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Development of Commercial Ready Processes for ADCs Xi Hu, Eric Bortell, Frank W Kotch, April Xu, Bo Arve, and Stephen Freese Org. Process Res. Dev., Just Accepted Manuscript • DOI: 10.1021/acs.oprd.7b00023 • Publication Date (Web): 10 Mar 2017 Downloaded from http://pubs.acs.org on March 10, 2017

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Organic Process Research & Development

Development of Commercial Ready Processes for ADCs Xi Hu1*, Eric Bortell1, Frank W. Kotch1, April Xu1, Bo Arve2, Stephen Freese1 1

Pfizer, Inc., Biotherapeutics Pharmaceutical Sciences, Worldwide R&D, Pearl River, NY 10965, USA

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Pfizer, Inc., Biotherapeutics Pharmaceutical Sciences, Worldwide R&D, Chesterfield, MO 63017, USA

ACS Paragon Plus Environment

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ABSTRACT: As Antibody Drug Conjugates (ADCs) move through clinical development, from phase 1 to pivotal studies, supported by clinical efficacy and appropriate safety, the development of late stage and commercial-ready processes and methods become a priority. During development of early stage proesses, the focus is on speed, consistency and quality. For later stage development, additional aspects need to be considered including process robustness, cost effectiveness, process and methods transferability and an integrated control strategy as a foundation for the regulatory filing. KEYWORDS: antibody drug conjugate; quality attributes; process development; technology transfer; process validation


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INTRODUCTION Late stage development of an ADC can be triggered by many factors. In a regular development program the ADC will go through the traditional clinical development stages, including Phase 1 and Phase 2. As the drug reaches Proof-of-Concept there is typically justification for making the significant investment in planning and execution of development of processes and methods to support Phase 3/Pivotal studies followed by Process Validation, launch and commercial production. With the introduction in 2012 of the Breakthrough Therapy Designation,1 it has become possible that an ADC demonstrating early signs of efficacy in disease areas with significant unmet medical need can move directly from Phase 1a/1b into Pivotal trials. Of course, completing all requisite development work under an accelerated timeline necessitates careful planning and coordination across multiple functional units since an ADC involves two drug substance intermediates (DSI), the linker/payload and the monoclonal antibody (mAb), one drug substance (DS), the ADC, and the drug product (DP). Also, the regulatory filing will require particular considerations and may involve multiple distinct Module 3 sections, one for each major intermediate, one for the drug substance and one for the drug product.2 In either case, accelerated or traditional drug development, the expectation is that complete Chemistry, Manufacturing and Controls (CMC) sections are submitted in the regulatory filing. Most drugs today are developed using a Quality by Design (QbD) approach with a view towards Continuous Process Verification (CPV).3 QbD methodologies are intended to drive deep process and product understanding. Important quality attributes that impact efficacy and safety are determined and, through systematic experimentation, process parameters that impact these attributes are identified. Once the process is optimized and established at lab scale, the manufacturing process operating ranges are determined using appropriately qualified scale down models. These studies typically involve design of experiments (DOEs) and statistical analysis. In certain cases, edge of failure for selected parameters may also be determined. Purification steps, such as chromatography and diafiltration, are typically optimized using standard bioprocessing approaches. Scale-up and technology transfer afford particular challenges and opportunities. A thorough understanding of the reaction kinetics, addition rates and other parameters is required to appropriately design and operate the large scale conjugation reaction vessel. Detailed process fit evaluations must be performed to ensure that existing equipment will meet the requirements of the process. In cases where new equipment will be acquired, a first decision about the use of disposable versus dedicated, reusable or multi-product equipment needs to be made. For dedicated and multi-product equipment, cleaning validation becomes a critical consideration. Scale-up, equipment changes and potential site changes also require the establishment of comparability for scaled-up and optimized processes. Along with the development of the commercial process, an integrated control strategy, including specifications, needs to be developed, that ensures established quality attribute ranges are achieved through appropriate control of important process parameters. Also, an integrated regulatory strategy is required linking the quality attributes of linker, payload, mAb, ADC (DS) and drug product.


