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L. Letendre et al.
13.7.3 Isolation ofN-Acetyl Calicheamicin
The isolation of N-acetyl calicheamicin from both Process 1 and Process 2 was
resource intensive (Fig.13.6). In Process 1, the reaction mixture was reduced in
volume prior to normal phase chromatography. Product-containing fractions were
evaporated to dryness and the product was then precipitated from ethyl acetate/
heptane. Process 2 relied upon an aqueous workup, extraction with dichloromethane, and evaporation of solvent followed by purication using reverse-phase chromatography with aqueous acetonitrile. Product-containing fractions were extracted
with dichloromethane followed by concentration and precipitation with ethyl acetate/heptane. Process 3 sought to reduce the number of manipulations, maximize
yield, and improve manufacturability. Having conducted the acetylation reaction in
mainly acetonitrile and quenched with water, all that was needed to prepare the
reaction mixture for purication by preparative reverse-phase chromatography was
to target the appropriate acetonitrile-water ratio for column loading. So, the aqueous
quench directly generated the chromatography loading solution. Fraction selection
was controlled by an in-process assay purity specication.
Fig. 13.6 Isolation of N-acetyl calicheamicin
13 Mylotarg: TheJourney toFDA Reapproval andBroad International Approval
391
In place of the Process 2 extractive workup of puried fractions, an improved
method was sought which reduced unit operations and solvent usage and ideally
eliminated dichloromethane. It was found that near quantitative recovery could be
achieved by solid-phase extraction. Using the same chromatography stationary
phase, the fractions could be loaded onto the column by appropriately choosing the
proportion of water in the load solution. Fractions were pooled, diluted to achieve a
higher water content, and loaded onto the column. The bound product was washed
with water (containing a small percent acetonitrile to prevent column dewetting)
and eluted with acetonitrile providing the product in essentially dry solvent. The
acetonitrile was evaporated from the high-concentration eluate. Finally, a tractable
solid was obtained by precipitation of the product from ethyl acetate/heptane and
drying under vacuum in a manner similar to Processes 1 and 2.
The combined effects of the process changes introduced in Process 3 compared
to Process 2 were a signicant reduction in unit operations, fewer days to complete
manufacture, and an approximately 30% increase in average yield.
13.7.4 Production ofActivated Calicheamicin Derivative
The conversion of N-acetyl calicheamicin to activated calicheamicin derivative
requires the addition of the activated linker. This conversion is the point where
Processes 1, 2, and 3 differ most signicantly, with each iteration decreasing the
number of steps used to install the activated linker. Reducing the amount of chemistry performed on the calicheamicin-containing molecule preserves the highly
valuable natural product and was likely to improve overall yield.
Process 1
Process 1 utilized three steps to add the activated linker: two steps to assemble the
cleavable linker on the calicheamicin core and one to activate the carboxylic acid for
reaction with the antibody (Fig.13.7, clockwise path). This approach rst reacts
half of the linker, 3-methyl-3-mercaptobutanoic acid hydrazide (often referred to as
DMH in publications [4], red in Fig.13.7), with the methyl trisulde of N-acetyl
calicheamicin. This addition was a relatively low-yielding step, which was nally
improved in Process 3. The addition was conducted in acetonitrile in the presence
of triethylamine followed by concentration and silica gel chromatography. The
selected fractions were concentrated, dissolved in methanol, and precipitated with
water to afford solid N-acetyl calicheamicin DMH.
The second portion of the linker, 4-(4-acetylphenoxy)-butanoic acid (often
referred to as AcBut in publications [4], blue in Fig.13.7), was then appended to
N-acetyl calicheamicin DMH under acidic conditions in methanol. After removal of
the solvent and silica gel chromatography to afford N-acetyl calicheamicin
DMH- AcBut, the linker payload needed only to be activated for subsequent addition to gemtuzumab. The acid moiety was converted to the N-hydroxysuccinimide
(NHS) ester by treatment with NHS (green in Fig. 13.7) in acetonitrile and
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Fig. 13.7 Addition of the activated linker by Processes 1, 2, and 3
L. Letendre et al.
N,N- dimethylformamide (DMF) in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). Subsequent silica chromatography and
precipitation from ethyl acetate/hexane led to the desired activated calicheamicin
derivative.
