Introduction
Some believe that the Chemistry, Manufacturing, and Controls (CMC) section of an IND is the “easy part.” In reality, underestimating its importance has often led to costly setbacks and regulatory pushbacks. Why? Because demonstrating adequate control of product quality at the time of IND submission is a critical requirement for regulatory authorities.
Regulators expect sponsors to prove that the product is well-characterized, safe, and consistently manufactured, and this assurance is built on the analytical testing package included in the CMC section. The FDA and other global agencies specifically require that key analytical methods are fit-for-purpose and that there is clear evidence that the most critical product attributes are measured, monitored, and controlled.
The Foundation of IND Success: Demonstrating Product Consistency and Safety
IND’s Primary Objective – Proving Safe Dosing in Humans
The primary objective of an Investigational New Drug (IND) application is to demonstrate that a new therapeutic agent can be safely administered to humans in early clinical trials. Although Phase 1 investigational drugs are exempt from full compliance with 21 CFR Part 211 current Good Manufacturing Practice (cGMP) requirements, they remain subject to statutory cGMP obligations under Section 501(a)(2)(B) of the Federal Food, Drug, and Cosmetic Act, which mandates that manufacturing facilities and controls ensure the drug’s identity, strength, quality, and purity The Food and Drug Administration (FDA) guidance for cGMP for Phase 1 Investigational Drugs reinforces this requirement to protect subject safety during first in human studies.
The IND framework balances regulatory flexibility for early-stage development with rigorous safety assurance, supported by adequate CMC data and nonclinical safety studies. Within the CMC section, analytical testing provides the key evidence linking product quality to patient safety, ensuring that investigational materials are properly identified, free from extraneous matter or adventitious agents, and correctly dosed in accordance with FDA recommendations for early phase clinical studies.
Analytical Testing as Your Strongest Evidence
Within an IND submission, analytical data serves as the most direct proof of product quality and consistency. Demonstrating that multiple manufacturing runs yield drug substance and drug product with reproducible identity, purity, potency, and safety profiles builds confidence with regulators. Agencies such as the FDA, European Medicines Agency (EMA), and International Council for Harmonisation (ICH) require that analytical procedures be accurate, reproducible, scientifically sound, and fit for intended use in accordance with ICH Q2(R2) and United States Pharmacopeia (USP) <1220> on Analytical Procedure Lifecycle. Compliance with these standards ensures the safety, efficacy, and reliability of investigational materials while mitigating regulatory risk and avoiding costly delays.
Sponsors must define drug substance and drug product specifications in the original IND submission, including the list of tests, analytical methods, and acceptance criteria that establish product quality as described in ICH Q6A and Q6B. Without robust, reliable analytical methods, manufacturers risk failing specifications and producing substandard materials that cannot withstand regulatory scrutiny under 21 CFR 211.165 (Testing and Release for Distribution).
Phase-Appropriate Analytical Method Qualification: What FDA Actually Expects
The IND-Stage Reality: Qualification vs. Full Validation
At the IND stage, regulatory expectation is phase appropriate qualification, not full validation. According to the FDA Guidance for Industry on Analytical Procedures and Methods Validation for Drugs and Biologics, early phase analytical methods should demonstrate fitness for purpose, even if not all validation parameters from ICH Q2(R2) are fully established. The approach recognizes that methods will evolve throughout development, with refinement driven by stability data and product understanding.
Phase appropriate qualification involves showing that methods are suitable for their intended use. Methods must reliably measure key safety and quality attributes for clinical material. Qualification studies typically assess:
Specificity for identity assays
Sensitivity for impurity detection
Precision for quantitative assays
Linearity for dose and potency determinations
As programs advance to later phases, these qualified methods are further developed and validated under ICH Q2(R2), integrated into a full lifecycle framework per USP <1220> and ICH Q14 Analytical Procedure Development, ensuring traceability, robustness, and long-term method control.
