# Presentation: 070255

## Immunogenicity of Therapeutic Proteins:A Case Report

**Fred Bader**
**Vice President **
**Process Sciences, Global Biologics Supply Chain**
**Johnson & Johnson**
** **Scientific Considerations Related to Developing Follow-On Protein Products
- FDA Public Workshop
- September 14 & 15, 2004

## Immunogenicity of Therapeutic Protein

- One common type of immunogenicity is the formation of antibodies against a protein.
- Nearly all therapeutic proteins induce antibodies in some percent of patients who receive the product.
- Frequency ranges from one case per million doses to over 50% of patients treated.
- Non-neutralizing antibodies generally have no clinical effect.
- Neutralizing antibodies inhibit the therapeutic effect of the protein.

## Mechanism and Factors Involved in IgG Immune Reactions

- All antibodies are produced by B cells (a type of white blood cell).
- IgG antibodies are a common type of antibody produced against therapeutic proteins.
- The production of IgG antibodies requires four factors that are relevant to this discussion.
**Protein**
**B cells** that have *Receptors* that can recognize the *Protein*
**T cells** that have a *Receptor* that can bind to a short peptide sequence from the *Protein*
**Adjuvant** that can activate the *Antigen* *Presenting* *Cell*  that presents the short peptide sequence to the *T cell*

## B Cells and T Cells are Patient Specific 

**B Cells**
- Receptors are _*randomly*_ generated during early development
- Bind to the 3 dimensional protein structure
- Are deleted if they bind to self proteins during B cell formation (Tolerance)

**T Cells**
- Receptors are _*randomly*_ generated during early development
- Bind to short peptides of ~9 to 15 amino acids that are presented by APCs
- Are deleted if they bind to self peptides without an adjuvant (Tolerance)

## Protein and Adjuvant are Possible Product Specific Factors 

**Protein**
- Has a primary sequence of amino acids that is genetically determined
- Is folded into a 3 dimensional structure
- Has post translational modifications, particularly carbohydrate
- Can be damaged and modified

**Adjuvant**
- A substance distinct from the protein that activates APCs and other white blood cells stimulating an immune reaction
- An adjuvant provides an initial signal to the immune system to become active

## Protein Factors Related to Immunogenicity

- Probability of an antibody reaction to a protein increases when:
- The protein is non-human
- The protein is human but with a slightly different sequence
- The protein is damaged such that it appears different than the self protein (may still be functional) and can be recognized by B and T cells
- Typical types of protein damage
- Oxidation		Deamidation		Clipping
- Misfolding	Disulfide Scrambling	Aggregation
- Deglycosylation	Chemical modification

## Potential Adjuvants 

- Host cell protein (particularly bacterial and yeast)
- Host cell DNA/RNA (particularly bacterial and yeast)
- Lipopolysaccharides
- Chemical impurities
- Excipients and formulants
- Manufacturing aides
- Leachates and extractables
- Trace chemicals and minerals

## EPREX and PRCA 

- Erythropoietin – a natural protein that stimulates the production of red blood cells (erythrocytes)
- Recombinant Erythropoietin – manufactured form of the natural protein (generic name is epoetin) used to treat anemia.
- EPREX **® **– ** **a brand of epoetin marketed outside of the U.S.
- Pure Red Cell Aplasia (PRCA) – severe and rare form of anemia

## Case: An Increase in PRCA Associated with EPREX® 

- Beginning in 1998, an increased, though still rare, incidence of PRCA was linked to epoetin products, particularly EPREX brand.
- Antibodies neutralize all rHuEPOs and patient EPO
- One case per 5,000 patient years
- Marketed in over 90 countries

