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ArticleCite This: ACS Omega 2019, 4, 20564−20570

http://pubs.acs.org/journal/acsodf

Good Manufacturing Practice Strategy for Antibody−Drug
Conjugate Synthesis Using Site-Speciﬁc Chemical Conjugation: FirstGeneration AJICAP
Yutaka Matsuda, Colin Clancy, Zhala Tawﬁq, Veronica Robles, and Brian A. Mendelsohn*
Ajinomoto Bio-Pharma Services, 11040 Roselle Street, San Diego, California 92121, United States

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S Supporting Information*

ABSTRACT: The development of antibody−drug conjugates (ADCs) is in great demand in the oncology ﬁeld. With
the goal of maximizing the therapeutic index, the conjugation
technology to produce ADCs has been shifted to a sitespeciﬁc manner; however, it is still challenging to establish
robust and scalable synthetic processes. We have developed achemical conjugation platform termed AJICAP for sitespeciﬁc ADC synthesis using IgG Fc-aﬃnity peptides. Here,
we report the preparation of site-speciﬁc ADCs based on ﬁrstgeneration AJICAP technology for use in good laboratory
practice studies. Analysis of the ﬁnal ADC product was
conducted using validated systems and good manufacturing practice. This work may not only prompt further biological studies
of AJICAP-ADC but also establish a strategy to provide well-documented manufacturing data to enable new drug application
ﬁlings (e.g., investigational new drug applications) for site-speciﬁc ADCs.

■INTRODUCTION
Industrial research and development activity for antibody−
drug conjugates (ADCs) has rapidly increased in recent years.
Five ADCs have been approved for clinical use by the Foodand Drug Administration (FDA): brentuximab vedotin(Adcetris), trastuzumab emtansine (T-DM1, Kadcyla),1−3
gemtuzumab ozogamicin (Mylotarg), inotuzumab ozogamicin(Besponsa), and very recently, polatuzumab vedotin (Polivy).4
To date, more than 80 ADCs are in clinical development.5,6
Current ADCs on the market have a stochastic distribution ofcytotoxic drugs linked across several diﬀerent sites of theantibody.7−9 This heterogeneous conjugation manner cancause diminished eﬃcacy and/or increased toxicity compared
to a homogeneous ADC. Hence, the therapeutic index ofheterogeneous ADCs is limited.10
The development of site-speciﬁc conjugation has become auseful technology in the ADC ﬁeld to overcome the limitationsof traditional ADCs.11 However, to date, no site-speciﬁc ADCs
have been commercially approved by the FDA. Thus, thedevelopment of scalable and robust ADC processes is still ahighly challenging task for drug developers and contractdevelopment and manufacturing organizations (CDMOs).12
We have developed direct chemical site-speciﬁc conjugation
technology for intact native antibody modiﬁcation using Fcaﬃnity compounds (Scheme 1).13,14 The ﬁrst-generation
iteration of this technology, termed AJICAP, has alreadyundergone initial process development, including the gramscale synthesis of site-speciﬁc ADCs,15 biological evaluations,14,15 and the establishment of appropriate analyticalmethods.16 These promising results, showing the scalability© 2019 American Chemical Society

and high cytotoxic eﬃcacy of site-speciﬁc ADCs produced by
AJICAP technology, prompted our group to attempt theapplication of AJICAP technology to relevant manufacturing
production scales. Herein, we report our recent eﬀorts to
produce AJICAP-ADCs appropriate for the speciﬁc research or
preclinical phase for use in good laboratory practice (GLP)
studies. The ADC synthetic approach described herein ismodeled as a technical transfer from the research and processdevelopment stages to clinical and commercial manufacturing
scales and methods. To begin, a foundational approach basedon a good manufacturing practice (GMP) strategy was used toadapt traditional ADC GMP production for site-speciﬁc ADC
production. Using this strategy, site-speciﬁc ADCs were
produced on a gram scale synthesized using AJICAP
technology. Analysis of the resulting ADCs was conductedby qualiﬁed analysts using validated equipment.
These studies demonstrate the reproducibility and robustness of the AJICAP technology to produce next-generation
ADCs, and we believe that our approach to producing sitespeciﬁc ADCs suitable for use in GLP studies, based on a
strategic assessment of appropriate regulatory frameworks, canserve as a model for others as they develop their own
approaches to the manufacture of materials for use in studies
that support clinical regulatory ﬁlings.
Received: July 30, 2019
Accepted: October 30, 2019
Published: November 26, 2019
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ArticleScheme 1. AJICAP Technology Overview■

