# Presentation: 417366

## The Value of State-of-the-Art Analytical Characterization in the Development and Evaluation of Follow-on Protein ProductsJoerg Windisch, Ph.D.Global Head Technical DevelopmentNovartis Biopharmaceutical Operations

## Introduction (1)

- Modern techniques **enable quicker, more predictable and less costly development** and regulatory decision-making, and allow industry to **avoid unnecessary or unethical duplication** of trials
**Regulatory requirements must evolve **in line with these developments and the state-of-the-art analytical techniques
**Rigorous scientific criteria** meeting the highest standards must be applied consistently to** both originator and follow-on products**
- Follow-on products that are **as safe and efficacious as the originator **can be developed by applying **state-of-the-art science **at all relevant levels – if the **appropriate strategy **is selected** **and the **science **is** conducted properly**

**Introduction ****(1)**

## Introduction (2)

- Recombinant biopharmaceuticals poorly understood
- Quality depended mainly on (consistency of) manufacturing process
- Safety and efficacy established in extensive clinical trials
**1990s**
- Major advances in analytical science and technologies
- Recognition of well-characterized biopharmaceuticals
**Present day**
- Analytical tools allow in-depth investigation of all relevant properties of a (glyco)protein (primary, secondary, tertiary, quaternary structure, posttranslational modifications incl. glycans)
- Protein product can be characterized and defined by a carefully selected panel of state-of-the-art analytical techniques

**Introduction (2)**

## Introduction (3)

- They have the **same mode of action** and are used for the **same indications** as the originator product
**State-of-the-art analytical techniques** can provide a better informed and more robust **understanding** of what is under consideration when industry and the Agency discuss FOPPs
- Development of FOPPs requires
- an extensive and **sound set of data generated in-house**(full CMC incl. bioassays, preclinical, and clinical) plus
- data derived from **comparator studies** with an originator product at all relevant stages

**Introduction (3)**

## Development Strategy for Follow-on Protein Products

**to innovator**

**Development ****Strategy for Follow-on Protein Products**

**Stand-alone product and process development**

## The Complexity of the Product Impacts the Extent of Studies Required for FOPP Development

- Protein pharmaceuticals exhibit** different levels of complexity**
- The choice of** methods and extent **of analytical studies** must match **the** complexity **of the product** **in order to achieve thorough characterization. The **ability to thoroughly characterize** protein products will continue to **increase** as physicochemical and bioanalytical **tools improve**, and this will enable ever more extensive comparisons to the reference product.
- The **extent of studies at the subsequent levels (preclinical, clinical) **will be determined** case-by-case **based on the level of** understanding gained from the initial physicochemical and biological analyses.**

## Value and Limitations of Comparator Studies with an Originator Product

- complete CMC development
- full physicochemical characterization
- full biological characterization
- full release testing
- basic non-clinical testing
- clinical trials
**Comparator studies** with an originator product **may** **allow **the follow-on sponsor to
- limit preclinical testing
- limit dose-ranging studies
- limit population(s) in clinical phase III testing
- extrapolate to broader indications based on sound scientific rationales
- Development programs must be **science- and data-driven** and will be different from **product to product.**

**Value and Limitations of Comparator Studies with an Originator Product**

## Comparing Physicochemical and Biological properties

## General Considerations for Physicochemical Comparisons

- Evaluate **complexity of molecule**
- Use **experience** gained with the innovator product **over many years**  as foundation for characterization and development plan
- Consider all **state-of-the-art analytical techniques**
- Consider the **relevance** of quality parameters **for clinical safety and efficacy**
- Discriminate between **product-related substances** and **product-related impurities** (ICH Q6B)

## Analytical Methods for Evaluation of Product AttributesIdentity and Purity

| Parameter | Test | Resolved species |
| --- | --- | --- |
| Primary structure incl. disulfide bridging | N-terminal Edman sequencing;peptide mapping with UV and MS detection; MS/MS sequencing | Mistranslations, post-translational modifications |
| Higher order structure | Circular dichroism, 1D-NMR, Bioassay | Folding |
| Mass | LC-ESI-MS, MALDI-TOF-MS | Covalent modifications |
| Size | SDS-PAGE, SEC, AUC, FFF, light scattering | Aggregates, folding |
| Charge | IEF, CE, IEX-HPLC | Charge variants incl. deamidation, N-terminal modifications |
| Hydrophobicity | RP-HPLC | Oxidized variants, covalent modifications |
| Binding | Immunological binding, SPR | Integrity of epitopes, binding properties |

**Analytical Methods for Evaluation of Product Attributes****Identity and Purity**

## Analytical Methods for Evaluation of Product AttributesIdentity and Composition of Glycans

| Parameter | Test |
| --- | --- |
| Mass | MALDI-TOF-MS; LC-ESI-MS |
| Charge | Asialo and sialo glycan mapping with HPAEC-PAD, CE, IEX-HPLC |
| Polarity | Asialo glycan mapping with NP-HPLC |
| Composition | HPAEC-PAD |
| Glycan sequence | Enzymatic degradation + MS/MS; LC-ESI-MS/MS |
| Sialic acids | HPAEC-PAD |
|  |  |

