA Methotrexate BioburdenIn Vitro Adventitious AgentsAb Quantitation by Protein A

SDS-PAGE Capillary IEFHPLC-SEC Protein AA280 DNALAL

SDS-PAGE Capillary IEFHPLC-SEC BioburdenA280 DNALAL

SDS-PAGE Capillary IEFHPLC-SEC BioburdenA280 DNALAL

SDS-PAGE Capillary IEFHPLC-SEC BioburdenA280 DNALAL

SDS-PAGE Capillary IEFHPLC-SEC BioburdenA280 LAL

HPLC-SEC BioburdenA280 LAL

SDS-PAGE Capillary IEFHPLC-SEC BioburdenA280 DNALAL

SDS-PAGE Capillary IEFHPLC-SEC BioburdenA280 DNALAL

SDS-PAGE Capillary IEFHPLC-SEC BioburdenA280 DNALAL

Refer to the Certificate ofAnalysis in Attachment 1

Refer to the Certificate ofAnalysis in Attachment 2

Bulk Sterility MycoplasmaIsoenzyme In vitro AVAIn vivo AVA MAPRetrovirus (RT) Retrovirus (TEM)Retrovirus Characterization forInfectivity to Human Cells

Bulk Sterility MycoplasmaIsoenzyme

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Reference Standard

Produced from a batch of material manufactured in 2003. FDA has accepted the specification of “conforms to

standard” for a number of attributes. This became a problem when comparing pre-2010 lots with

the most recent lots. Quantitative specifications were developed as PD moved

along. Potency assays continue to be reported as the ED50 as well

as the percentage of the reference standard.

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In the Interest of Time

A large menu of standard assays (SDS-PAGE, SEC, etc.) showed no differences between the old and new lots

So – how about potency?• Binding to GD-2 expressing cells?• C’ mediated killing (Fc structure/function)• Binding to FcγIIIa receptor (effector-cell receptor)• ADCC (performed by Jackie Hanks/Paul Sondel, University

of Wisconsin)

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Relative Activity of Ch14.18 Lots (Potency Tests)

Lot Number GD2 Binding(Cell-Based ELISA)

Complement Dependent Cytotoxicity (CDC)

Human CDC Rabbit CDC

L0302007 (Reference Lot)

100% 100% 100%

L1001002 (Bulk Lot)

126% 70% 92%

L1001001(FVP Lot)

122% 118% 89%

L0512003 110% 86% 134%

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Comparison of KDs

ch14.18 Samples

Equilibrium Dissociation Constant KD (µM)

Flow Cell 2 Flow Cell 3 Flow Cell 4

Run 1 Run 2 Run 1 Run 2 Run 1 Run 2

Clinical Lot(Lot # L0512003)

0.58 0.56 0.62 0.77 0.57 0.54

FVP(Lot # L1001001)

0.41 0.45 0.44 0.47 0.36 0.39

Reference Lot(Lot # L0302007)

0.47 0.43 0.50 0.53 0.55 0.52

FSB(Lot # L1001002)

0.55 0.59 0.41 0.51 0.49 0.47

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Everything Looked Great

ADCC – not QC assay – also normal No issues until…..

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Early Lots of CH14.18 Show Comparable cIEF Profile

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Current Lots of Ch.14.18 Show Quantitative Differences in Relative Peak Intensities Compared to

Earlier Reference Lot

pl

sign

al

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MS of Intact Ch14.18Reference Lot (Green) vs. Old Clinical Lot L0512003

(Blue) = Similar

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MS of Intact Ch14.18Reference Lot (Green) vs. New Clinical Lot L1001001

(Blue) = Not Similar

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Relative Percent of Four Major N-Oligosaccharides by CE

Lot Number G0F G1F.3 G1F.6 G2F

Ref Std L0302007

34 36 12 18

L1001001(new lot 1)

62 25 8 5

L1003008(new lot 2)

62 24 9 5

L0512003(old clinical lot)

36 37 12 15

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Reason for the Change?

Too many years and changes in raw materials to pinpoint precise root cause.• No new charge species or glycoforms – only a change in

relative distribution Important to recall that there was no evidence of any change

to the product by routine tests and no differences were reflected in 4 different potency tests.

Product made in same facility and by same people – still a change.

Time will tell if any of these changes have any impact on efficacy.

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Consequences?

FDA allowed the product to be used transparently to the earlier lots but there was enhanced monitoring for AEs, stability changes.

Health Canada initially viewed the changes as a new product but also allowed the use of the new material in the ongoing trial.• Enhanced reporting.• Required that there be a new reference standard that looked

more like the new clinical material and its stability be monitored under a formal stability protocol.

• Many technical reports, ad hoc meetings to bridge/explain the differences and how to handle them both in the lab and, more importantly, in the clinic.

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Take-Away Message

Biologicals are complex and, therefore, have unique CMC challenges that must be understood and anticipated long before the formal product development phase begins in earnest = Knowledge Management.

All data (productivity, stability, activity) can inform decision making down the road and have to be of the highest quality and retrievable.

Not having a useful (think QC friendly) potency assay, reference material, or an understanding of a product’s stability issues can have a profound impact on PD time and costs.

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Take-Away Message (cont.)

AskWhat am I doing NOW that can affect the safety, identity, purity, potency, and overall quality of a drug I may want to make LATER from the cells, etc. I am handling in the lab (traceability of genetic material and contact with animal-derived RMs, cross-contamination?)?

Is this really the concept candidate I want to take forward into expensive tox and clinical trials? Is it “druggable?”

The consequences of not planning backward from the first-in-man research goal can be….

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Consequence of Poor Planning

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And then..

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SAIC’s Virtual Pharmaceutical and Product Development Support Service Capabilities

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• CMC Characterization• Product Development

Plans• Facility Audits

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Repository

Project Planning

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Thank You

Opinions expressed in this presentation are mine and do not represent those of SAIC, NIAID, FDA, or other regulatory bodies.

Thanks to the organizers.