including good documentation practices.”22 Implementation ofcGMP requirements can be resource-prohibitive for early
development activities. One of the areas where early
development is lacking is in the presence of an independent
Quality Management System (QMS) to provide oversight ofthe project. Per the ICH, “The elements of [a Pharmaceutical
Quality Management System] should be applied in a mannerthat is appropriate and proportionate to each of the productlifecycle stages, recognizing the diﬀerences among, and thediﬀerent goals of each stage.”23 The inclusion of a QMS should
be gradually applied to product development to ensure greater
eﬃciency and a seamless transition from early development tolate-phase clinical and commercial operations.
Our intent is to use phase-appropriate cGMP processes to
produce a material that is suitable for use in GLP studies.
Producing these GLP-ready materials with solid documentation packages will enable the ﬁling of investigational new drug
applications (INDs) and eventually new drug applications(NDAs). Documentation of early-phase development work is
key to the success of any development project.
An emerging area of focus in the current regulatoryenvironment is data integrity, which refers to the completeness,consistency, and accuracy of data. The minimum requirements
for data integrity include the fact that all data documented forcGMP activities should be attributable, legible, contemporaneously recorded, original, and accurate (ALCOA).24 Early
implementation of systems to comply with data integrity
standards ensures any information included in regulatory
submission meets data integrity requirements and will not
result in deﬁciencies in the submitted data.
FDA regulations for GLP studies provide guidance for
conducting nonclinical laboratory studies to support newpharmaceutical product applications, such as an IND or
NDA.19 The majority of the guidance is focused on executionof the study itself and does not elaborate on the methods orprocesses needed to produce the test articles that supply thestudy. The requirements stated in the regulations for test
article characterization require identity, strength, purity,
compositions, or other characteristics to be documented andthe methods of production to be documented.19
Ajinomoto Bio-Pharma Services (Aji Bio-Pharma) has
developed an internal procedure to ensure that productdevelopment of a client/sponsor material is included within
the overall QMS and in a regulatory framework to allow for
appropriate data integrity. The requirements of this procedure
were developed from regulatory guidance documents andrepresent the implementation of a regulatory framework to
support early product and process development. Materials

