NBEs vs NCEs: an overview

Luigi Colombo

29NOV2017

Herbert Boyer

Robert Swanson

Robert Swanson and Herbert Boyer: Giving Birth To BiotechA venture capitalist and a scientist changed how drugs are made

Everything started here...

Genentech, South San Francisco campus, the birthplace of biotechnology

The Pharma Biotech World1976 On 7th April Robert Swanson and Herbert Boyer founded Genentech.1978 Genentech researchers produce human insulin in cloned bacteria.1980 Genentech shares are floated at a price of USD 35; an hour later they have risen to USD 88.1982 Human insulin becomes the first recombinant medicine to be approved for use in the USA1985 For the first time, a recombinant medicine produced by a biotech company is approved for use:

Protropin, produced by Genentech (active ingredient: somatrem, a growth hormone forchildren).

1986 Genentech licenses Roferon-A to Roche.1990 Roche acquires a majority holding in Genentech and by 1999 has acquired all the company’s

shares.1987–97 Major new drug approvals: Activase (1987; active ingredient: alteplase, for dissolving blood

clots in myocardial infarction); Actimmune (1990; interferon gamma-1b, for use in chronicimmunodeficiency); Pulmozyme (1992; dornase alfa, for use in asthma, cooperative projectwith Roche); Nutropin (1993; somatropin, a growth hormone); Rituxan (1997; rituximab, foruse in non-Hodgkin’s lymphoma, cooperative project with Idec).

1998 The humanized monoclonal antibody Herceptin (trastuzumab) is approved for use against aparticular type of breast cancer.

Why a biotech drug?

1. Cytokines (interferons, Ils, CSFs,...)2. Enzymes (alteplase, dornase alpha,...)3. Clotting Factors4. Hormones (insulin, EPO, hFSH, hGH,...)5. Vaccines6. Monoclonal Antibodies

Types of Biotech Pharmaceuticals

• A biologic is manufactured in a living system such as amicroorganism, or plant or animal cells. Most biologics are very large,complex molecules or mixtures of molecules. Many biologics areproduced using recombinant DNA technology.

• A chemical drug is typically manufactured through chemical synthesis.

• Chemical drugs generally have well-defined chemical structures, and afinished drug can usually be analyzed to determine all its variouscomponents. By contrast it is difficult, and sometimes impossible, tocharacterize a complex biologic by testing methods, and some of thecomponents of a finished biologic may be unknown..

How do Drugs and Biologics Differ? I

Therefore, for biologics, "the product is the process." Because the finishedproduct cannot be fully characterized in the laboratory, manufacturers mustensure product consistency, quality, and purity by ensuring that themanufacturing process remains substantially the same over time. By contrast, adrug manufacturer can change the manufacturing process extensively andanalyze the finished product to establish that it is the same as before themanufacturing change.

The living systems used to produce biologics can be sensitive to very minorchanges in the manufacturing process. Small process differences can significantlyaffect the nature of the finished biologic and, most importantly, the way itfunctions in the body. To ensure that a manufacturing process remains the sameover time, biologics manufacturers must tightly control the source and nature ofstarting materials, and consistently employ hundreds of process controls thatassure predictable manufacturing outcomes.

How do Drugs and Biologics Differ? II

Pros and cons of expression hosts

E.coli

Pichia pastoris

CHO cells

+ -Easy manipulationRapid growthLarge-scale fermentation simplemediaHigh yield

Proteins may not fold correctly or may aggregateAlmost no posttranslational modifications

Relatively rapid growthLarge-scale fermentationPerforms some PTM

PTM may differ from humans (especially gycosylation)

May grow in suspensionPerform all required PTM

Slow growthExpensive mediaDifficult to scale-upSerum dependent

The product is the process

B

A

C

• What Are Possible Differences between NCE and Biotechnolgy-based APIs?

• Is process development following similar path?• Is the analytical dev. strategy comparable?• Why will different approaches be necessary for some

biotechnology issues?• …• …

NCEs vs. NBEs

Bioprocessing

NCE Biopharmaceutical

• Chemistry Biology• Isolation Harvest & Capture • Crystallization Purification• Drying/Milling Formulation/Freezing • API API (DP)

Typical API Manufacturing Process Stages

Attribute NCE Biopharm Effect

Structural basis Covalent Covalent & weak Delicate structures Raw Materials Defined chemicals Some complex Less control

materials, cellsSolvent Organics WFI Viral safety Process High temp Physiological Viral safety

& pressureHomogeneity >99% single Microhetero Power of

chemical entity geneity Analytical tools Process Fewer unit ops More unit ops Complexity of

Pro Val, QRM, & QbD

Key Differences between NCEs & Biopharmaceutical APIs

• NCEs are typically composed of >99% single chemical entity; analyticaltools can define every atom, impurities, contaminants

• Biopharms are microheterogenous, and even simple ones have 10E4different chemical entities in API (complex ones may have 10E8 to 10E9);analytical tools cannot usually define every atom, and definition ofproduct-related substances, product-related & process-relatedimpurities, & contaminants can be very complex & imperfect

• Combination of delicate structure and microheterogeneity also preventaccelerated & stressed conditions from accurately predicting stability ofbiopharmaceuticals, which typically rely on real time/storage conditiondata for product expiry & handling labeling

• Measurements of purity/impurities are relative to both the method ofmeasurement and the process used to produce the API

Homogeneity

Microheterogeneity of non-glycosylated proteins hGHA 191-amino acid protein with 2 S-S bridges

