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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5608_Библиотеки_им_академика_М_И_Перельмана.pdf
Chapter 13
Mylotarg: TheJourney toFDA Reapproval
andBroad International Approval
LeoLetendre, DurgeshNadkarni, andFrankKotch
Abstract Mylotarg (gemtuzumab ozogamicin) was the rst cytotoxic antibody-
drug conjugate approved by the FDA.After its approval, it was withdrawn from the
US market in 2010 although it continued to be marketed in Japan. As new clinical
data were amassed, an effort to have it reapproved by the FDA and approved in
other major markets was initiated. The efforts to bring the Chemistry, Manufacturing,
and Controls portion of the regulatory lings up to contemporary standards while
still maintaining the original product prole provide an interesting journey through
the advances in chemistry, analysis, and regulatory science since the late twentieth
century.
Keywords Mylotarg · Calicheamicin · Gemtuzumab ozogamicin · Registration ·
Amino acid substitution · Antibody-drug conjugate
13.1 Introduction
Mylotarg® (gemtuzumab ozogamicin) is an antibody-drug conjugate (ADC) [1, 2]
used to treat CD-33 positive acute myeloid leukemia (AML) in adults and pediatric
patients [3]. Gemtuzumab ozogamicin consists of a recombinant humanized
Dedication: This chapter is dedicated to the memory of Tok Han who skillfully shepherded both
the reintroduction of Mylotarg and the introduction of Besponsa through technology transfer, manufacturing, and regulatory inspections driven by his empathy toward the patients who would benet from these medicines. His loss at a young age was felt by all who worked with him.
L. Letendre (*)
Leo Letendre Consulting, LLC, Oakdale, CT, USA
D. Nadkarni · F. Kotch
Bioprocess Research & Development, Biotherapeutics Pharmaceutical Sciences, Pzer Inc.,
Chestereld, MO, USA
381© The Author(s), under exclusive license to Springer Nature Switzerland AG 2024
K. Gadamasetti, S. A. Kolodziej (eds.), Bioprocessing, Bioengineering
and Process Chemistry in the Biopharmaceutical Industry,
https://doi.org/10.1007/978-3-031-62007-2_13
382
L. Letendre et al.
anti- CD33 IgG4 antibody (hP67.6) covalently linked to the cytotoxic agent N-acetyl
calicheamicin [4]. Mylotarg originally received accelerated approval from the US
FDA in 2000 and later received Japanese approval in 2005. With the original accelerated approval from the FDA came a requirement for the completion of a conrmatory clinical trial. In 2010 Pzer voluntarily withdrew Mylotarg from the US market
when the conrmatory clinical trial failed to demonstrate its clinical benet [5]. The
product was not withdrawn from the Japanese market at the request of the Japanese
authorities.
During this initial period of marketing as well as after the US withdrawal, independent researchers continued to evaluate Mylotarg for the treatment of AML.These
studies focused on the use of Mylotarg with other anticancer agents as well as variations in the dosing regimens. The resulting phase 3 ALFA-0701 trial, which became
the registrational trial, concluded that a “fractionated” Mylotarg dosing regimen in
conjunction with daunorubicin and cytarabine provided an overall benet [6, 7].
The fractionated dosing regimen consists of splitting the typical original dose of
9mg/m3 into three smaller doses over 1week followed by lower doses during continuation therapy. This dosing regimen led to a decrease in side effects while maintaining efcacy [6]. In September 2017, the FDA reapproved Mylotarg for the US
market after a positive recommendation by the Oncologic Drugs Advisory
Committee (ODAC). Subsequently, Mylotarg has been approved in most major
markets.
With the decision to pursue reapproval, an assessment of the state of the original
Chemistry, Manufacturing and Controls (CMC) section of the original application
was conducted. Mylotarg was the rst antibody-drug conjugate to be approved by
the FDA [1]. The application, therefore, was breaking new ground for both Wyeth/
Pzer (the applicant) and the agency. One note of the difference between today’s
antibody-drug conjugate lings and Mylotarg’s is that the original application was
led as a New Drug Application (NDA) while current applications are in the form
of a Biological License Application (BLA). While CDER was responsible for the
review of both the NDA and the BLA, the additional requirements for approval of a
BLA were followed. During the intervening years much had changed in the regulatory expectations for new drugs and biologics as analytical methods, in particular,
and medical knowledge have progressed. The rst question to be addressed after the
decision to seek reapproval was whether to go forward with the existing CMC
development and characterization data or to attempt to bring it up to contemporary
standards. (Drug substance and drug product historical data, all 17 or so years’
worth, was always assumed to be included.) It was in this context that the assessment of our knowledge about Mylotarg and the gaps between what was acceptable
for the initial ling and that for the reling was conducted. In the end, the decision
was made to supplement the information within the original application with
increased knowledge of the manufacturing process in order to contemporize and
make it consistent with BLA expectations.
