APR Sept/Oct 2023 - 46

» FORMULATION & DEVELOPMENT
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Procedures: Text and Methodology " provides guidance that Quality
Control Laboratories can avail of in consideration of which analytical
method characteristics are applicable for method validation. Despite
the information available, validation of analytical methods for
Advanced Therapy Medicinal Products (ATMPs) can be even more
challenging. This is due to the inherent variability in starting materials,
complex biological features and associated manufacturing process.
Other factors are the limited batch history and sample availability
due to low batch sizes and high manufacturing costs, lack of available
assay references and assay controls.
Unlike more mature biological pharmaceutical products such as
monoclonal antibodies, which like ATMPs such as AAVs, are highly
heterogeneous, analytical techniques and process manufacturing
workflows are still the subject of intense development optimization
and change. The analytical methods used to support ATMP drug
development and release can be thought of in terms of three
categories related to their level of maturity and expected analytical
development and validation time costs. Firstly, " fully mature " drug
characteristics that are analogous to those seen in more established
molecule types can be analyzed using established methodologies
used for other biological molecules. Examples include excipient
testing for antifoam clearance, Host Cell Protein (HCP) and Host Cell
(HC) DNA impurity. This makes them relatively simple to implement
from the earliest phases of development, often with kit-based assays
and fully GMP analytical systems and software.
A second group of assays include those which are commonly found
in GMP QC release laboratories. Therefore, they can take advantage
of the fully developed analytical platforms and software used for this
purpose, but with some additional considerations which may require
further analytical development effort and special attention during
validation. Examples in this second group include the measurement of
characteristics such as Post Translational Modifications (PTMs) of capsid
proteins by peptide mapping, analysis and relative quantification of
capsid proteins by LC-UV/FLD or CE UV/FLD, SEC for aggregate analysis
and protein impurity determinations can be expected to require
further development if based on equivalent assays such as those used
in mAb products. PTMs measured at the peptide or protein level must
consider the likely impact of sample preparations used to disrupt the
capsid. SEC and other particle sizing methods have to be suitable for
larger size monomers and aggregates, with AAV molecules being >30
times larger than monoclonal antibodies and protein concentrations
are also likely to be significantly lower and thus sensitivity may be a
consideration. Monoclonal antibodies are often manufactured to
concentrations greater than 100mg/mL whereas AAVs are likely to
have protein concentrations of <0.05mg/mL and are manufactured
at significantly lower batch volumes. Protein impurity assessments
are also likely to be required to perform at protein concentrations
significantly less than those seen for antibody methods and may be
required to perform at a level too low for many existing procedures.
To continue with AAV molecules, several characteristics such as
empty/full assessment, infectivity and potency rely on techniques
that are uncommon in pharmaceutical release settings. By their
nature AAV molecules are highly complex and therefore, methods to
measure this complexity may require significant development and
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optimization in order for them to be appropriate for use in a GMP
setting. As a result, this type of method analysis could likely change
the most during the development process, leading to significant
extra work in proving comparability of the analytical method over
the course of the assay lifecycle. It can also lead to uncertainty
during specification setting in early phases. Techniques such as AUC
and cryoEM would be more than capable of evaluating empty, full
and partial AAV species, but are not routinely used in a GMP setting.
Therefore, these methods do not have commercially available Annex
11 or 21 CFR 11 compliant software that could be used as part of
the system's qualification in a GMP environment. This can lead to
significant time investments for later validation activities closer to
the commercialization stages of the product.
As described previously, the amount of material that is manufactured
is often in very small quantities. In some cases, these bespoke
medicines are limited to just the amount that serves as the dose
given to the patient. Due to the novelty and complexity of ATMPs,
reference materials are not always available, thus making it more
difficult to demonstrate that the procedures and the tests developed
are suitable for their intended use. The use of interim references
can be an option in these circumstances and their use could be
recommended to provide a level of continuity and confidence in the
analytical methods applied. In particular, as these interim references
are developed through their analytical lifecycle where this is a
requirement. Reference standards should be representative of the
manufacturing process as much as is possible and may need to be
replaced with associated bridging studies as the process develops.
Where reference standards are not available, in assay controls can
help to demonstrate assay consistency and support in proving
representativeness during the drug development lifecycle.
Data from reference standards and analytical controls should be
used to set assay acceptance criteria. These criteria may also be wide
compared to other modalities at the clinical phase stage. However,
as the drug development progresses the results from these assays
should also provide assurance of manufacturing consistency.
Trending results over time should confirm release criteria and, where
assay variability is present, studies should be performed to ascertain
the cause and establish understanding of inter and intra assay
variability. In line with ICH Q14, Design of Experiment (DoE) studies
could be useful in designing these studies and can help conserve
limited sample amounts.
Throughout the development process, it is recommended that
continued and frequent dialogue with regulatory agencies is
maintained. Communications should include details of draft analytical
strategies. This should help focus efforts for continued analytical
development and validation. It also builds an understanding and
justification for areas where sample and data availability may be
limited. With agreement from regulatory agencies, it may also allow
for the gradual reduction of release criteria once it is clear that these
criteria are either not relevant to safety and efficacy or not required
due to process robustness such as with process excipients.
The storage and prudent use of retain samples from all key process
lots could likely be critical in establishing assay comparability or for
APR Sept/Oct 2023

Table of Contents for the Digital Edition of APR Sept/Oct 2023