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LATE STAGE ADC DEVELOPMENT Late stage development of ADCs in preparation for production of material for pivotal clinical trials, process validation, regulatory approval and commercialization presents an unusual combination of challenges.4 These challenges arise from several sources: •

•

•

•

ADCs are multi-component conjugates linking large and small molecules. Depending on the chemistry used, ADCs can be heterogeneous, consisting of a population of closely related species or, when site-specific technologies are used, more homogeneous with a specified number of payloads per mAb.5 Late-stage development work is directed toward understanding and controlling the parameters that effect the linker-payload distribution and other important attributes of the ADC. ADCs are manufactured using process steps that need to combine aqueous processing of biologics with solvent processing of small molecules. This can sometimes lead to unexpected outcomes, in particular as processes are optimized towards higher concentrations and during scale-up. The highly toxic materials must be handled in appropriate facilities during development and manufacture.6

Production of ADCs involves the combination of a cytotoxic drug, a reactive linker, and an antibody. The antibody is either intrinsically reactive via the amino acids or is modified to render it reactive. Because of the polyvalent nature of antibodies, heterogeneity may be an important issue for the development chemist to monitor. Depending on the conjugation chemistry used, the conjugate may vary from a single homogeneous product to a highly heterogeneous mixture of related conjugates. The distribution of conjugates that defines the product is driven by the chosen conjugation chemistry and is established during preclinical and early stage clinical studies. The challenge for late stage development is the optimization of the commercial process that maintains the product quality attributes and ensures comparability to early clinical stage material. This requires that process parameters have been properly optimized to ensure that the process consistently delivers the target quality attributes. For complex and heterogeneous ADCs, understanding how process parameters impact the reactivity of individual conjugation sites may be a challenge. While the site-specific chemistries largely obviate the issue of heterogeneity, endogenous cysteine and lysine chemistries result in conjugates with modifications to a number of sites. Chromatographic purification may be included in the process to reduce product heterogeneity, although these mixtures can be difficult to separate preparatively. Traditional protein purification is largely devoted to removing host cell proteins, nucleic acids, high molecular weight species and process related impurities, while small molecule purification focuses on isolation of the unique desired species and eliminating closely related non-product molecules. On the other hand, ADC purification may have the additional challenge of controlling heterogeneity of the target product. Heterogeneity in ADCs arises, in large part, from the co-existence of conjugate species with different drug/antibody ratio (DAR). DAR is generally a critical quality attribute that can have an effect on safety, efficacy and stability. The goal of chromatography, then, is to control the heterogeneity within defined limits. On the other hand, because both the mAb and linker-payload are purified intermediates,


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upstream impurities are not present to a significant extent, while reaction side-products, residuals, and degradant are a larger concern than is typical for bioprocesses. A prerequisite for understanding the effect of process parameters on quality attributes is to have assays in place that are sufficiently sensitive to the product quality. Two important attributes which do not have direct parallels in other fields are conjugation site and DAR. Assays for these attributes will take different forms for different chemistries. In addition to these quality attributes, which can only exist and be monitored after conjugation, some quality attributes inherent to the protein or linker/payload may be obscured in the conjugate. Quality attribute, such as deamidation or conjugatable drug impurities, may be changed following conjugation, and yet cannot be directly monitored, it is necessary to have a comprehensive control strategy. The control strategy includes understanding of starting material quality attributes, effect of conjugation conditions on the drug substance quality attributes, which is gained through laboratory model systems, and fate and purge of impurities in the downstream process. A successful control strategy will demonstrate that control of impurities is achieved in a scientifically sound fashion even in the absence of specific release assays. A final challenge in developing ADCs is the high toxicity of the drug species. Drugs used in ADCs typically have IC50 values (half maximal inhibitory concentration) in the low nM to pM range,7 toxicity that is outside the experience of most chemists. Therefore laboratory work needs to be performed with strict regard to procedural, engineering, and protective equipment controls for exposure and contamination. These controls must also be implemented in the manufacturing area, a requirement that necessitates specialized facilities.6 This review will examine these issues in detail in the context of the development of a lysineconjugated ADC. Case Study for Inotuzumab Ozogamicin Inotuzumab ozogamicin (InO) is an antibody-drug conjugate comprised of a humanized monoclonal antibody (inotuzumab) covalently linked to the antibiotic calicheamicin (ozogamicin).8 Inotuzumab selectively binds to CD22 receptors, which are present on B lymphocytes. Calicheamicin is a potent enediyne cytotoxic agent that causes double-strand breaks in DNA, leading to cell death.9 InO binds to CD22-expressing cells and upon internalization, releases calicheamicin which causes cytotoxicity. It is currently in phase 3 clinical trials in acute lymphoblastic leukemia (ALL). InO was granted Breakthrough Therapy Designation for this indication. InO is produced using conventional lysine conjugation where succinimidyl-activated calicheamicin derivative reacts with the ε-amino group of lysines on inotuzumab to form a covalent bond. A representation of InO is presented in Figure 1. Approximately 6 calicheamicin derivative molecules are conjugated to each inotuzumab molecule.