RESULTS AND DISCUSSION
Approaches to the implementation of Current Good
Manufacturing Practice (cGMP) principles in early development activities vary widely across the entities conducting thesestudies. While mature quality systems are in place for larger,
established pharmaceutical companies, the majority of thesmaller, independent, and research-oriented organizations
currently lack these controls. What follows is the developmentof a process for the production of materials suitable for use inearly development and GLP preclinical studies used to support
regulatory ﬁlings for pharmaceutical products. This process
was developed using guidance from multiple regulatory
agencies to develop a phase-appropriate cGMP process,which will help ensure acceptance of these ﬁlings withregulatory agencies.
Regulations regarding the manufacture of pharmaceuticalproducts for late-phase clinical and commercial use are wellestablished by regulatory agencies, including the FDA cGMP
Regulations17 and European Union GMP Annex 1.18 Guidance
for the manufacture of preclinical materials to support GLP
studies,19 however, is less well deﬁned, which can lead tocomplications when entities move into early- and late-phasemanufacturing where the regulatory requirements ramp up to
full cGMP. All activities associated with development ofpharmaceutical products are ultimately included in any
submission for commercial approval by regulatory agencies,
and a lack of early application of a GMP strategy in early
product development can be an area of weakness in theseﬁlings.
As outlined in the International Conference on Harmonisation of Technical Requirements for Registration of
Pharmaceuticals for Human Use (ICH), “The aim ofpharmaceutical development is to design a quality productand its manufacturing process to consistently deliver theintended performance of the product”,20 and “The goal of
manufacturing process development for the drug substance isto establish a commercial manufacturing process capable ofconsistently producing drug substance of the intended
quality.”21 It is critical that the early development work is
documented to ensure that the required data is able to be
included in submissions to support regulatory approval.
This does not mean that all early development work must becompleted according to cGMP standards; as explained by the
FDA Guidance Documents for Process Validation, “Generally,
early process design experiments do not need to be performed
under the cGMP conditions required for drugs intended forcommercial distribution. They should, however, be conductedin accordance with sound scientiﬁc methods and principles,
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ArticleScheme 2. Gram-Scale Synthesis of Trastuzumab-AJICAP-MMAE (5)Manufacturing Controls. All activities, including manufacturing data, equipment use, processing steps, and deviations,are documented in an electronic laboratory notebook (ELN)system. All manufacturing-related studies to be executed
require a protocol that is subject to pre-approval by the PD
owner and quality unit. This protocol describes the intent andpurpose of the study and includes a detailed batch record todeﬁne the process steps necessary to complete the manufacturing activity. The batch record includes supporting information,
including a bill of materials, list of equipment, in-processcontrol testing, and required sampling steps. During executionof the protocol batch record, each step is signed oﬀ by theoperator performing the activity and veriﬁed by a second
operator in accordance with GMP principles.17 Any deviationfrom the pre-approved batch record is recorded and justiﬁed toensure that it does not have an adverse impact on theformulation.17 Once execution is complete, the batch record is
subject to review by the PD owner and quality unit to address
any documentation deﬁciencies or deviations. Operations anddocumentation related to these manufacturing processes are
subject to an internal audit program administered by thequality unit.
Laboratory Controls. The testing and analysis of thedevelopmental material are executed using analytical methods
demonstrated to be suitable for their intended use. Method
qualiﬁcation and validation are generally required for the
analysis of GMP material to ensure that the performance of thetest method is appropriate for the test article.25 Analyticalmethods used to support developmental formulations do not
require method qualiﬁcation or validation; however, the test
method should be proceduralized prior to use to ensure thatthe test execution is as intended.
Samples are taken at deﬁned steps in the process and areentered into a laboratory information management system totrack the lifecycle of the sample. All analyses of test materials
are performed using GMP-compliant equipment and reagents.
Data generated during analysis is reviewed by the laboratory
personnel and quality unit to ensure adherence to the study
protocols. Given the developmental nature of these studies,most materials do not have predeﬁned acceptance criteria tocompare with the analytical results. Data is then assembled
into a certiﬁcate of testing (COT), which is reviewed by the