13 Mylotarg: TheJourney toFDA Reapproval andBroad International Approval
383
13.2 Gemtuzumab Ozogamicin
Gemtuzumab ozogamicin (the Mylotarg drug substance) consists of a recombinant
humanized IgG4 antibody linked to the modied cytotoxic natural product
γ-calicheamicin using an acid-labile linker [4].. The structure of gemtuzumab ozo-
gamicin is shown in Fig.13.1. γ-Calicheamicin is present in the form of its N-acetyl
derivative, which provides some additional stability to that portion of the molecule.
The antibody is linked to the N-acetyl calicheamicin through a linker, which attaches
to the antibody through several of its lysines and to the N-acetyl calicheamicin
through a disulde moiety. The hydrazide portion of the linker provides acid lability
allowing the antibody drug conjugate to release the calicheamicin payload in the
acidic environment of the lysosome once it is internalized [8].
As Mylotarg was developed during the early days of seeking modied antibody
drugs, it was designed using the simplest chemistry available for connecting the
payload to the antibody, amide linkage through the lysine side-chain amines. Since
the gemtuzumab antibody contains 44 lysines, a kinetically controlled distribution
of the linker attachment is expected. Indeed, the attachment of the linker payload is
predominated by four lysine sites [9]. However, the distribution of linker payload is
still more complicated than a simple explanation of kinetic control would describe.
With an average drug-to-antibody ratio of approximately 2.5, the expectation would
be that typically two or three calicheamicin moieties would be attached to each
antibody with a normal distribution of species with higher and lower loading. What
has been observed, however, is a bimodal distribution; approximately half of the
antibodies bear twice the average loading while the other half are unconjugated or
have very low loading [9]. An explanation for this result still awaits convincing
experimental results.
Fig. 13.1 Gemtuzumab ozogamicin (Mylotarg)
384
L. Letendre et al.
13.3 Gemtuzumab Antibody
The gemtuzumab antibody (also referred to as hP67.6) is a humanized murine anti CD33 IgG4 monoclonal antibody. The antibody sequence comprises a murine
complementarity- determining region (CDR) grafted onto a human IgG4 kappa
framework [4, 10] to avoid undesired immune response. Gemtuzumab does not
have either antibody-dependent cell-mediated cytotoxicity (ADCC) or complement
dependent cytotoxicity activity. Additionally, the antibody’s effector function was
shown to not be part of the mode of action.
13.4 Calicheamicin
γ-Calicheamicin (also known as calicheamicin γ
I
) is an enediyne-containing anti-
1
biotic produced by the fermentation of Micromonospora echinospora ssp. calichen-
sis, which was isolated from a sample of caliche soil from Kerrville, Texas [11, 12].
γ-Calicheamicin is one of several enediyne antibiotics produced by this organism
(Fig. 13.2). Other calicheamicins include δ-calicheamicin and β-calicheamicin,
both active compounds, and the inactive ε-calicheamicin. The rst isolated calicheamicin compounds were brominated on the aromatic ring, including calicheami-
Br
cin γ
. During optimization of the fermentation conditions, it was found that
1
supplementing the media with sodium iodide resulted in a signicant increase in
yield and afforded the iodinated derivative, calicheamicin γ
I
, as the major product
1
and core structure for linker-payload development [12].
γ-Calicheamicin is attached to the amine of lysine residues on the gemtuzumab
antibody as an N-acetylated derivative through an acid-labile linker. The linker was
designed to be hydrolyzed in the lysosome [8] (low pH) once the antibody-drug
conjugate has been internalized, releasing the toxin from the antibody as N-acetyl
calicheamicin DMH [4] (Fig.13.3). Mediated by the aromatic containing sugar portion of the molecule (“Glycan” in Fig.13.3), calicheamicin binds to specic sites
within the minor groove of DNA [13, 14]. Cleavage of the disulde bond leads to an
intramolecular hetero-Michael addition followed by a Bergman cyclization [15] to
Fig. 13.2 Major calicheamicins produced by M. echinospora ssp. calichensis fermentation
13 Mylotarg: TheJourney toFDA Reapproval andBroad International Approval
Fig. 13.3 Mode of action of calicheamicin-based ADCs
385
give a p-benzyne diradical. When bound to DNA, the p-benzyne diradical causes
double-strand cleavage ultimately leading to cell death [13] (Fig.13.3).
13.5 Assessing theRegulatory andCommercial Needs
An analysis of the state of knowledge about the production of Mylotarg identied a
number of areas where the knowledge obtained for the initial approval and current
expectations was different. Additionally, even though Mylotarg was withdrawn
from the US market, it has remained in the Japanese market since its approval in
2005. Therefore, unlike developing a drug de novo, any redenition of the processes
used to manufacture Mylotarg based upon enhanced understanding would have to
maintain comparability with the product produced over 17years of commercial
manufacture.