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Figure 1. Structure of Inotuzumab Ozogamicin

A major challenge in the development of a robust, scalable and commercial manufacturing process for InO is the control of product heterogeneity to ensure comparability to early clinical and toxicology material. Due to the non-site-specific nature of lysine conjugation chemistry, InO is a heterogeneous mixture of conjugates with different loads of linker payloard (calicheamicin derivative) attached to different lysine sites on inotuzumab. However, due to the optimized process conditions developed, the drug load distribution of InO is controllable and very reproducible and the conjugation occurs at preferred lysine sites. Of the more than 80 lysines on the mAb, more than 90% of conjugation occurs on 8-10 kinetically preferred lysine sites (4-5 on each half of the antibody). Hence, InO has a characteristic loading distribution and positional isomer profile. To ensure the consistency of this profile throughout the late-stage process development, a systematic and risk-based approach was employed for carrying out development studies. First, the quality attributes of InO were screened and the ones related to the conjugate heterogeneity were identified. These heterogeneity-related quality attributes include: • • • • •

total drug load drug load distribution unconjugated antibody unconjugated linker payload sequence position of conjugated lysines

Figure 2 shows an example of the InO drug load distribution by imaged capillary isoelectric focusing (iCE). Due to the charge difference, species with different DAR migrates differently in the electropherogram.


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Figure 2. Drug Load Distribution by iCE

Having established analytical test methods for the quality attributes listed above along with the target specifications, these quality attributes were used to measure the heterogeneity for conjugates generated from process characterization studies. The next step was to evaluate the process steps that influence these quality attributes. The process for making InO drug substance consists of four major steps: 1) Conjugation of inotuzumab with activated calicheamicin derivative 2) Purification of crude conjugate using Hydrophobic Interaction Chromatography (HIC) 3) Buffer exchange using ultrafiltration/diafiltration 4) Formulation to make the final formulated drug substance

An initial risk assessment was conducted by a multi-disciplinary team to identify the process parameters with potential to impact InO drug substance heterogeneity. These process parameters include: • •

Conjugation reaction: conjugation pH, time, temperature, drug input, co-solvent and surfactant input HIC step: column challenge, loading and eluting buffer concentrations and pH, and fraction pooling

Process characterization studies were executed using a combination of multivariate DOE and univariate experimental strategies to characterize the relationship between these process input parameters and the quality attributes. Results showed total drug load (DAR) and drug load distribution were mainly controlled by calicheamicin input in the conjugation reaction and fraction pooling in the HIC purification step. For the conjugation reaction, increasing calicheamcin input led to DS with higher DAR, and corresponding drug load distribution shifted toward the higher loaded species, as indicated in the iCE spectra (Figure 3) and the corresponding LC-MS (Figure 4). Following HIC purification step, free calicheamicin and free


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antibody were removed from conjugate due to their different mobility on the HIC column. The collected HIC fractions were then pooled based on the criteria regarding to protein concentration, drug load and aggregation level, which further refined the conjugate drug load and its distribution and ensured the desired conjugate quality attributes were met before proceeding to the next process step.

Figure 3. Comparison of Drug Load Profile by iCE


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Figure 4. Comparison of Drug Load Profile by LC-MS

Acceptable ranges for calicheamicin input and HIC fraction pooling criteria were then established based on the target specifications for drug substance heterogeneity. As experimental results and further scale up experience were obtained, the risk assessment was revisited and a criticality assessment performed. Both calicheamicin input and HIC fraction pooling criteria were designated as critical process parameters (CPPs) for the control of conjugate heterogeneity. Another challenge for late-stage process development is scale up of the conjugation process from laboratory scale to manufacturing production scale. Unlike typical biological processes, the conjugation reaction of inotuzumab with activated calicheamicin derivative is very rapid. Judged by the amount of unconjugated mAb remaining in the reaction, the conjugation reaction at 10mg/mL mAb concentration reached completion within 2 minutes, as shown in Figure 5. The fast reaction kinetics demands adequate mixing of the reaction mixture at all scales. Furthermore, the addition rates and method of addition are also parameters that need to be considered for a reaction with fast kinetics.