As can be readily surmised from the Process 1 description above, this synthesis
is lacking since it performs several steps on the valuable calicheamicin core, involves
multiple chromatography steps, and several steps involve solvent swaps. All of
these limitations were to various degrees addressed in Processes 2 and 3.
Process 2
Process 2 (Fig.13.7, diagonal path) was initially implemented in order to supplement the supply of activated calicheamicin derivative to support the clinical development of inotuzumab ozogamicin with the intention of subsequently adding the
preparation to the gemtuzumab ozogamicin supply chain. The primary improvement included in Process 2 was the assembly of the linker prior to attachment to
N-acetyl calicheamicin. Using conditions similar to Process 1, the linker (DMHAcBut in Fig. 13.7) was added followed by a precipitative workup to provide
N-acetyl calicheamicin DMH-AcBut. Conversion to the NHS ester remained the
same as in Process 1. However, the isolation of activated calicheamicin derivative
was modied to rst purify the material by reverse phase chromatography, extraction with dichloromethane, and purication by silica gel chromatography and concentration. As was done in Process 1, the material was precipitated from ethyl
acetate/hexane. While this process was more convergent, it still suffered from the
13 Mylotarg: TheJourney toFDA Reapproval andBroad International Approval
393
NHS activation step involving the penultimate calicheamicin intermediate and the
need for two chromatographic steps, a less than optimal purication process for
activated calicheamicin derivative. Process 3, developed some 20years after Process
1, aimed to address these issues with contemporary knowledge and equipment.
Process 3
Process 3 (Fig.13.7, left direct path) became the manufacturing process for the US
relaunch of Mylotarg and the launch of Besponsa. The primary improvement was
the attachment of the activated linker to N-acetyl calicheamicin in one step. Along
the way, novel chemistry was discovered that boosted the crude yield which, coupled with an optimized purication procedure, provided a signicant increase in
overall yield. Preparation of the activated linker is not discussed here other than to
say that it proceeds smoothly with the major improvements arising from replacing
acid chlorides with less reactive analogs for the formation of the hydrazide and the
NHS ester [21].
Attachment of the activated linker to N-acetyl calicheamicin was originally
investigated with the relatively crude activated linker. The yields were signicantly
higher than what had been achieved at the time for the reaction of DMH-AcBut with
N-acetyl calicheamicin. However, as the preparation of the activated linker improved
with concomitant increase in its purity, the yield of activated calicheamicin derivative decreased. Contemplating upon what could cause this, the development group
posited that there was a remaining coupling agent (carbodiimide) in the activated
linker from the NHS ester-forming step. The hypothesis was tested and proven to be
correct by including EDC in the coupling reaction mixture leading to an improved
yield of activated calicheamicin derivative.
A mechanistic understanding of this observation was aided by the work of Myers
etal. [22]. The reaction of the sulfur on the activated linker with the trisulde of
N-acetyl calicheamicin is a complicated mix of equilibria and dead-end reactions.
As shown in Fig.13.8, the linker sulfur could, in theory, attack any of the three
sulfurs of the trisulde giving four different products of which only one is desired.
Work by Evans and Saville [23] indicated that the two reactions which displace a
leaving group from the central sulfur are slow which is consistent with our observation of relatively low levels of ε-calicheamicin, the ultimate product of cleaving the
sulfur–sulfur bond between the calicheamicin moiety and methyl disulde. The formation of ε-calicheamicin results from the reaction of the allylic sulfur via a Michael
addition into the enone followed by Bergmann cyclization [22] (Fig.13.8, lower
left). The second product of attack on the central sulfur is the linker trisulde
(Fig. 13.8, lower right). Of the two remaining displacements, one provides the
desired disulde affording activated calicheamicin derivative (Fig. 13.8, upper
right) while the other produces a dead-end disulde (Fig.13.8, upper left). As shown
in Fig. 13.9, a carbodiimide (blue, EDC for this case) can convert the dead-end
N-acetyl calicheamicin disulde to an activated disulde, allowing another
molecule of activated linker to attack the allylic sulfur to produce the desired
product [21].