Regulatory Framework and Guidelines
The FDA Guidance for Industry on Analytical Procedures and Methods Validation for Drugs and Biologics establishes clear expectations for analytical validation parameters while recognizing reduced requirements during early development. The updated ICH Q2(R2) reinforces this principle by introducing the Analytical Target Profile (ATP), a forward-looking tool defining what an analytical method must achieve at each development stage. At the IND stage, the ATP emphasizes safety relevant measurements such as identity, purity, and potency rather than commercial release testing.
Complementary standards such as USP <1220> Analytical Procedure Lifecycle and USP <1225> Validation of Compendial Procedures advocate a lifecycle approach covering method design, qualification, continued verification, and ongoing monitoring. Together with ICH Q14, these frameworks support the evolution of analytical methods from IND qualification to commercial validation, ensuring consistency, control, and scientific integrity throughout the product lifecycle.
Defining Essential Analytical Attributes for IND Submission of Protein and Peptide-based Therapeutics
Identity Testing – Confirming You Have What You Think You Have
Peptide Mapping (LC–MS) confirms the amino acid sequence and detects chemical modifications. At IND, qualified peptide mapping should show high specificity and broad sequence coverage (>95%) for proteins such as antibodies.
Mass Spectrometry (MS) verifies molecular weight and detects post-translational modifications (PTMs) like glycosylation, oxidation, and deamidation that can affect potency and immunogenicity.
Orthogonal Identity Methods (cIEF, SDS-PAGE) provide independent confirmation of identity. At IND, at least two orthogonal, qualified methods are expected to confirm specificity.
Purity and Impurity Profiling
Size Exclusion Chromatography (SEC) is the primary tool for aggregate analysis, ensuring clear resolution between monomer and aggregate peaks to control immunogenicity risk.
Capillary Electrophoresis–SDS (CE-SDS) detects size variants, fragments, or incomplete assembly, confirming molecular integrity under reducing and nonreducing conditions.
Host Cell Protein (HCP) ELISA quantifies residual process impurities, confirming effective purification. Acceptance limits are typically 1–100 ng/mg of drug substance.
Residual DNA (qPCR) ensures genetic impurity levels remain below the safety threshold (<10 ng per dose for parenteral products).
Potency — Demonstrating Biological Activity
Binding Assays (ELISA, SPR) measure target-binding capability, often supportive rather than primary potency assays unless binding is the direct mechanism of action.
Cell-Based Bioassays quantify biological activity relative to a reference, reflecting the product’s mechanism of action in a disease-relevant system. Critical for biologics to confirm functional potency.
Gene and Cell Therapy Potency Assays evaluate transduction efficiency (AAV, LVV) or cytotoxic function (CAR-T), confirming both gene transfer and biological efficacy.
Strength and Dosing Accuracy
UV Spectrophotometry (A280) is the standard for determining protein concentration, requiring qualification for interference and linearity.
Orthogonal Quantification Methods (Amino Acid Analysis, RP-HPLC) provide confirmatory accuracy, particularly when applied to spiked placebo formulations.
Safety-Related Testing
Sterility Testing (USP <71>) is mandatory for aseptically filled products and verifies absence of microbial growth after a 14-day incubation.
Particulate Matter Testing (USP <788>) evaluates both visible and subvisible particles, ensuring compliance with injectable product specifications.
Stability-Indicating Testing
Real-Time Stability Studies are conducted at intended storage conditions (e.g., 2–8 °C or −20 °C/−80 °C), monitoring appearance, potency, purity, and safety at defined intervals (0–12 months).
Accelerated Stability Studies: Performed at elevated temperatures to predict shelf life and identify degradation pathways impacting critical quality attributes.
General Physicochemical Testing
Includes assessments of appearance, color, clarity, pH, osmolality, and reconstitution properties (for lyophilized products). These simple yet essential tests can reveal degradation, contamination, or formulation issues that would compromise clinical usability.
Ready to Strengthen Your IND Analytical Strategy?
Building a comprehensive, phase-appropriate analytical testing program requires deep expertise in biologics development, regulatory requirements, and analytical technologies. Many companies struggle with determining which methods are truly necessary at IND, how to design qualification studies that satisfy reviewers, and how to establish analytical infrastructure supporting both current needs and future growth.