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Figure 5. Amount of Unconjugated Inotuzumab versus Reaction Time

To identify the parameters that control mixing efficiency in the reactor systems at various scales from 0.1 L to 10L, the blend time of each reactor was determined using salt-spiking experiments in which the conductivity of the solution inside the reactor was monitored upon addition of concentrated sodium chloride solution. Blend time data demonstrated that consistent mixing profiles were achieved in each reactor with comparable agitation settings. Therefore, the labscale reactor (0.1 L) was considered an appropriate scale-down model for conjugation. In addition, computational fluid dynamics simulations were used to characterize mixing at various scales. Next, mixing studies were performed using the lab scale reactor to assess the impact of mixing conditions on conjugate quality attributes. The mixing conditions included agitation rate, above or below surface addition method and addition time of the activated calicheamicin derivative solution. The experiments showed that low agitation rate and above surface addition resulted in poor mixing as evident from incomplete conjugation. On the other hand, when the calicheamicin solution was added subsurface, the conjugation reaction proceeded efficiently even at low agitation rates. After the addition rate was defined, the acceptable operation ranges for mixing inputs were tested at the intermediate scale (1 L) for process consistency and scalability. The results in Figure 6 indicate that the drug load profile of the intermediate-scale run is comparable to that of the reference standard prepared from an early-stage clinical run. The process has been further scaled-up to commercial scale and comparison of yield and quality attributes demonstrates that the established process is robust and scalable for commercial-scale production, as shown in Figure 7. In principal, shear-dependent aggregation may limit the use of high mixing rates, although in this case aggregation as a function of mixing was not observed.


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Figure 6. Drug Load Profile by iCE

Figure 7. Comparison of Conjugation Attributes at Different Scales 10.0 9.0

70

8.0 7.0 6.0

60 50 40

5.0 4.0 3.0 2.0

30 20 10

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0 Lab Avg. (n=4)

Clin. Avg. (n=5)

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DAR or % Agg

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% Yield

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% Yield DAR

Technology Transfer to Manufacturing Once the commercial scale process was defined, technology transfer of the process to manufacturing was initiated. Information sharing and technical discussions between development and manufacturing teams occur throughout the development cycle, but it is near the conclusion of process characterization studies that technology transfer documents are finalized in preparation for manufacturing.


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Below, the process fitting, manufacturing equipment and single-use systems, supply of drug substance intermediates, raw material considerations, and the handling of cytotoxic materials will be discussed as they relate to transfer of the process to manufacturing. Manufacturing equipment and manufacturing process The main manufacturing equipment for ADC drug substance production includes reactors and purification systems. The selection of appropriate manufacture equipment for each manufacturing process is based on multiple factors. This section describes single-use systems, reactors, chromatography equipment, buffer exchange equipment, and accompanying considerations. Single-Use Systems ADC production may make significant use of single use systems. Several vendors supply completely disposable manufacturing systems, including chromatography skids, chromatography columns, tangential flow filtration skids, and membranes. Single use bioprocess containers are frequently used for product transfers, dilutions and filtrations. In addition to the benefit of eliminating cleaning and cleaning validation, single use systems reduce the potential for exposure of workers to the cytotoxic agents. Designs allow for completely closed modules that do not have to be dissembled after use. To the extent that disposable systems minimize handling of contaminated surfaces, the process will be safer. To realize safety improvements from minimal handling, attention should be paid to ensure that connections between modules are leakfree during use and after use. Reactors In the case of the InO process, a glass reactor was chosen because of its mixing capabilities and scale-up performance. The reactor was characterized by computational fluid dynamics (CFD) simulations using ANSYS software 14.0. The CFD result (Figure 8) showed that adequate mixing was achieved within a short period of blend time. This mixing profile is similar to that observed in smaller development reactors. In addition, CFD simulations were used to evaluate multiple designs including 1) single impeller vs dual impeller, 2) angle of blades (45, 60 and 90o) and 3) height of blades. Salt spiking experiments, addition rates and various addition location experiments were performed to confirm the CFD models prior to the scale-up runs. These results demonstrated that appropriate mixing could be achieved without the use of baffles. In depth studies of reactor mixing is driven both by changes in fill volumes of a particular reactor and changes in reactor design. In the inotuzumab ozogamicin case, a five-fold change in scale necessitated an evaluation of mixing dynamics.


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Figure 8. CFD Simulation of Commercial Scale Reactor

While there are no disposable reactors that are equivalent to glass or stainless steel reactors in terms of mixing performance, mixing bags and disposable bioreactors have been evaluated and in certain cases used for bio-conjugation reactions. CFD simulations of a disposable bioreactor design were conducted using ANSYS software 13.0. The results indicated that slow mixing occurred at the top of the vessel and there were micro-eddies formed (Figure 9). Hence this disposable reactor design needed for cell growth limits blend time and may not provide adequate mixing for ADC conjugation reactions, in particular for reactions with fast kinetics. Figure 9. CFD Simulation of a Disposable Bio-reactor