The primary commercial need was identied as a capacity issue in the production of the linker payload, activated calicheamicin derivative. Two factors were
important. First, the yield of the linker payload from a contract manufacturing organization had decreased over the history of the production to an unsatisfactory level.
The second factor complicating this issue was that the imminent introduction of
another ADC, Besponsa® (inotuzumab ozogamicin), which contained the same
linker payload, would signicantly increase demand for activated calicheamicin
derivative [16]. Clearly, an improved activated calicheamicin derivative supply
chain was necessary.
386
L. Letendre et al.
13.6 Production ofGemtuzumab Antibody
The gemtuzumab antibody was originally developed by Celltech Ltd. [10]. The
antibody was produced from mammalian myeloma NS0 cell lines and then puried.
In planning to re-register Mylotarg, it was anticipated that the production of the
gemtuzumab antibody would not require extensive work but that the characterization and release methods would need to be brought to contemporary standards. In
the process of scrutinizing the characterization data, a discrepancy in a protein
digest indicated that the antibody had contained an amino acid substitution (AAS)
at multiple sites. Further analysis indicated that the AAS was present in the initially
registered product at low levels and that the level had increased signicantly at multiple sites in the contemporary antibody. After determining that the issue was not
one of clonal purity, efforts were undertaken to determine both the cause of the
substitution and the effects upon efcacy.
Comparison of retained antibody samples with contemporary samples indicated
that the amino acid substitution had always been present but that its level had signicantly increased and had done so in a stepwise manner [17]. The substitution
occurred on both the light and heavy chains including the CDR.Eventually, the root
cause of the amino acid substitution was traced to a change in the supplier of a complex raw material used in the gemtuzumab cell culture. Given the nature of this raw
material, it was not possible to return to using the original raw material source. This
discovery led to an extensive re-investigation of the effects of various components
used in the production of gemtuzumab as well as the process parameters controlling
the cell culture. Eventually, a control strategy was established to produce the antibody in a robust manner with the amino acid substitution level comparable to that
obtained prior to the change in the raw material supplier.
The discovery of amino acid substitution in gemtuzumab was not without its
regulatory impact and required additional quality data to be submitted to address
concerns raised by regulatory authorities. The concerns raised related to the AAS
levels of the materials used in the clinical studies, the control strategy, the potential
impact of the AAS on functional activity on Mylotarg, and the overall comparability
throughout development. The clinical basis for returning Mylotarg to the world
market was largely dependent upon the results of numerous studies run by independent investigators which, in aggregate, took a number of years to conduct. Over this
time period, most of the Mylotarg used in these studies did not contain elevated
amino acid substitution. Thus, the European Medicines Agency, for example, found
that the amino acid substitution led to multiple major deciencies [17] including
joint deciencies within the Quality and Clinical sections of the application to conrm the comparability of the clinical material with the proposed commercial
material.
In order to address these deciencies, the development team pursued a number
of additional studies that further conrmed that the amino acid substitutions did not
affect the efcacy of Mylotarg. The team demonstrated that no new sites of conjugation to calicheamicin were introduced with the amino acid substitution and the
13 Mylotarg: TheJourney toFDA Reapproval andBroad International Approval
387
antibody glycosylation was found to be comparable before and after the amino acid
substitution increased. Biophysical and biological activity was also addressed. The
level of cytotoxicity before and after increased substitution was demonstrated to be
comparable and, supporting this conclusion, binding data were also found to be
comparable for both CD33 and FcRn. Taken together, this provided a strong data
package for submission to regulatory agencies, which supported the conclusion
that, while the amino acid substitution affected the composition of Mylotarg, conjugates with variable amino acid substitution were functionally equivalent and the
clinical results were acceptable.
The regulatory response to the amino acid substitution and its correction was
varied. With Pzer submitting the data discussed above, several agencies next
sought to understand how quickly a change in the antibody manufacturing conditions could be implemented. As part of the review, Pzer indicated that process
improvements were already in development, but that it would take longer than the
regulatory review process to produce antibody under the restorative conditions.
Based upon the overall comparability data, the proposed control strategy to affect
remediation, and the realities of commercial antibody production, agencies approved
the application with the understanding that Pzer would subsequently introduce the
restored antibody. Post approval, an improved gemtuzumab process which resulted
in reduced amino acid substitution was implemented and approved. As part of this
submission, Pzer communicated its intent to continue to monitor the level of amino
acid substitution as part of the control strategy.
