Related-Impurities Assessment Considerations for

APIs in Generic Complex Peptide Products

SBIA 2020: Advancing Innovative Science in Generic Drug Development Workshop
Session 1: Method Development/Validations for Non-traditional Analytical Methods
Topic 1: Complex Active Pharmaceutical Ingredients (APIs) Including Peptide Products

Mani Ethirajan, PhD

Chemist
Division of life cycle API/Office of New Drug product
OPQ/CDER/U.S. FDA
September 29, 2020
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Pharmaceutical Quality
A quality product of any kind consistently
meets the expectations of the user.

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Pharmaceutical Quality
A quality product of any kind consistently
meets the expectations of the user.

Drugs are no different.

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Patients expect safe and effective
medicine with every dose they
take.

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 Pharmaceutical quality is
assuring every dose is safe and
effective, free of contamination
and defects.

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 It is what gives patients
confidence in their next dose of
medicine.

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 Learning Objectives
• Describe the components that are required to demonstrate
API sameness for peptides with respect to Drug Master File
(DMF)

• Know the common deficiency and Importance of the impurity

Comparability Studies in DMF

• List a few comparability study scenarios

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 Relevant Terminology for Peptides

• Peptide — FDA considers a polymer composed of 40 or fewer amino

acids to be a peptide regulated as a drug under the FD&C Act*

• Protein — FDA interprets the term “protein” to mean any alpha amino
acid polymer with a specific defined sequence that is greater than 40
amino acids in size

• Only Synthetic Peptides are eligible to be approved as generics under

section 505(j) of the FD&C Act†

* FDA Guidance for Industry: New and Revised Draft Q&As on Biosimilar Development and the BPCI Act (Revision 2), December 2018, pp 13-14.
† Section 505 of the Federal Food, Drug, and Cosmetic Act (FD&C Act) (21 U.S.C. 355)

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 Examples of Therapeutic Peptides
8 AAs

9 AAs

20 AAs

32 AAs

34 AAs
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 Peptide Guidances
• No FDA general peptide guidance
• Draft Guidance for Industry: ANDAs for Certain Highly Purified
Synthetic Peptide Drug Products that Refer to Listed Drugs of rDNA Origin
[Liraglutide, Glucagon, Nesiritide, Teriparatide, and Teduglutide]

• Product-specific guidance (PSG)

– Glatiramer Acetate Injection
– Semaglutide

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 Draft Guidance for Industry:
(ANDAs for Certain Highly Purified Synthetic Peptide Drug Products that Refer
to Listed Drugs of rDNA Origin)

i. Active ingredient sameness:

• Primary sequence and physico-chemical properties Drug Substance (DMF)

• Secondary & Higher order structures

• Oligomer/Aggregation states; and Drug Product (ANDA)
• Biological activities (by in vitro or animal studies).

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 Draft Guidance for Industry:
(ANDAs for Certain Highly Purified Synthetic Peptide Drug Products that Refer
to Listed Drugs of rDNA Origin)
ii. Related Impurities
• For the impurities that are common between your DS and the RLD, the
acceptance criteria should be not more than those observed in the RLD (at the
end of the shelf-life).

• For any new impurities in your DS, but not observed in the RLD, they should not
exceed 0.5%. Furthermore, each of these new impurities present at 0.10% or
greater should be identified and justified for not affecting the safety and efficacy.

• Utilize sensitive and high resolution analytical methods (e.g., UHPLC-HRMS*) to

detect and characterize peptide-related impurities in a proposed generic synthetic
peptide in comparison to RLD. Identify and report each specified and unspecified
peptide-related impurity that is 0.10% of the drug substance or greater. (LOQ is
less than 0.10%)
www.fda.gov *Zeng et al. AAPS J. 2015, 17, 643-651 12
 API Sameness & Related Impurities in DMF
• Structure confirmation or comparative structural signature
analysis
– Primary structure of peptide; structural features and fingerprints, etc.

• Comparative physicochemical property analysis

– spectroscopic analysis, etc.

• Comparative impurity profile analysis

– peptide-related impurities in synthetic peptides

Totality of Evidence approach

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 Origin of Impurities (DMF)
Differences in manufacturing process may result in differences in
related impurities:
Chemical Synthesis Recombinant Synthesis

Starting materials (AAs) Fermentation & cell culture media

components
Reagents,
Catalysts, Residual DNA & cellular proteins
Solvents
Bacteria, fungi, mycoplasma, viruses,
Intermediates etc.

By-products Column materials

Other Degradation products Other Degradation products

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 Peptide Related-Impurities: SPPS

SPPS:

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Adopted from Bachem Solid-Phase Peptide Synthesis white paper
 Potential Related-Impurities (DMF)
• Impurities may result from the insertion, deletion, or modification of
amino acid sequences or residues; can be process or degradative in
origin or both
– Proteolysis (e.g., peptide hydrolysis to form fragments) Degradative
– Deamidation (hydrolysis of primary amide to carboxylic acid) impurities
– Oxidation (e.g., oxidation of methionine sulfur to sulfoxide/sulfone)
– Reduction (e.g., reduction of cystine to cysteine)
– Racemization (e.g., epimerization of amino acid residue α-stereocenter)
– Deletion (incomplete coupling)
– Truncation (missing amino acids)
– Insertion (additional amino acids) Process
– Incomplete deprotection (attached protective groups) impurities

– Disulfide exchange (e.g., cystine isomerization)