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Chromatography Hydrophobic interaction chromatography (HIC) purification plays a critical role in the production of InO to ensure that predetermined critical quality attributes are met. HIC is analogous to reverse chromatography in that retention is driven by hydrophobicity. However, HIC resins are designed to work with an elutropic series that runs from a strong aqueous buffer (up to ~4 M, the weak solvent) to water or weak buffer (~20 mM, the strong solvent) and, for protein work, generally does not extend to organic solvent/water mixtures. Because the calicheamicin conjugated to inotuzumab imparts greater hydrophobicity to the conjugate, this step is effective in removing unconjugated antibody, aggregates, free drug and excipients from the product. Since this step includes fraction collection, the drug loading of the ADC can also be affected depending on how the fractions are pooled. For InO each fraction is tested for protein concentration, drug/antibody ratio and aggregates and criteria have been established which ensure that only those fractions that result in a final product that meets specifications are pooled. To establish the fraction pooling criteria, it is important that studies performed using the smallscale model translate to manufacturing scale. Figure 10 shows a typical lab-scale chromatogram. A useful attribute of calicheamicin conjugates is the presence of the 310 nm chromophore. This results in a significant signal at 310 nm from the conjugate, whereas any residual unconjugated antibody does not have a 310 nm absorption. After the initial binding of crude conjugate with the column, a wash with a low phosphate concentration elutes unconjugated antibody, followed by a step wash with buffer solution containing no phosphate that elutes the product. Late eluting fractions contain more highly conjugated species, which may be included depending on their effect on the drug/antibody ratio. Figure 10. Lab-Scale HIC Chromatogram

Scale up of the column to manufacturing scale was accomplished by keeping the column height constant and increasing the cross-sectional area proportional to the reaction amount, while maintaining constant linear velocity. All other operations and buffers within the chromatography step were kept constant. Based on the comparability between scales of the UV traces, yield and quality attributes of the product, the chromatography step was shown to be scalable and robust.


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Final Ultrafiltration/Diafiltration and Filtration of Drug Substance The primary purposes of the ultrafiltration/diafiltration (UF/DF) step are to adjust the concentration and to replace the chromatography buffer with the drug substance buffer. UF/DF systems use pressure to force small molecule buffer components and water through a semipermeable membrane to concentrate the solution (UF phase of process) and buffer-exchange the product at constant volume (DF phase). The InO UF/DF process uses a stainless steel skid, however the retentate vessel is a disposable bag system that was designed to provide adequate mixing during the process. Dye mixing studies were performed on the production scale bag to confirm that there were no unmixed regions during the process. In addition, the dye experiment was used to determine the optimal positions of the return and permeate lines, since the return and permeate flow is used to augment the mixing. The UF/DF operation was directly scaled from laboratory scale models using the same membrane material. Normal flow filtration is performed after each process step to reduce the level of particulates. Vmax and Pmax studies10 were performed at small scale to determine the required filter area at manufacturing scale for each of the process steps. Confirmation of filter area was determined during scale-up runs. Since this filtration is performed during transfer from one single-use bioprocess container (BPC) to another single-use BPC, a disposable peristaltic pump system was used.

Supply of Drug Substance Intermediates For ADCs, there are two drug substance intermediates (DSIs): the monoclonal antibody (mAb), which is a biologic, and the linker/payload, which is typically a small molecule. Planning for the supply of these two very different intermediates is essential for ADC manufacturing, and each presents its own challenges. Manufacturing of mAbs in the pharmaceutical industry has a 30-year history going back to the approval of Orthoclone OKT 3 in 1986, the first approved monoclonal antibody.11 Since then, processes for the production of mAbs have been improved and optimized and there is an in-depth knowledge of these processes. However, important attributes of the mAb used as a DSI en route to an ADC may be different from those when the mAb is the drug substance. One example of such an attribute is the level of aggregates; a higher threshold may be acceptable for a mAb DSI, since further aggregate removal may be achieved by the ADC process. The attributes required for the mAb must be evaluated for each ADC. Another difference for mAbs manufactured as DSIs is the scale of production. ADCs are potent drugs and are typically administered at lower doses than therapeutic antibodies. Hence mAb DSIs may require facilities and scales of operations that are smaller than for therapeutic mAbs or the campaigns can be significantly shorter. The linker/payloads (L/P) for most ADCs currently in development are semisynthetic or fully synthetic small molecules, and therefore have regulatory requirements that differ from a biologic2. Furthermore, the payload is generally a complex molecule, relative to many drugs, and has a correspondingly challenging manufacturing process. And lastly, the L/P typically contains a cytotoxic drug, which requires special facilities and handling. The manufacturer must have the capability to handle high potency active pharmaceutical ingredients (APIs).