## The Mechanism of PRCA is Known

- “In all cases, there was evidence of the presence of strong neutralizing antibodies...it is clearly related to the treatment with rHuEPO”1
- Data from Dr. Casadevall2, and confirmed by Johnson & Johnson and Amgen, indicated the following antibody characteristics:
- Neutralizing
- Predominantly IgG, no IgM (T cell mediated B cell response)
- Oligoclonal (T cell mediated B cell response)
- Bind to the protein and not the carbohydrate
- Bind to conformational epitopes and not denatured EPO
- 1. Nicole Casadevall, Nephrology Dialysis Transplantation (2002) 17 Supp 5: 42-47
- 2. Nicole Casadevall, Nephrology Dialysis Transplantion (2003) 18 [Suppl 8]: viii37- viii41

## Finding the Cause

More than 100 people involved in the extensive investigations
Clinical, Pre-Clinical, Manufacturing, Process Sciences, Logistics, Quality, Analytical, Regulatory
Comprehensive lab investigations and epidemiology 
Investigation took four years
Advisory Board of expert immunologists
Worked closely with Health Authorities
Considerable cost

- More than 100 people involved in the extensive investigations
- Clinical, Pre-Clinical, Manufacturing, Process Sciences, Logistics, Quality, Analytical, Regulatory
- Comprehensive lab investigations and epidemiology
- Investigation took four years
- Advisory Board of expert immunologists
- Worked closely with Health Authorities
- Considerable cost

**Case: An Increase in PRCA ****Associated with EPREX****®**** **

## Product Factors Investigated 

- EPO Molecule
- Aggregation
- Oxidation
- Deamidation
- Denaturation
- Carbohydrate
- Change of sequence
- Clipping
- Exposing new epitopes
- Disulfide shuffling
- Folding
- Activity
- Impurities
- Others
- The EPO molecule is intact and consistent over time

- Possible Adjuvants
- Silicone oil
- Polysorbate 80
- Polysorbate 80 Micelles
- CHO Host Cell Protein
*Leachates*
- The evidence points strongly to the leachates
- Significant level (~1μg)
- Not in other EPOs
- Timing (1998)
- Consistent with Immunology
- Animal data (positive)
- Clinical data (positive)

## New Tryptic Map Profiles Show Extractables from Older Prefilled Syringes

**New Tryptic Map Profiles Show Extractables from Older Prefilled Syringes**

- Trypsin

- No Trypsin

**Extractable Non-Peptides**

- EPO Monomer

## HPLC of EPREX polysorbate 80 formulations with rubber and FluroTec® coated syringe stoppers

**min**

**25**

**30**

**35**

**40**

**45**

**50**

**55**

**60**

**65**

**70**

**mAU**

**0**

**20**

**40**

**60**

**80**

**100**

**120**

**140**

**Leachates only occur with uncoated rubber stopper syringes**

**EPREX s****yringe ****with rubber stoppers ****stored at 5****o****C for 18 months**** :**
**multiple extra peaks**

**EPREX syringe with coated stoppers stored at 5****o****C for 18 months: no extra peaks**

**1**

**2**

**3**

**4**

**5**

**6**

**7**

**8**

**9**

**10**

**EPO**

**Polysorbate 80**

## Leachates Are the Most Probable Cause 

**      ****ONLY THE LEACHATES PROVIDE A FIT TO ALL SIX CRITERIA**

- Yes

- Clinical results

- Yes

- Animal results

- Yes

- Absence from other EPO products

- Yes

- Magnitude of the change

- Yes

- Principles of immunology

- Yes

- Timing of the increase of PRCA in 1998

- _Leachate_

- _Criteria_

## The Leachates Theory

- Leachates are released by exposure of uncoated rubber syringe stoppers to the polysorbate 80 that was added to the EPREX formulation in 1998.
- We believe that these leachates act as an adjuvant and stimulate the formation of anti-EPO antibodies in CRF patients when administered subcutaneously. (Supported by animal data)
- Elimination of leachates by use of FluroTec coated stoppers decreased the frequency of PRCA to baseline.
- We believe that leachates are the most probable cause of the increase in EPREX associated PRCA.

## Final Key Messages

- Patient safety must be of paramount importance when considering follow-on biologics.
- Immunogenicity is an important consideration.
- Only clinical trials can assess the immunogenicity potential in patients.