• Impurities may form during storage
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 General Considerations for
Impurity Comparability Studies (DMF)
• Conduct a comparative impurity profiling of the RLD and proposed generic DS to
i. Demonstrate that impurities common to both the proposed DS and the RLD
are present in the proposed DS at the same or lower levels than in the RLD
ii. Analyze and characterize new impurities in the proposed DS that are not
common to the RLD
• Conduct each study on a statistically meaningful number of batches (generally at
least three) of both the proposed drug substance and the RLD
• It is recommended the proposed DS be tested on or near release and at the end
of the proposed shelf life, and RLD batches of different ages be tested prior to
expiry (as available). Provide sample ages for the dates of all studies
• Use multiple orthogonal validated methods
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 Orthogonal Analytical methods (DMF)
Related Impurities Complementary methods*
Deletion LC-HRMS(MS)
Insertion LC-HRMS(MS)
Truncation LC-HRMS(MS)
proteolysis LC-HRMS(MS)
Substitution LC-HRMS(MS)
FG modification LC-HRMS(MS)

Disulfide modification LC-HRMS(MS)

Deamidation LC-HRMS(MS)
Acetylation of amino functions LC-HRMS(MS)

*Zeng et al. AAPS J. 2015, 17, 643-651

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 Scenario: 1 (in DMF)
Generic DS Impurities RLD impurities
Specified Impurities AC Specified Impurities AC
A NMT 1.0% A NMT 0.50%
B NMT 0.20%
B NMT 0.20%
C NMT 0.15%
C NMT 0.15%
D NMT 0.20%
D NMT 0.15%
Any unspecified NMT 0.10% Any unspecified NMT 0.10%
Total Impurity NMT 2.0% Total Impurity NMT 2.0%
Impurities-A, B, C, & D are common impurities (may be Process and /or degradants)

If the common impurity A is higher than the RLD, you propose to control
it at the same or lower levels than in the RLD.

Limits of Common Impurities ≤ RLD impurity levels

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 Scenario: 2 (in DMF)
Generic DS Impurities RLD impurities
Specified Impurities AC Specified Impurities AC
A NMT 0.50% A NMT 0.50%
B NMT 0.20% B NMT 0.20%
C NMT 0.15% C NMT 0.15%
D NMT 0.20% D NMT 0.20%
E NMT 0.40% Any unspecified NMT 0.10%
Any unspecified NMT 0.10% Total Impurity NMT 2.0%
Total Impurity NMT 2.0%
Impurities-A, B, C, & D are common impurities (may be Process and/or degradants)
Impurity –E could be specific for Generic process

New Impurities ≤ 0.50%

Additional data to qualify impurity-E may be requested from

an ANDA applicant referencing this DMF.
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 Scenario: 3 (in DMF)
Generic DS Impurities RLD-DS impurities
Specified Impurities AC Specified Impurities AC
A NMT 0.50% A NMT 0.50%
B NMT 0.20% B NMT 0.20%
C NMT 0.15% C NMT 0.15%
D NMT 0.20% D NMT 0.20%
Any unspecified NMT 0.10% Any unspecified NMT 0.40%
Total Impurity NMT 2.0%
Total Impurity NMT 2.0%
Impurities-A, B, C, & D are common impurities (may be Process and/or degradants)

The ‘any unspecified impurity’ limit, in your DS, is recommended to be

controlled at the level of 0.10% (or based on MDD) or else justification should
be provided.
This general approach may be considered when developing other peptide API products.
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 Typical Deficiency (DMF)
If you provide insufficient information regarding related-impurities…
Deficiency: Please provide one-time comparison of the impurity profile of your Drug Substance (DS) with the RLD Drug
product (DP) and provide data. Please perform these studies taking into account the following.
a. Demonstrate that impurities common to both the proposed DS and the RLD DP are present in the proposed
DS at the same or lower levels than in the RLD.
b. In general, any new impurities not common to the RLD that are present in your proposed DS at a level above
0.10% should be identified and reported by RRT and their limit should be justified.
c. Samples should be analyzed using at least two orthogonal validated analytical methods. Use of UHPLC-
HRMS/MS should be considered to facilitate peak identification and matching between the RLD DP and
proposed DS samples and to ensure peak purity (see Liquid Chromatography-High Resolution Mass
Spectrometry for Peptide Drug Quality Control by Zeng et al., AAPS J. 2015, 17, 643-651).
d. Ensure that sample analysis dates and batch manufacturing or expiry dates are provided for all studies to
facilitate calculation of sample ages.

Justification: Risk analysis data, safety mitigation data, based on manufacturing

capability, literature data, statistical analysis, existing specifications, etc.

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 Challenge Question #1
Which one of the following Amino acid polymers is
considered by the FDA to be regulated as a peptide drug
under the FD&C Act?
A. Amino acid polymer contains <100 & >40 amino acids
B. Amino acid polymer contains ≤40 amino acids
C. Amino acid polymer contains >100 amino acids
D. Amino acid polymer with the size of >15,000 Da.
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 Challenge Question #2

Which one of the following is not considered to be

synthetic peptide related-impurities :
A. Amino acid Insertion impurities
B. Amino acid Deletion impurities
C. Residual DNA & cellular protein impurities
D. Incomplete deprotection impurities

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 Summary

• Impurity profile comparability studies on the proposed peptide

drug substance with RLD is a key part in qualifying related
impurities in the Peptide API (in DMF).

• Each peptide API has its own challenges, DMF applicants

need to evaluate individual situation and apply these
recommendations accordingly.

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Thank you