Schedule a complimentary 30-minute consultation with our CMC specialists to discuss your analytical readiness for IND filing. We’ll assess your current analytical strategy, identify potential gaps, and provide actionable recommendations tailored to your product and development stage.
References
- 21 CFR 211.165. (n.d.). Testing and release for distribution. Code of Federal Regulations.
- Federal Food, Drug, and Cosmetic Act, 21 U.S.C. § 501(a)(2)(B) (1938).
- International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use. (1999a, March). ICH Q6B: Specifications: Test procedures and acceptance criteria for biotechnological/biological products. https://database.ich.org/sites/default/files/Q6B_Guideline.pdf
- International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use. (1999b, October). ICH Q6A: Specifications: Test procedures and acceptance criteria for new drug substances and new drug products: Chemical substances. https://database.ich.org/sites/default/files/Q6A_Guideline.pdf
- International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use. (2023a, June). ICH Q2(R2): Validation of analytical procedures. https://database.ich.org/sites/default/files/ICH_Q2%28R2%29_Guideline_2023_1130.pdf
- International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use. (2023b, November). ICH Q14: Analytical procedure development. https://database.ich.org/sites/default/files/ICH_Q14_Guideline_2023_1130.pdf
- U.S. Department of Health and Human Services, Food and Drug Administration. (2008, July). Guidance for industry: cGMP for Phase 1 investigational drugs. https://www.fda.gov/media/70975/download
- U.S. Department of Health and Human Services, Food and Drug Administration. (2015, May). Guidance for industry: Analytical procedures and methods validation for drugs and biologics. https://www.fda.gov/files/drugs/published/Analytical-Procedures-and-Methods-Validation-for-Drugs-and-Biologics.pdf
- United States Pharmacopeial Convention. (2023a, May). General chapter <1220>: Analytical procedure lifecycle. In United States Pharmacopeia–National Formulary (USP 46–NF 41).
- United States Pharmacopeial Convention. (2023b, December). General chapter <1225>: Validation of compendial procedures. In United States Pharmacopeia–National Formulary (USP 47–NF 42).
External Reference Links:
USP General Chapter <71>: Sterility Tests
- USP General Chapter <71>: Sterility Tests. URL: https://www.usp.org/harmonization-standards/pdg/general-methods/sterility-test
- USP General Chapter <85>: Bacterial Endotoxins Test. URL: https://www.usp.org/harmonization-standards/pdg/general-methods/bacterial-endotoxins
- USP General Chapter <788> Particulate Matter Testing. URL: https://www.usp.org/harmonization-standards/pdg/general-methods/particulate-contamination
- EMA: Guideline on Development, Production, Characterisation and Specification for Monoclonal Antibodies. URL: https://www.ema.europa.eu/en/development-production-characterisation-specifications-monoclonal-antibodies-related-products-scientific-guideline
Appendix
Acronyms Table
| Acronym | Definition |
| Acronym | Full Term |
| AAV | Adeno Associated Virus |
| ATP | Analytical Target Profile |
| CAR-T | Chimeric Antigen Receptor T Cell |
| CE-SDS | Capillary Electrophoresis–Sodium Dodecyl Sulfate |
| cGMP | Current Good Manufacturing Practice |
| cIEF | Capillary Isoelectric Focusing |
| CMC | Chemistry, Manufacturing, and Controls |
| EMA | European Medicines Agency |
| ELISA | Enzyme Linked Immunosorbent Assay |
| FDA | Food and Drug Administration |
| HCP | Host Cell Protein |
| ICH | International Council for Harmonisation |
| IND | Investigational New Drug |
| LC-MS | Liquid Chromatography–Mass Spectrometry |
| LVV | Lentiviral Vector |
| MS | Mass Spectrometry |
| PTM | Post Translational Modification |
| qPCR | Quantitative Polymerase Chain Reaction |
| RP-HPLC | Reversed Phase High Performance Liquid Chromatography |
| SDS-PAGE | Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis |
| SEC | Size Exclusion Chromatography |
| SPR | Surface Plasmon Resonance |
| USP | United States Pharmacopeia |
| UV | Ultraviolet |