produced using this approach are acceptable for use in GLP
preclinical studies used to support pharmaceutical development eﬀorts.
Facility and Equipment. All activities related to development formulations are conducted in the Process Development(PD) laboratory. The PD laboratory is located in a segregatedarea of a GMP manufacturing facility to ensure no crosscontamination of test articles with other GMP materials.
Segregation of the PD laboratory is achieved using dedicatedutilities, including heating, ventilation, and air conditioning(HVAC); deionized water; required gases; separate accesscontrolled entry; and personal protective equipment.
Equipment used in the production of development
formulations is not subject to the full requirements of GMP
qualiﬁcation and validation; however, all equipment undergoes
routine preventive maintenance (PM) and calibration toensure adequate operation. Analytical equipment used for the
analysis of development formulations is maintained as GMPequipment with supporting qualiﬁcations, including installationand operational qualiﬁcation (IOQ), documented PM andcalibration schedules and written procedures for operation andmaintenance.
Laboratory manufacturing equipment and materials are
single use and disposable wherever possible to eliminate therisk of cross-contamination. Where single use is not possible,
materials are product-dedicated.
Control of Raw Materials. All raw materials (RMs) andexcipients used in the manufacture of development formulations are classiﬁed as GMP materials. GMP materials follow aquarantine, sampling, testing, and quality unit release process
to ensure compliance with approved product speciﬁcations,which deﬁne identity, strength, purity, and quality characteristics.17 Lot release packets are maintained by the quality unit
for a designated record retention timeframe after release. Test
articles often do not have pre-established criteria to allow for
the creation of product speciﬁcation to support the GMP RM
process described above. Qualiﬁcation and release of such test
articles are at the discretion of the product sponsor, who
assumes responsibility for ensuring that the materials meet therequirements of the desired study. At a minimum, an identity
test is performed upon receipt of the test article to ensure that
it is the correct material for the protocol.
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ArticleFigure 1. HIC analysis for IPC: (a) trastuzumab; (b) trastuzumab conjugated to peptides (2); (c) linker cleavage compounds (3); (d) site-speciﬁc,thiol-modiﬁed product (4); and (e) trastuzumab-AJICAP-MMAE (5).

Figure 2. RP-HPLC analysis for IPC: (a) trastuzumab; (b) trastuzumab conjugated to peptides (2); (c) linker cleavage compounds (3); (d) sitespeciﬁc, thiol-modiﬁed product (4); and (e) trastuzumab-AJICAP-MMAE (5).

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Articlewith one thiol-containing group, which was derived from thepeptide reagent. For RP-HPLC, all samples were pretreatedwith DL-dithiothreitol (DTT) to cleave the disulﬁde bonds.
This reduction also caused cleavage of the linkage between the
peptide moiety and trastuzumab in compound 2. As a result of
this cleavage, the heavy chain modiﬁed by the newly formed
thiol was observed in the chromatogram. Additionally, twopeaks (retention times, 8.3 and 11.7 min) matched theretention time for reduced trastuzumab. From these results, theheavy-chain selectivity of the conjugation of peptide 1 totrastuzumab was conﬁrmed, showing the compatibility of this
synthesis with the reactor vessel system with stirring.
After tangential ﬂow ﬁltration (TFF) puriﬁcation, compound 2 was treated with tris(2-carboxyethyl)phosphinehydrochloride (TCEP) under previously optimized conditions
using slightly acidic buﬀer.15 Compound 3 was eluted with aretention time close to that of the naked antibody in the HIC
chromatogram, indicating that the peptide moieties had been
removed from compound 2 (Figure 1c). Theoretically,
compound 3 should retain the internal noncovalentinteractions holding the complex together, even if all thecovalent interchain disulﬁde bonds are cleaved; therefore, theoverall surface hydrophobicity of compound 3 should notchange signiﬁcantly, and this is indeed reﬂected in theretention time in the HIC chromatogram. RP-HPLC was notuseful for the IPC analysis of this reaction because the RPHPLC chromatogram of compound 3 was identical to that ofcompound 2 (Figure 2c). Therefore, we tested another IPC
analytical method to support the HIC result. Ellman’s assay ofcompound 3 showed an average of 10.2 free sulfhydryl groups
per antibody, indicating that all the interchain disulﬁde bonds
and the linkage between the peptide and antibody were cleaved
to form free thiol groups (Table S2, Supporting Information).
Puriﬁcation by the TFF system gave puriﬁed 3, which wasconverted to compound 4 by exposure to mild oxidative
conditions. For the monitoring of this reaction, Ellman’s assay
was a useful method of analysis, showing that there was anaverage of 1.87 free sulfhydryl groups per antibody (Table S3,
Supporting Information). HIC and RP-HPLC were not viableoptions to analyze this reaction step because the retention timeof compound 4 did not shift from that of compound 3 in either
chromatogram.
Conjugation of MC-VC-MMAE with compound 3 waseasily monitored by both HIC and RP-HPLC due to thehydrophobicity of this drug linker. Both chromatograms
matched with previous studies [drug antibody ratio (DAR) =1.6], and 1.7 g of trastuzumab-AJICAP-MMAE (5) was
obtained after TFF puriﬁcation.28,29
In addition to reaction monitoring, the RP-HPLC results
described in Figure 2 indicate the heavy-chain speciﬁcity of
AJICAP conjugation as no light-chain conjugates wereobserved in the RP-HPLC chromatogram. In our previous
report, a peptide mapping analysis of intermediate 4 (that iscorresponding to the precursor of ﬁnal ADC 5) indicated that
AJICAP technology can perform in a site-speciﬁc manner tofunctionalize a single lysine in the Fc region of an antibody.14
These results support the conclusion that trastuzumabAJICAP-MMAE (5) retains site-speciﬁc conjugation. Further
analytical investigations including peptide mapping of 5 are
now underway.