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L. Letendre et al.
Fig. 13.8 Thiolate attack on the methyl trisulde. (Adapted from Myers etal. [22])
With improved reaction conditions in hand as well as a mechanistic understanding of the chemistry available, the nal efforts focused on optimizing the purication and isolation of the product.
The activated calicheamicin derivate contains both acid-sensitive (hydrazide)
and base-sensitive (NHS ester) groups. Thus, any chromatographic purication of
the molecule must take this into account. Also included in γ-calicheamicin are a
number of ionizable groups which, depending upon the pH of any mobile phase
used in reverse phase chromatography, could affect the ability to separate the desired
product from impurities. An extensive study of the stability of activated calicheamicin derivative in aqueous acetonitrile identied the optimal pH which allowed for
efcient purication of the product. However, the elution conditions could not fully
prevent degradation at ambient temperature and therefore fractions were immediately sampled as they were collected and frozen during in-process analysis. Fractions
that met the purity requirements were combined and additional water was added to
decrease the solvent strength. The solution was then concentrated using solid phase
extraction on a reverse phase column similar to the isolation of N-acetyl calicheamicin. Once the material was loaded, the column was washed with water and then
13 Mylotarg: TheJourney toFDA Reapproval andBroad International Approval
Fig. 13.9 Role of EDC in the addition of activated linker
395
eluted with acetonitrile. The early portion of the product peak eluted in acetonitrile
containing a small amount of water, which could degrade the product if allowed to
stand. Therefore, the rst few percent of solvent was isolated separately and immediately reduced in volume relying on the acetonitrile/water azeotrope to dry the
material. After all of the solvent was removed, the material was precipitated from
ethyl acetate/hexane to provide a tractable solid after drying under vacuum.
Manufacture of activated calicheamicin derivative by Process 3 has many
improvements compared to Process 2. Changes made in Process 3 have greatly
improved manufacturability, reduced the number of unit operations by almost 50%
including the removal of multiple time-intensive chromatography steps, removed
chlorinated solvents from the process, and doubled the average yield. Compared to
Process 1, the Process 3 yield improvement approached an order of magnitude and,
to date, the yield and quality of the product have been consistent demonstrating that
the process is well controlled.
13.7.5 Regulatory Challenges Involving Activated
Calicheamicin Derivative
Two major challenges were presented by the activated calicheamicin derivative.
One was chemical and the other was based upon the application of the ICH guidelines. The former presented itself as we attempted to fully understand the fate of the
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L. Letendre et al.
various impurities encountered during the preparation. The latter presented itself in
our efforts to reach uniformity in the designation of the regulatory starting material.
The study of the purge and fate of impurities in the preparation of activated calicheamicin derivative began, of course, with the crude calicheamicin fermentation
product. Using mainly LC-MS, most of the previously known analogs were identied as well as about a dozen new related compounds. As reported previously, the
major analogs were β-calicheamicin and δ-calicheamicin, which differ from
γ-calicheamicin by the alkyl group on the nitrogen of the amino sugar, and
ε-calicheamicin which is derived from γ-calicheamicin having undergone the
Bergmann cyclization [22]. Following these various impurities through the preparation, we were gratied to learn that essentially all impurities were removed during
the purication of N-acetyl calicheamicin and those that remained were removed
during the purication of activated calicheamicin derivative.