**Analytical Methods for Evaluation of Product Attributes****Identity and Composition of Glycans**

## Advances in Analytical Science (1)

| Technique | Properties | Advances | Added value |
| --- | --- | --- | --- |
| HPLC | Size, charge, hydrophobicity | Stationary phases, instrumentation;
miniaturization | Increased resolving power, improved precision; 
increased sensitivity, hyphenation to MS |
| ESI-MS
MALDI-MS | Mass of intact biomolecules
 | Ionization modes, mass analyzers
 | Direct and sensitive analysis of proteins and glycans |
|  | Primary structure
Glycan structure | MS/MS, MSn
Instrumentation
Data evaluation
Sample preparation (enzymes) | Full sequence coverage 
Structural characterization of main structure and of modifications
 |

## Advances in Analytical Science (2)

| Technique | Properties | Advances | Added Value |
| --- | --- | --- | --- |
| Capillary electro-phoresis | Charge, isoelectric point | Capillary format, automation, detection modes | Ultimate separation performance; sensitivity, quantitation |
| NMR(combined with CD) | Secondary/ tertiary structure | Instrumentation; 2D/3D techniques | Determine and control folding of intact protein in solution |
| Light scattering detection | Aggregation | Instrumentation; data evaluation | Characterization of aggregates |
| AUC | Aggregation | Data evaluation | Evaluate soluble and non-soluble aggregates |
| FFF | Aggregation | Separation format, instrumentation | Soluble and non-soluble aggregates;complement to SEC |

## Acceptance Criteria for the Comparison at the Physicochemical Level

- Upon comparison to the reference material and literature data, the FOPP must be **indistinguishable** with respect to:
- Primary structure
- Higher order structure
- Size and mass
- Charge
- Hydrophobicity
- Immunological binding

**Acceptance Criteria for the Comparison at the Physicochemical Level**

## Acceptance Criteria for the Comparison at the Physicochemical Level

- Subtle differences in the quantitative composition of product-related substances and impurities will be observed
- Criteria for comparison: Case-by-case decision depending on
- Level 1: Amount of variants and batch-to-batch variability of the originator product
- Level 2: Classification of product variants
- Product-related substance (identical to main product regarding safety and efficacy) or impurity (properties not investigated or different from main product regarding safety and/or efficacy)
- Understanding of the relevance of a difference on clinical safety/efficacy

**Acceptance Criteria for the Comparison at the Physicochemical Level**

## Evaluation of Identity, Purity, and Potency Results from the Synthesis of Data from Orthogonal Methods

**No single method** can completely characterize **all aspects** of a product
- The scientifically **sound selection of** **orthogonal parameters** and methods can
- Expand the analytical window
- Reduce the risk of ‘blind areas’, where no sufficient knowledge of the product is available
**Derive the complete picture of a product**

**Evaluation of Identity, Purity, and Potency Results from the Synthesis of Data from Orthogonal Methods**

## Special Considerations

## Special Considerations for Glycoproteins

**Identify and characterize all glycan structures**
- Site occupancy – site specificity
- Composition
- Antennarity
- Type of linkage
- Anomery
- Sialylation
- Absence of immunogenic structures
**2.  Show consistency and stability**
**• ****	**Batch to batch – release
- In process control – process development
- • 	Sialic acid content – charge/glycoform distribution
- • 	Oligosaccharide profile

## Special Considerations for GlycoproteinsGlycan Specific Analysis on Different Levels

**Special Considerations for Glycoproteins****Glycan Specific Analysis on Different Levels**

**Structural **
**Characterization**
**(Mass spectometry**
**Enzymes**
**NMR)**

## ImmunogenicityControl of potential sources

- Thorough **definition and control** of the product
- Extensive analytical characterization
- Special **focus on**
- Aggregation
- Product-related impurities
- Potentially immunogenic glycans
- Host cell proteins and other process-related impurities
- Excipients, primary packaging materials
- Leachables/leachates
- Preclinical assessment
- Clinical studies for final confirmation
- Post-approval pharmacovigilance and risk management programs

## Protein AggregatesComplementary Methods for Control and Characterization

## General Considerations for Comparisons of Biological Activity

- Design studies that will **mimic mode of action** in humans*:  In vitro* (cell or tissue culture) or *in vivo* (animal) bioassays
**Multiple bioassays** should be performed for products with **different modes of action** in different indications; these bioassays should be reflective of these different modes of action
- Establish **link between physicochemical properties and PK/PD** profile, especially if both *in vitro* and *in vivo* assays are employed
- Bioactivity is a first key parameter to predict efficacy

## The Relevance of Biological Assays

- Ligand/receptor assays (not cell based)
- SPR
- Modern cell based bioassays (early read out)
- Receptor binding
- KIRA
- PACE
- Reporter gene assay
- Classical cell based bioassays (late read out)
- Proliferation assay
- Death of cells as read out
- In vivo bioassays
- Includes in vivo clearance event

**Variability**

**Speed**

**Relevance**

**The Relevance of Biological Assays**

**?**

## Conclusions

## Conclusions on the Value and Limitations of Analytical Comparisons

**Much more is known today** about each particular protein product than at the time of its original development
- Extensive knowledge gained by original sponsor about process and product development (some but not all of which is public)
- Information available from the scientific literature and other public domain sources
- Extensive experience through clinical use, specific risk assessments, and subsequent development of expanded indications
**A combination** **of** **multiple orthogonal methods** can help overcome limitations of single methods to obtain **complete picture**
**Improved analytical methods and designs **will allow **expedited yet complete development programs**
**Rigorous standards **of ensuring product **safety and efficacy must be maintained** while at the same time allowing industry to **avoid unnecessary or unethical duplication of trials**