PD owner and quality unit, and used to document acceptanceof the material produced in the study and release of thematerial to the project sponsor.
Packaging, Labeling, and Distribution. Packaging andlabeling, including sample aliquoting, container closure, andsecondary packaging of the test material, are at the discretionof the sponsor. The minimal labeling requirements include a
product description, batch number, date of manufacture,storage conditions, and a “Not for Human Use” statement.
Release of the material is documented on the COT, whichaccompanies the shipment to the sponsor.
Record Keeping. All documentation, including the preapproved protocol, executed batch record, analytical raw data,
and reports and COT associated with the manufacture of thetest material, is maintained in the ELN system indeﬁnitely.
This system is compliant with 21 CFR Part 11 requirements
for electronic records to ensure that the documents areavailable for further review.26
Antibody−Drug Conjugate Synthesis Using SiteSpeciﬁc Chemical Conjugation. Next, we applied this
GMP strategy for AJICAP-ADC synthesis (Scheme 2). From aprevious gram-scale ADC synthesis,15 we optimized several
processes aimed at future manufacturing. First, to check thecompletion of each reaction, characteristic IPC analysis wascompleted. All reactions, including peptide conjugation, wereconducted in glass reactor vessels. This stirring system is awell-known technique, which enables the later application ofthis synthesis to the larger batch vessels that are often used for
kilogram-scale ADC preparation in a manufacturing facility.27
To the best of our knowledge, the chemical modiﬁcation ofproteins by an aﬃnity reagent using a glass reactor vial andstirrer has not been reported, and there was concern that thismixing methodology may trigger undesired nonspeciﬁc
conjugation or reduce the quality of the proteinaceous material
produced.
Peptide reagent 1 was added to a trastuzumab solution, andthis reaction mixture was stirred using the Chemglass system.
IPC analysis of this reaction was conducted using several
analytical techniques. Hydrophobic interaction chromatography HPLC (HIC-HPLC), which is well known as the goldstandard method for the analysis of antibody-related products,
provided clear visual results (Figure 1a,b). The retention timeof the trastuzumab−peptide conjugate (2, Figure 1b) was
longer compared with that of trastuzumab (Figure 1a),indicating that the peptide conjugation was almost complete
within 1 h and trastuzumab modiﬁed by two peptides was the
major product. However, two additional peaks were also
observed in the HIC chromatogram. The smallest peak(retention time, 8.0 min) was attributed to trastuzumab
conjugated with one extra peptide, implying that an undesiredside reaction had occurred. The other peak, with a retention
time of 6.3 min, was assigned as trastuzumab conjugated with
only one peptide. This HIC result was identical to the previous
results,14 and quadrupole time-of-ﬂight mass spectrometry (QTOF MS) analysis of compound 2 after puriﬁcation also
supported this observation (Figure S16, Supporting Information).
Reverse-phase HPLC (RP-HPLC) is also commonly used
for IPC analysis; in particular, the site selectivity of AJICAP
conjugation was previously conﬁrmed by this methodology(Figure 2).16 The RP-HPLC chromatograms of reduced 2
showed three peaks (Figure 2b). The highest peak (retention
time, 12.0 min) corresponded to the heavy chain conjugated20568