Despite our detailed understanding of the purge of impurities, HPLC analysis of
activated calicheamicin derivative always showed a small peak eluting just before
the desired product (Fig.13.10, left) even though the chromatographic fractionation
during purication excluded this peak. Believing that the peak must be from some
related compound, an LC-MS analysis across both the minor and major peaks provided mass spectral data that were identical. An additional HPLC experiment provided an explanation for the phenomena. The major peak was isolated and reinjected
immediately followed by several more injections in sequence thereafter. The resulting chromatograms demonstrated that the major peak was being converted to the
minor peak over time. Understanding that the major peak did not completely convert to the minor and that the minor peak always represented the same proportion of
the major, we concluded that the two peaks represented isomers in equilibrium.
The source of the isomerization is not known with certainty. The activated linker
alone demonstrated similar behavior thus pointing to the isomers originating from
the linker portion of activated calicheamicin derivative. The acyl hydrazone core of
the activated linker was identied as the likely isomerization center (Fig.13.10).
Fig. 13.10 Activated calicheamicin derivative isomers
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397
Interconverting isomerization of acyl hydrazone compounds has been observed by
HPLC [24], NMR [25, 26], and IR spectroscopy [27]. The HPLC observation with
activated calicheamicin derivative could be explained by dynamic isomerization
around the hydrazone bond, the hydrazide bond, or a combination of the two
(Fig.13.10, right). Luckily, narrowing the mechanistic explanation down to one was
not required because it had been demonstrated that the two peaks were from the
same molecule albeit from different, interconverting forms. Since the hydrazone is
hydrolyzed to release the toxin invivo, the released toxin would be the same regardless of the isomer’s origin.
The purge and fate experiments also included the fate of the impurities in the
activated linker. In a manner similar to that of N-acetyl calicheamicin, it was demonstrated that none of the impurities in the activated linker contributed to impurities
in the activated calicheamicin derivative. The ICH Q11 guideline [28] speaks to
several points relevant to the selection of starting materials for the preparation of
activated calicheamicin derivative. Among others, these include:
• The activated calicheamicin derivative is a semisynthetic material. In selecting a
starting material Q11 states: “However, if it can be demonstrated that one of the
isolated intermediates in the synthetic process complies with the principles out-
lined above for the selection of starting materials for synthetic drug substances,
that isolated intermediate can be proposed as the starting material.”
• The inclusion of manufacturing steps in Section 3.2.S.2.2 of an NDA or BLA is
guided by: “Manufacturing steps that impact the impurity prole of the drug
substance should normally be included in the manufacturing process described
in Section 3.2.S.2.2 of the application.”
A regulatory strategy was thus developed proposing starting materials for activated calicheamicin derivative. The strategy included:
• that the preparation of the semi-synthetic activated calicheamicin derivative ful-
lled the requirement to be treated as a standard synthetic compound
• that the impurities in both N-acetyl calicheamicin and the activated linker did not
contribute to the impurities found in the activated calicheamicin derivative
• that activated calicheamicin derivative was not a drug substance but an interme-
diate in the production of antibody-drug conjugates.
Pzer therefore approached the various regulatory agencies proposing N-acetyl
calicheamicin and the activated linker as starting materials. The initial responses
from the agencies were varied. Some accepted the proposal. Some indicated that the
activated linker and crude γ-calicheamicin fermentation product would be acceptable. And some agencies on nal review changed their requirements, differing from
Pzer in their interpretations of the ICH guidelines on starting materials. During the
review of the nal submission, a number of agencies indicated that the calicheamicin starting material should be earlier in the synthesis than N-acetyl calicheamicin.
The argument for requiring an earlier starting material, reminiscent of the propinquity arguments of draft versions of ICH Q11, was cited by some agencies. In the
398
end, the regulatory starting material ranged from the bacterial cell banks in a few
markets to N-acetyl calicheamicin in most markets. The activated linker was
accepted everywhere as a starting material.
L. Letendre et al.
13.8 Production ofGemtuzumab Ozogamicin
Drug Substance
Conjugation of the activated calicheamicin derivative to the gemtuzumab antibody
followed by purication constitutes the drug substance process. While the drug substance process for this ADC, the rst on the market and the only one for over a
decade, may seem unsophisticated by contemporary standards, it has provided consistent drug substance for over two decades.