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For InO, the activated calicheamicin derivative L/P is manufactured in a built-for-purpose facility designed to conduct fermentation, isolation, and synthetic modification of calicheamicin (see “Handling of Cytotoxic Materials” below). Figure 11 shows the steps for manufacturing of activated calicheamicin derivative. 9, 12 Activated calicheamicin derivative is purified by reverse-phase high performance liquid chromatography (RP-HPLC). RP-HPLC provides highly efficient separation of the product from impurities, and is coupled with an in-process assay for fraction purity. This purification step therefore allows tight control of impurities in the DSI. Although the activated ester in activated calicheamicin derivative is susceptible to hydrolysis under the aqueous/organic conditions of RPHPLC, hydrolysis can be controlled by HPLC mobile phase pH and therefore minimized. Residual solvents are controlled by precipitation and vacuum drying steps. The sequence of RPHPLC followed by precipitation/drying affords DSI with suitably low impurity and residual solvent content for the ADC conjugation process. Figure 11. Manufacturing Steps for Activated Calicheamicin Derivative H3C S

Fermentation Isolation

CH3

I

O

S

Working Cell Bank O H3C HO H3CO

O

O

HO

S

CH3 O

OCH 3 OCH 3

OH

S O CH3 O HN HO O

HN H3CO

OH

NH

O

OCH 3 O H

O

-Calicheamicin

Acetylation O O N O

O

H3C

O

S

Activated Linker Addition

O N CH3 O

CH3

I

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S O H3C HO H3CO

O

HO

N H

OCH3 OCH 3

OH

NH

S O CH3 O HN HO O N H3CO

OH

O

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O H

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H3C HO H3CO

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OH

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NH

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OCH3 O H

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O

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N-Acetyl Calicheamicin

Activated Calicheamicin Derivative

Setting specifications for the L/P that are appropriate for the conjugation process is an important aspect of the L/P process and analytical control strategy development. Once specifications are drafted, the L/P process can be developed to meet those specifications and enable a successful conjugation process. Analogous to small molecule processes, the strength of the L/P is critical to ensure an accurate charge into the conjugation, so appropriate levels of impurities and residual solvents must be achieved. Also important for L/P is the level of conjugatable and nonconjugatable impurities. Conjugatable impurities should be minimized, since these compounds can conjugate to the antibody and become part of the DS. For the case of the activated calicheamicin derivative process, the activated ester of a calicheamicin homolog is formed from a low-level impurity in N-acetyl calicheamicin; this conjugatable impurity is purged by the RP-


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HPLC purification. Non-conjugatable impurities must be removed by the ADC process, and appropriately sensitive assays are needed to monitor the clearance of these compounds. Development and Validation Strategies Process development for the mAb and L/P may be executed in parallel with conjugation process development for the ADC drug substance. Such parallel development efforts must be managed properly and governed by the end goal. Communication between development teams is paramount, since attributes of the mAb and L/P can directly impact the conjugation process. Ideally the development and scale-up of the L/P, mAb and ADC processes are sequenced and timed such that development of all three processes can be performed almost concurrently. Figure 12 shows an overview of the timing and steps for development through validation for the L/P, mAb and ADC. This approach represents the most conservative approach with the least risk. It should be noted that providing L/P that is representative of the commercial process for validation of the ADC process is likely sufficient. Therefore, the timing of the validation of the L/P process can be moved out to occur concurrent with or after the ADC process validation. A requisite for this approach to be successful is the necessity to demonstrate equivalency between material from the final commercial process and that used in validation of the ADC. Following a small molecule paradigm would allow the validation of the L/P process to take place concurrently with the filing of the BLA. Recent filings have for the most part included the process validation package in the BLA. An additional complexity of multicomponent validation is the risk that the antibody or L/P may not have sufficient demonstrated stabilities to support the entire validation program. Furthermore, while maintenance of quality attributes of the components will be demonstrated during their respective stability programs, the control strategy must ensure that the component quality attributes will be maintained throughout the drug substance and drug product lifetimes.

Figure 12. Schematic for Linker/Payload, mAb and Drug Substance Process Development, Evaluation, and Validation


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For InO, the biologics approach for process validation (i.e. validation prior to filing) was also applied to the L/P process, both to align the validation strategy for L/P with that of the mAb and ADC and to facilitate the desired flow shown in Figure 12. Batch Size Batch size and stability are essential process considerations for the supply of DSIs. The ADC conjugation process scale is determined by projected commercial need, and for an ADC, this scale often requires DSI quantities that are below the amount manufactured by the respective DSI processes. To optimize resources and time, it may be desirable to run the DSI processes at a larger scale to prepare supplies of the DSIs for multiple DS batches. The scale for mAb and L/P manufacturing will depend on the process, equipment, and manufacturing facility available, but scale will also be governed by the ADC batch size and the long term stability of the DSIs. For the manufacturing of InO, the typical yield of the mAb process provides sufficient material for three ADC batches and the typical yield of the L/P process is sufficient for five ADC batches. Both mAb and L/P are stable for several years, and the projected number of ADC batches per year will consume both DSIs before their expiry.