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ArticleCONCLUSIONSA GMP strategy, including the regulatory framework andguidance, was established for use in GLP preclinical supply of amaterial. For the demonstration, AJICAP-ADC synthesis wasachieved. All reactions were conducted in appropriate scalable
glass reactor vessels, enabling future scale-up in a manufacturing facility. Several IPC analytical methods based on thecompound or intermediate characteristics were also conducted.
The total product yield for the four steps was increased to 90%
from a previous study, and ﬁnally, 1.72 g of trastuzumabAJICAP-MMAE was obtained. These results demonstrated the
reproducibility and robustness of AJICAP synthesis, enabling
its application for future manufacturing.30 The establishmentof our strategy to produce materials suitable for GLP studies
and incorporation in relevant regulatory ﬁlings is based on acarefully considered review of relevant regulatory guidance andmanufacturing logic and can serve as a model for future
biomolecular pharmaceutical development.

mM EDTA, pH 7.4) as DF buﬀer at an antibody concentrationof 6.6 mg/mL to aﬀord linker cleavage product 3 (1.79 g, 96%
yield) in conjugation buﬀer.
Experimental Procedure for Reoxidation (Step 3 of
AJICAP Technology). To a solution of linker cleavage
product 3 (6.6 mg/mL, 1.79 g) in conjugation buﬀer wasadded a 50 mM dimethyl sulfoxide solution of DHAA (40equiv, 8.60 mL), and the mixture was incubated at room
temperature. After 3 h, a small amount of reaction mixture (0.5mL) was sampled for IPC analysis. After checking the IPC
analysis, the reaction mixture was puriﬁed by a TFF systemusing a Sartocon Slice 200 ECO Hydrosart membrane (30kDa; Sartorius) and conjugation buﬀer as DF buﬀer at an
antibody concentration of 6.8 mg/mL to aﬀord linker
reoxidation product 4 (1.78 g, 100% yield) in conjugation
buﬀer.
Experimental Procedure for Payload Conjugation(Step 4 of AJICAP Technology). To a solution ofreoxidation product 4 (6.8 mg/mL, 1.78 g) in conjugation
buﬀer were added dimethylacetamide (DMA) (11.5 mL) and a
10 mM DMA solution of MC-VC-MMAE (10 equiv, 13.3mL), and the mixture was incubated at 20 °C. After 2 h, a small
amount of the reaction mixture (0.5 mL) was sampled for IPC
analysis. After checking the IPC analysis, the reaction mixture
was quenched by an excess amount of a 50 mM aqueous
solution of N-acetyl cysteine and incubated at 25 °C for 15
min. This reaction mixture was puriﬁed by a TFF system usinga Sartocon Slice 200 ECO Hydrosart membrane (30 kDa;
Sartorius) and conjugation buﬀer as DF buﬀer at an antibody
concentration of 20 mg/mL. Next, the buﬀer exchange of thissolution was conducted by a TFF system using a Sartocon
Slice 200 ECO Hydrosart membrane (30 kDa; Sartorius) andformulation buﬀer as DF buﬀer at an antibody concentrationof 6.8 mg/mL to aﬀord trastuzumab-AJICAP-MMAE 5 (1.73
g, 95% yield) in formulation buﬀer.
General Procedure for IPC. Gel ﬁltration of several
reaction mixtures was conducted for removal of impurities toobtain a puriﬁed protein, which was used for several analyses.
Instruments/Analytical Method. The concentration ofproteins was determined by the Slope Spectroscopy methodwith a Solo-VPE system.14
HIC-HPLC and RP-HPLC analysis were performed aspreviously reported.16
Q-TOF MS analysis was performed as previously reported.14■