One interesting aspect of gemtuzumab ozogamicin which engendered a number
of questions from knowledgeable colleagues as well as others in the industry is
whether we would attempt to adjust the drug loading prole to align more closely
with contemporary ADCs. Mylotarg contains about 50% unconjugated or “low conjugate” antibody [9, 29]. This was not realized until a number of years after its initial launch when improved analytical methods were able to identify this fraction.
Pzer did not and could not easily change the composition of Mylotarg because the
original composition was the basis of the clinical trial experience as well as the US
commercial experience and the product remained on the market in Japan. All work
to further our understanding of the manufacturing process must ultimately keep the
unconjugated fraction unchanged.
The manufacturing process involves the addition of activated calicheamicin
derivative in an alcoholic solvent to an aqueous solution of the gemtuzumab antibody, a surfactant, and a buffering system. The process development used for the
original approval of Mylotarg explored the process space using one factor at a time
(OFAT) experiments. While OFAT justication for some process parameters is
acceptable if shown to be unaffected by other process parameters, it is not surprising
that many conjugation processes are best described using multivariate analyses.
While the number of ingredients used in the conjugation reaction may seem few,
there are multiple parameters associated with them. Complicating the development
of this conjugation chemistry, the addition of the activated calicheamicin derivative
to the lysine amines is analogous to the Shotten-Baumann reaction, which is known
to be both rapid and very exothermic. In the present case, the NHS ester, a less reactive acid derivative, replaces the acid chloride of the Shotten-Baumann reaction but
is still quite reactive and the kinetics can be nearly as fast. Therefore, a number of
additional reaction parameters such as antibody to activated calicheamicin derivative ratio, antibody concentration, and reaction temperature, which are related to the
conjugation kinetics and drug substance quality attributes, needed to be included in
the re-examination of the process parameters.
13 Mylotarg: TheJourney toFDA Reapproval andBroad International Approval
399
Several parameters were studied for their effect on the level of aggregate and the
average drug loading (drug to antibody ratio, DAR) with target values set based
upon the capability of the subsequent purication to bring the material within the
drug substance specication.
The parameters were investigated in a series of multivariable experiments (design
of experiments or DOEs), which demonstrated that several parameters either did not
affect the outcome or their effect was not correlated with changes to some other
parameter. Given that it had been demonstrated that the kinetics of the addition of
the NHS ester of activated calicheamicin derivate was fast, the decision was made
that mixing could be set such that it would be sufcient for all subsequent experimental conditions and thus not included in any DOE analysis. In order to determine
an acceptable blend time and translate it to the scaled-down model used in the DOE,
the blend times and concentration distributions within the commercial scale and lab
scale reactors were modeled both experimentally and with in silico uid dynamics
calculations. The modeling exercise rst determined that which could be achieved
in the commercial setting then the results were scaled down to the lab scale and held
constant for the OFAT and DOE experiments. The remaining process parameters
were tested in a multivariable environment, which demonstrated some interaction
between several of the factors. These interactions would not have been discovered
if the parameter space had been investigated as they were originally with OFAT
experiments. The goal of these experiments was to determine a set of parameters
that would keep the drug substance attributes, including the amount of unconjugated antibody and drug-to-antibody ratio, within the historical ranges dating back
to the initial introduction of Mylotarg to the market. Thus, the operating conditions
were redened while keeping specications unchanged. The practical effect of this
deeper knowledge was to adjust the manufacturing instructions to meet the better
understood requirements.
Purication of the drug substance reaction mixture to produce bulk drug substance was performed by size exclusion chromatography using the nal excipient
solution as the eluting buffer. Since the quality of the crude drug substance after the
conjugation reaction was well within the historical range, the chromatographic purication process was not modied.
13.9 Production ofMylotarg Drug Product
The Mylotarg drug product is a lyophilized powder, which is reconstituted with
water for injection. Since the bulk drug substance contains all of the excipients in
the drug product, no changes to the drug product were necessary.