Raw Materials For biologics processes, endotoxin and bioburden levels are typically monitored throughout the process, and appropriate limits in raw materials are determined during development. Examples of raw materials that may be tested for endotoxin and bioburden are buffers, excipients, chromatography resins, filters, and processing containers. The amount of heavy metals in raw materials must be controlled so as not to introduce these potentially toxic impurities into the drug substance and drug product. Heavy metals can originate from a wide range of materials, so it is important to carefully assess all materials in the process. Extractables and leachables must be understood and controlled for containers, bags, and tubing used in the process. A variety of impurities can be introduced through product contact and knowledge of potential impurities and their sources will improve process understanding and control. An example of a raw material attribute that is controlled for the L/P is the water content of ethanol used for L/P dissolution. As shown in Figure 11, activated calicheamicin derivative contains an activated ester for reaction with lysine residues on the mAb. These activated esters are susceptible to hydrolysis, and for this reason, the specification for water in ethanol is set to ≤ 0.2%. Prior to conjugation, activated calicheamicin derivative is dissolved in ethanol and this solution is charged into the conjugation reaction. Hydrolysis of the activated ester is detrimental to the conjugation reaction since the L/P will not react once it is hydrolyzed. To minimize hydrolysis, water in ethanol used for dissolution is tightly controlled by the raw material specification.


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It is important for the development team to evaluate the required attributes of each raw material and set specifications accordingly. Compendial specifications in force during the development phase may, or may not, be appropriate. However, since compendial specifications are subject to change, the requirements of the process are used to determine raw material specifications.

Handling of Cytotoxic Materials The proper handling of cytotoxic materials is paramount for operator safety6. Standard operating procedures (SOPs) must be in place for receipt, shipping, handling, and storage of cytotoxic compounds. Properly qualified equipment must be available for handling and storage of cytotoxic compounds, such as isolators/glove boxes for weighing and preparing solutions. Appropriate personal protective equipment (PPE) is essential, and PPE requirements may be different in each area of the manufacturing facility. Areas where cytotoxic compounds are handled and stored may require restricted access, and proper signage must be posted and maintained to alert all personnel entering these areas. Activated calicheamicin derivative is manufactured in a built-for-purpose facility that was designed to produce, handle, and store calicheamicin products. The fermentation and -calicheamicin isolation operations are conducted in closed systems, between which materials are transferred with no operator contact. All synthetic steps in the manufacture of activated calicheamicin derivative are conducted inside large isolators (glove boxes) constructed to both isolate operators from calicheamicin contact and allow for ease of manipulation. Lastly, drug substance manufacturing, where activated calicheamicin derivative is conjugated to the antibody and the ADC is purified, is conducted in a dedicated suite that facilitates safe charging of the L/P solution into the conjugation reaction and handling of the ADC through purification and UF/DF operations. The drug substance is then frozen and stored until progressing to drug product. For the InO process, drug product is produced in a separate suite in the same facility as calicheamicin and drug substance. This arrangement allows the entire process, from raw materials to sealed vial, to occur in the same building and helps to ensure proper handling and control of the cytotoxic materials.

Analytical Challenges for Late Stage Development There are multiple analytical activities occurring during late stage ADC development which may include the following: • • • • • • • •

determination of criticality of quality attributes development and validation of all the assays for release for critical quality attributes development of additional characterization methods for attributes important to product and process understanding analytical testing support for process characterization establishment and characterization of reference materials release and stability testing for clinical materials comparability studies for potential process and manufacturing site changes development/validation of in-process assays for critical process steps


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• •

analytical testing support for residual clearance for process validation determination of analytical control strategies