EXPERIMENTAL SECTION
Materials. Human IgG1 trastuzumab (Herceptin) was
purchased from Roche Pharmaceutical Company (Switzerland). Maleimide-C6-valine-citrulline-monomethyl auristatin E(CAS#: 646502-53-6; MC-VC-MMAE) was purchased fromNJ Biopharmaceuticals LLC (USA). Peptide reagent 1 wasprovided from Ajinomoto Co., Inc. All other chemical reagentswere purchased from Sigma-Aldrich (USA).
Experimental Procedure for Peptide Conjugation(Step 1 of AJICAP Technology). To a solution of
trastuzumab (10 mg/mL, 1.90 g) in 10 mM AcONa buﬀer(pH 5.5) was added a 22 mM dimethylformamide (DMF)
solution of peptide reagent 1 (9 equiv, 5.15 mL) and stirred at
25 °C using the Chemglass system. After 1 h, a small amountof reaction mixture (0.5 mL) was sampled for IPC analysis.
After checking the IPC analysis, the reaction mixture was
puriﬁed by a TFF system using a Sartocon Slice 200 ECO
Hydrosart membrane (30 kDa; Sartorius) and 10 mM AcONa
buﬀer (pH 5.5) as diaﬁltration (DF) buﬀer at an antibody
concentration of 20 mg/mL. Next, the buﬀer exchange of thissolution was conducted by a TFF system using a Sartocon
Slice 200 ECO Hydrosart membrane (30 kDa; Sartorius) andformulation buﬀer (20 mM histidine containing 5% trehalose,
pH 5.2) as DF buﬀer at an antibody concentration of 6.9 mg/
mL to aﬀord trastuzumab−peptide conjugate 2 (1.92 g, 98%
yield) in formulation buﬀer.
Experimental Procedure for Linker Cleavage (Step 2of AJICAP Technology). To a solution of trastuzumabpeptide conjugate 2 (6.9 mg/mL, 1.92 g) in formulation buﬀer
was added 0.25 M aqueous solution of ethylenediaminetetraacetic acid (EDTA) (12 equiv, 0.572 mL, pH 7.4), 100 mg/mL
polysorbate 20 (0.0265 mL), and 0.5 M aqueous solution of
TCEP (20 equiv, 0.476 mL) at 37 °C. The resulting mixture
was measured by a pH meter, showing that the ﬁnal pH was
5.2. After 1 h at 37 °C, a small amount of reaction mixture (0.5mL) was sampled for IPC analysis. After checking the IPC
analysis, the reaction mixture was puriﬁed by a TFF systemusing a Sartocon Slice 200 ECO Hydrosart membrane (30kDa; Sartorius) and 10 mM AcONa buﬀer (pH 5.5) as DF
buﬀer at an antibody concentration of 20 mg/mL. Next, thebuﬀer exchange of this solution was conducted by a TFF
system using a Sartocon Slice 200 ECO Hydrosart membrane(30 kDa; Sartorius) and conjugation buﬀer (50 mM PBS, 10■

ASSOCIATED CONTENT

S Supporting Information*

The Supporting Information is available free of charge athttps://pubs.acs.org/doi/10.1021/acsomega.9b02419.
Tables comparing research and development, GMP, andour approach; chromatographic ﬁgures; and DAR
calculation and Ellman’s assay results, as described in
the text (PDF)■

AUTHOR INFORMATION

Corresponding Author*E-mail: Brian.Mendelsohn@US.AjiBio-Pharma.com.
ORCID

Yutaka Matsuda: 0000-0003-2801-4706
Brian A. Mendelsohn: 0000-0002-6339-2377
Notes