Trisulfide182-189

Gln66 Exchange vs LysGln66 Exchange vs Val

Lys70 Dimethylationor Exchange vs Arg

Met14 Exchangevs Val

N-CarbamylationN-Succinylation

N-terminal SequenceLength Variation(des-Phe, des-Phe-Pro)

Disulfidescrambling

Asn12, 149, 152, 159 DeamidationAsp or IsoAsp Formation

• Amino acidsubstitutionsobserved in ~2%of the expressedprotein

• May lead to functional and/or antigenic differences

• Variants found in different products are not identical

Arg127 Exchangevs. Lys or Gln

Oxidation of Met14, 125,

125, 170 Exchange vs. Norleucine (or Ile) Met14,

Total possible variants: > 106

A. Eshkol, Serono & L. Fryklund, Pfizer

Microheterogeneity of glycosylated proteins t-PAA 527 -amino acid protein with 17 S-S bridges and 3 glycosylation sites

N-terminal sequence length variation

Proteolysis at Arg-X

Additional O-Glycosylation

Deamidation of Asn residues

Oxidation of Cysor Met residues

Variability in N-linkedcarbohidrate sidechains

Single-chain andtwo-chain forms

NH2 COOH

Total possible variants: 1.09 x 109

How many details?

CONTROLS: specification for NBEs-ICH Q6B

✓ efficacy

Why?

Patient safety first!

Efficacy

When? Who?

Process Development(IPC’s, Spec.s panel) Analytical Development

Dept./QC lab

Manufacturing(IPC’s, MCB/WCB validation)

Plant/QC lab/AD Dept.

When? Who?

Release (routinary) QC lab + QP (Spec.s)

Parameter Specification Test method

Appearance White powder visual

Identity (IR) Conforms with reference MRK-TM-001

Strenght (UV 280) 95-105 % (w/w) MRK-TM-002

Purity (HPLC) ≥ 98% (area %) MRK-TM-002

Heavy metals ≤ 20 ppm Ph.Eur. 2.4.8

Bioassay (in-vivo) 90-115 IU/mg USP <111>

----- ---- ----

When? Who?

Post release - StabilityStudy (ad hoc)

QC lab + QP (Spec.s)

Parameter Specification Test method

Appearance White powder visual

Assay (HPLC-IEX) 96-103% (w/w) MRK-TM-001

Strenght (UV 280) 95-105 % (w/w) MRK-TM-002

Purity (HPLC-RP) ≥ 98% (area %) MRK-TM-002

Impurities (CZE) r.t. 2.35 min NMT 0.2% MRK-TM-003

Bioassay (in-vivo) NLT 90% (w/w) USP <111>

Methanol NMT 0.5 (w/w) USP <467>

When? Who?

Una tantum (i.e change in process) full characterization

AD dept/QC lab + QP

Parameter Specification Test method

Identity Conforms to reference NMR

Sequence Conforms to reference MS

Glycoforms Conforms to reference MS

PTM Conforms to reference MS

Isoforms Conforms to reference CZE

AA composition Conforms to reference RP-HPLC

Bioassay Conforms to reference MRK-TM-023

When? Who?

Una tantum: housereference/workingstandard qualification

AD dept/QC lab + QP

Analytical technology

Relevant regulatory guidance http://www.ich.org

Growth Factor (I)amino acids: 181, Av. Molecular weight: 21163.3

Growth Factor (II)

Relevant species may be limited (e.g., NHP)• Non-human primate (NHP) such as cynomolgus and rhesus

monkeys• Limitations for repro tox, carcinogenicity studies• More expensive, can be harder to obtain• Ethical issues

GLP requirements for studies are the same

Tissue cross-reactivity studies needed for monoclonal antibodies –ability to bind to target and non-target tissues

Preclinical studies on Biologics I

May not be required:• Metabolism• Limited safety pharmacology• Genotoxicity• Carcinogenicity

Highest dose in toxicology studies:• Scientifically reasonable multiple of the highest projected clinical dose • Maximum feasible dose• Dose reflective of a pharmacodynamic marker e.g. saturation of antigen

Toxicity is usually due to exaggerated pharmacology

Calculate Minimal Anticipated Biological Effect Level (MABEL)• From animal efficacy/PK data and in vitro data• This dose may be lower than the lowest dose initially used in the clinic

Preclinical studies on Biologics II

Immunogenicity of Therapeutic Proteins

„ All therapeutic proteins are potentially immunogenic“(Dr. Amy Rosenberg, 2004, FDA)

❖ Two major mechanism:

- Reaction to neo-antigens:- Proteins of non-human origin (e.g. mouse proteins) in humans- No tolerance to hypervariable regions in proteins (e.g. antigen binding sites in

antibodies)- No tolerance to administered therapeutic proteins in patients with missing

endogenous counterpart (replacement therapy)- New epitopes in fusion proteins

- Breaking of immune tolerance:- Formation of aggregates

Factors relevant in immunogenicity

• No effect (majority of cases)• Hypersensitivity (pre-existing ADAs, e.g. to foreign sequences)

– Anaphylaxis (type 1 hypersensitivity)– Deposition of immune complexes in tissues (type 3 hypersensitivity)

• Altered PK– Increased clearance („clearing antibodies“ => reduced efficacy)– Decreased clearance (problematic for agonistic drugs => “overstimulation”)

• Neutralizing antibodies– Inhibition of binding => reduced efficacy– Neutralization of endogenous counterpart => immunedeficiency syndrom

(e.g. PRCA caused by neutralizing antibodies against EPO)

Immunogenicity Testing – Why?

Anti-Drug Antibodies (ADAs) might lead to the following consequences:

Something can always go wrong...

Viral contamination

Viral clearance studies