13.7 Production oftheCalicheamicin Linker Payload
A number of forces were in play when the decision was taken to revisit the production of the linker payload, activated calicheamicin derivative. With the desire to
return Mylotarg to the US market, and expansion to the European Union and Canada
as well as the rest of the world, regulatory success would increase the demand signicantly. Additionally, success in the registration of Besponsa would even further
increase the demand. As mentioned earlier, the original preparation was conducted
by a third party. Pzer determined that the third party would not be able to meet the
anticipated combined demand and that the preparation of activated calicheamicin
derivative needed to be revisited. Lastly, Pzer had built a facility in anticipation of
bringing the manufacturing in-house and any new preparation would be run in this
new facility. The new facility was constructed with a series of large isolators to
protect workers from exposure to the highly toxic material [18] which has an occupational exposure limit (OEL) in the tens of ng/m3. While the isolators were relatively large, any new equipment would need to be compatible with the oor and
bench space available.
Activated calicheamicin derivative has been manufactured by three different production routes. The rst and last, (Processes 1 and 3 respectively) have been used in
the preparation of gemtuzumab ozogamicin. Process 2 was practiced during the
388
ProCEss 1
L. Letendre et al.
development of Besponsa and was being considered for use with the re-launched
Mylotarg in addition to the commercial production of Besponsa. The comparison of
these routes follows.
13.7.1 Isolation ofγ-Calicheamicin
The preparation of the activated calicheamicin derivate begins with fermentation of
M. echinospora ssp. calichensis. A mixture of calicheamicin-related compounds is
found in the fermentation media and is isolated by a multi-step process. The fermentation and isolation could not be changed due to the limited availability of alternate
equipment in the dedicated Pzer facility. Also, the existing process was quite efcient and alleviated any push to do so. Thus, no changes were instituted.
γ-Calicheamicin was delivered from the fermentation as an oil consisting of the
various calicheamicin-related compounds and fermentation materials largely consisting of antifoam [19].
The isolation of γ-calicheamicin from the fermentation isolate differed slightly
from Process 1 to Process 2 (Fig.13.4). The desired calicheamicin was obtained
largely free of the fermentation-related byproducts by precipitation from a large
volume of di-isopropyl ether, a solvent we wished to remove for safety reasons.
Process 1 followed the precipitation with a normal phase chromatography step to
further purify the fermentation product. While this chromatography step may have
removed some calicheamicin-related products, it did not remove the major ones
(Fig.13.2). Process 2 did not include the chromatography and thus indicated that
ProCEss 2
ProCEss 3
Fig. 13.4 Isolation of γ-calicheamicin
g
g
g
13 Mylotarg: TheJourney toFDA Reapproval andBroad International Approval
389
downstream processes could remove the calicheamicin-related impurities. With the
major goal of removing di-isopropyl ether in mind, alternative precipitation solvents were investigated. A mixture of methyl tert-butyl ether (MTBE) and hexanes
was found to give equivalent or better recovery of γ-calicheamicin when compared
to the original di-isopropyl ether and was therefore utilized in Process 3.
13.7.2 Formation ofN-Acetyl Calicheamicin
The rst chemical step in the preparation of activated calicheamicin derivative is the
acetylation of the ethyl amino sugar (Fig.13.5). The challenge in adding an acetyl
group to the secondary amine is to prevent over-acetylation by reaction with the ve
alcohols and the alkoxyamine. During structure elucidation of the calicheamicins,
Lee et al. demonstrated that conducting the acetylation in methanol allowed for
highly selective acetylation on nitrogen [20]. Processes 1 and 2 essentially used the
method of Lee by adding acetic anhydride to a methanol solution containing triethylamine. The methanol effectively competes with the hydroxy groups on the sugars;
however, running the reaction in methanol required a large excess of both acetic
anhydride and triethylamine. On a production scale, this led to issues controlling
signicant exotherms during triethylamine addition and the aqueous quench due to
heat generated by methanol-water mixing. Both triethylamine addition and water
quenching had to be done very slowly to maintain the temperature and minimize the
degradation of the product. The Process 3 development team recognized the need to
reduce reagent amounts, prevent over-acetylation of γ-calicheamicin, and reduce or
avoid the exotherms. Acetonitrile was chosen as the reaction solvent and provided
multiple benets. Acetylation in acetonitrile with only a few percent of methanol
allowed signicantly lower amounts of acetic anhydride and triethylamine, affording N-acetyl calicheamicin with only trace formation of over-acetylated products.
Also, due to the reduced reagent amounts, triethylamine addition could be accomplished almost instantaneously without increasing the reaction temperature. Lastly,
the mixing of acetonitrile and water is endothermic, so the water quench could be
done rapidly and resulted in a decrease in temperature, eliminating any concerns of
product degradation during an exotherm.
Fig. 13.5 Preparation of N-acetyl calicheamicin