In addition, for ADCs, similar analytical activities need to be performed for both DSIs (mAb and L/P) as well as the ADC drug substance and drug product. In this section, the discussion will be focused on analytical strategies and challenges for late stage conjugation process support. There are several considerations for analytical support to late stage process development of ADCs. One consideration is that methods for determination of attributes important for both process and product understanding need to be established early. Methods from early stage development may need to be further evaluated to ensure suitability and robustness to support the late stage development. Additional methods and control may be needed to support the enhanced process/product understanding. Potential quality attributes for a typical ADC may include size heterogeneity (such as aggregation and fragmentation), charge variants, drug to antibody ratio (DAR), protein concentration and conjugated drug concentration, drug load distribution, sites of conjugation and occupancy (for lysine ADCs), and residual drug related species. For InO, since it is a calicheamicin based ADC, special precautions need to be taken during sample storage, preparation and analysis to prevent the un-intended degradation of drug due to the existence of an acid labile linker and potential photosensitivity of calicheamicin. Another consideration is to assess the impact of conjugation on those critical quality attributes in mAbs which may impact binding to the antigen. If there is a potential “hot spot” for deamidation in the CDR region where the antigen-antibody binding occurs, a method for measuring deamidation in the mAb/ADC needs to be developed to monitor the impact of the conjugation and purification process on this particular quality attribute. In the case of InO, it was determined that the conjugation and purification process does not have a significant impact on deamidation, so the control strategy reflects that deamidation is controlled at the mAb DSI stage, not on the ADC. One of the often encountered challenges for process support is that methods used for the final drug substance testing may not be applicable to in-process sample testing, as in-process samples are typically more complex due to different sample matrices and the existence of multiple components. For example, for DAR determination in the conjugation reaction mixture, the release UV method is not applicable as the free drug in the reaction mixture will interfere with the determination of conjugated drug concentration. This can result in an overestimation of DAR. Either a different method needs to be developed, or a modified method with subtraction of the free drug will be used. In other cases, matrix interference needs to be evaluated. Often spike/recovery studies need to be performed to ensure that the method is suitable as a quantitative assay. For methods which are sensitive to initial sample buffer conditions such as iCE, ion-exchange HPLC and HIC, sample preparation procedures may need to be adjusted to ensure that the method is still capable of resolving and accurately quantitating species of interest. In the InO case, the iCE method involves dilution of the original sample to reduce salt concentration to the level which can be tolerated. Another common strategy for process support is to use analytical methods that are different from the release methods, but to ensure equivalency through bridging studies. For InO process characterization studies, an ELISA assay was used for residual drug determination in the final drug substance and drug product. However, ELISA assays suffer from some limitations: more


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assay variability compared to a typical LC method, tedious procedures, and maintenance of critical reagents. For process characterization studies, an RP-HPLC assay was developed to determine the residual drug, in place of the ELISA assay. A spike/recovery at the HPLC limit of quantitation (LOQ) level was performed to demonstrate similar assay performance and to bridge the two methods. Additional methods may be needed to measure additional quality attributes which were not determined at the early stages of process development. For InO there was not an assay for drug loading profile at the earlier stage of development. Therefore, for process characterization studies both an iCE method and a HIC method were used to monitor the drug loading profile. In addition, peptide mapping was used to monitor the sites of conjugation. Another point worth noting is that not all of the attributes need to be tested for each in-process or process characterization sample. Particularly for process characterization studies, where process parameter ranges are not very wide, biological assays (including cytotoxicity) are typically not performed since these results may not be meaningful due to assay variability. To support process validation, all critical in-process assays need to be validated. In addition, suitable methods need to be developed to demonstrate clearance for all relevant process impurities, such as residual L/P, process buffers and residual organic solvents. This work is typically performed in conjunction with the process validation, using the materials generated from different stages of the process, to show clearance at each stage. Some of the process impurity methods can be challenging due to the requirement for very low assay LOQ in the presence of large amounts of protein conjugates. Often, different analytical techniques are needed for quantifying residual impurities. For example, for residual solvent in the ADC, a GC method with head-space sampler and an FID detector is typically needed for quantification. For some process impurities, detectors other than UV (such as ELSD, MS) are often utilized.

SUMMARY Several activities have contributed to the successful advancement of the inotuzumab ozogamicin program to late stage development. Key among these are the development of a control strategy that focuses on control of quality attributes from raw materials through drug product, attention to proper scaling of unit operations, detailed understanding of the product through application of appropriate analytical tools, and studies of the effect of process parameters on quality attributes. In addition, planning for the complex supply chain and facility requirements for handling cytotoxic materials has been crucial. This ADC is particularly complex, but the approaches used are applicable universally. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]


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ACKNOWLEDGEMENTS We acknowledge the following individuals who made great contribution to the Inotuzumab Ozogamicin project described herein: Ryan Manetta, Wesley Swanson, Vimal Patel, Nataliya Bazhina, Chunchun Zhang, Lawrence Chen, Julius Lagliva, Bhumit Patel, Eric Jin, Sen Zhang, Tok Han, Sonal Shah, and David Merkoolof.

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