The authors declare no competing ﬁnancial interest.
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ArticleChemical Conjugation: AJICAP 1st Generation. Org. Process Res.
Dev. 2019, DOI: 10.1021/acs.oprd.9b00316.
(16) Matsuda, Y.; Robles, V.; Malinao, M. C.; Song, J.; Mendelsohn,
B. A. Comparison of Analytical Methods for Antibody−Drug
Conjugates Produced by Chemical Site-Specific Conjugation: FirstGeneration AJICAP. Anal Chem. 2019, 91, 12724−12732.
(17) Title 21 CFR Parts 210 and 211cGMP in Manufacturing,
Process, Packing, or Holding of Drugs and Finished Pharmaceuticals.
http://academy.gmp-compliance.org/guidemgr/ﬁles/1-1-1.PDF.
(18) Annex 1 Manufacture of Sterile Medicinal Products. EudraLex The
Rules Governing Medicinal Products in the European Union Volume 4
EU Guidelines to Good Manufacturing Practice Medicinal Products for
Human and Veterinary Use. 25 November 2008, https://ec.europa.eu/
health/sites/health/ﬁles/ﬁles/eudralex/vol-4/2008_11_25_gmpan1_en.pdf.
(19) Title 21 CFR Part 58Good Laboratory Practice for NonclinicalLaboratory Studies. 18 February 2004, http://academy.gmpcompliance.org/guidemgr/ﬁles/CFR_2019/CFR-2019-TITLE21VOL1-PART58.PDF.
(20) ICH Harmonized Tripartite Guideline. Pharmaceutical Development. Q8 (R2). Current Step 4 version, August 2009, https://
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(21) ICH Harmonized Tripartite Guideline. Development andManufacture of Drug Substances (Chemical Entities and Biotechnological/Biological Entities). Q11. Current Step 4 version, 1 May 2012,https://database.ich.org/sites/default/ﬁles/Q11_Guideline.pdf.
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(24) FDA Guidance for Industry Data Integrity and Compliance with
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R1__Guideline.pdf.
(26) Title 21 CFR Part 11Electronic Records; Electronic Signatures.
https://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/
CFRSearch.cfm?CFRPart=11&amp;showFR=1.
(27) Stump, B.; Steinmann, J. Conjugation process development andscale-up. Methods Mol. Biol. 2013, 1045, 235−248.
(28) In the case of conjugation with maytansinoid-type drug linker,
DAR = 1.9 ADC was obtained (see ref 14). This DAR diﬀerence
between MMAE and maytansinoid was rationalized by their reactivity
and hydrophobicity. Typically, conjugation with higher hydrophobic
drug linkers gives lower DAR ADCs. See representative example: Xu,Y.; Jiang, G.; Tran, C.; Li, X.; Heibeck, T. H.; Masikat, M. R.; Cai, Q.;
Steiner, A. R.; Sato, A. K.; Hallam, T. J.; Yin, G. RP-HPLC DAR
Characterization of Site-Specific Antibody Drug Conjugates Produced
in a Cell-Free Expression System. Org. Process Res. Dev. 2016, 20,1034−1043.
(29) Our research group has completed optimization studies toimprove DAR, and these advancements will be discussed in the future
literature.
(30) Our research group has reported an initial study of peptide
conjugation and labeling for a variety of IgGs including IgG1, IgG2,
and IgG4 (see ref 14). ADC synthesis from these intermediates is acurrent focus of work in our group.

ACKNOWLEDGMENTS
The authors wish to thank our colleagues from Ajinomoto BioPharma Services, Inc., and Ajinomoto Co., Inc., as follows:
Monica Leung, Shawn Littrell, and William Wittbold fortechnical assistance for the GLP study; Melody Batuyong forreviewing batch records; Samuel Janssen, Wen Chen, andKeenan Moi for technical assistance with the ADC analysis;
Steve Folio, Darwin Richardson, Bert Barbosa, Michiya
Kanzaki, and Tatsuya Okuzumi for many helpful discussion
and suggestions in this study; and Kei Yamada for peptide
reagent supply and many helpful initial studies on AJICAP
technology.

■

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(13) Our research group has completed optimization studies tostreamline the conjugation reaction sequence, and these advancements will be discussed in the future literature. For this reason, we
termed the site-speciﬁc conjugation technology described in thismanuscript as “ﬁrst-generation AJICAP”.
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Gram-Scale Antibody-Drug Conjugate Synthesis by Site-Speciﬁc
20570

DOI: 10.1021/acsomega.9b02419
ACS Omega 2019, 4, 20564−20570