Process Development and Structural Characterization of an Anti-Notch

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Process Development and Structural Characterization of Anti-Notch 3 Antibody Drug Conjugate Durgesh Nadkarni, Qingping Jiang, Olga Friese, Nataliya Bazhina, He Meng, Jianxin Guo, Robert Kutlik, and Jeffry Borgmeyer Org. Process Res. Dev., Just Accepted Manuscript • DOI: 10.1021/acs.oprd.7b00337 • Publication Date (Web): 30 Jan 2018 Downloaded from http://pubs.acs.org on January 30, 2018

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Organic Process Research & Development

Process Development and Structural Characterization of Anti-Notch 3 Antibody Drug Conjugate Durgesh V. Nadkarni*¶, Qingping Jiang†, Olga Friese‡, Nataliya Bazhina¶, He Meng¶┴, Jianxin Guoǁ, Robert Kutlik‡, and Jeffry Borgmeyerǁ ¶

Bioprocess Research and Development, †Analytical Research and Development, Pfizer Inc., 401

N. Middletown Road, Pearl River, NY 10965 ‡

Analytical Research and Development, ǁ Pharmaceutical Research and Development, Pfizer Inc.,

700 Chesterfield Parkway West, Chesterfield, Missouri, 63017.

ACS Paragon Plus Environment

Organic Process Research & Development 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Table of Content Graphic


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ABSTRACT: The development of a process for the preparation of a conventional anti-Notch 3 antibody drug conjugate (ADC) is described. The initial reaction conditions used for the conjugation of an auristatin payload to an anti-Notch 3 monoclonal antibody led to the formation of an ADC mixture with a significant level of aggregates. Further process optimization studies resulted in the identification of reaction conditions for the formation of conjugate with low level of aggregates. The temperature of the antibody reduction step was found to have an impact on the formation of aggregates in the ADC mixture. Differences in the antibody reduction temperatures also caused changes in the distribution of conjugated payload on the ADC species. Stability studies of an anti-Notch 3 ADC prepared by two processes differing in the antibody reduction temperature showed subtle differences in their aggregation propensities. The aggregates produced in the crude ADC reaction mixture could be separated from the desired monomer on the hydroxyapatite column under mild conditions without significantly impacting the average drug loading of the purified ADC.

KEYWORDS: Notch 3, Antibody Drug Conjugate, Cysteine Conjugate, Auristatin ■ INTRODUCTION Antibody drug conjugates (ADCs) are a novel class of targeted anticancer agents that are composed of a monoclonal antibody that is covalently conjugated to a small molecule cytotoxic drug. The conjugates are typically formed by conjugating amino acids such as lysine, glutamine or cysteine on the antibody. Several antibody drug conjugates (ADCs) with a range of conjugation chemistries are currently undergoing evaluation in various phases of clinical trials. To date one cysteine conjugated ADC, brentuximab vedotin (Adcetris) and three lysine conjugated ADCs adotrastuzumab emtansine Kadcyla), gemtuzumab ozogamicin (Mylotarg) and inotuzumab ozogamicin (Besponsa) have received regulatory approval in some countries.1-3 The novel anti-Notch 3 ADC



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1(Figure 1) is targeted for triple negative breast cancer and ovarian cancers.4 The ADC is composed of an IgG1 monoclonal antibody that is conjugated via interchain cysteines, produced after partial reduction of interchain disulfides, to an auristatin containing linker-payload 2.5 The ADC 1 is a mixture of cysteine conjugated species with drug loading potentially ranging from 0 – 8 species (Figure 1). The conjugation of linker-payloads to antibodies can lead to formation of undesired aggregates in the conjugation mixture.6 Several factors such as the amino acid sequence and post-translational modifications of antibodies, hydrophobicity of linker-payloads, conjugation conditions, the site of conjugation, drug loading, and the ADC formulation can impact the formation of aggregates in the conjugation reaction.7 The conventional cysteine conjugation of the auristatin linker-payload 2 to an IgG1 antibody leads to the formation of aggregates under thermally stressed condition.8 Due to possible immunogenicity issues associated with the aggregates, it is important to either minimize their formation during conjugation or develop efficient purification processes for their removal from the conjugation mixture.

1

2


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Figure 1. Chemical structures of ADC 1 and the linker-payload 2. Due to promising early preclinical results, a significant quantity of 1 was required for phase I clinical trials. The process that was used for the preparation of small quantities of preclinical ADC supplies was not suitable for its preparation on larger manufacturing scale. In this paper we report our efforts towards development of the process for preparation of 1 that was amenable for scale up and detailed structural characterization to enable process development.

■ RESULTS AND DISCUSSION The process used for the preparation of 1 in early discovery research during screening studies is shown below (Figure 2).4 Due to significantly larger material needs to support further clinical trials, this process was not suitable for the preparation of 1 for scale up and further product development (Figure 2).

Conjugation Step TCEP Reduction of mAb, pH 7 Conjugation with the linker-payload

Dialysis with phosphate buffer

SEC column purification with phosphate buffer

Formulation

Figure 2. Procedure for preparation of 1 in early discovery research. For early nonclinical studies we initially prepared 1 by using similar TCEP reduction-conjugation chemistry but eliminated the size exclusion chromatographic step. The IgG1 antibody was partially reduced at 37 oC with 2.2 equivalents of TCEP. The partially reduced antibody was mixed with a



DMSO solution of the linker-payload 2 at 25 oC. The conjugation mixture was purified by ultrafiltration-diafiltration to perform buffer exchange and to remove organic solvent and residual free drug related species. Although most of the residual solvent was removed by the UF-DF process, the free drug related species could not be removed completely from the isolated conjugate 1. Attempts to improve clearance of free drug related species by evaluating several membranes and diafiltration conditions proved ineffective. This perhaps was due to the hydrophobic nature of the linker-payload 2. In order to render 2 more hydrophilic, several thiol based quench agents were screened for post-conjugation derivatization of 2 by Michael addition on the maleimide ring. Quenching of the conjugation mixture with L-cysteine resulted in efficient removal of the cysteine adduct through the UF-DF process (Figure 3).9 N-Ac Cysteine quench

µg of free drug species / mg of protein

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Cysteine quench 2-(Dimethylamino)ethanethiol quench

10.00

No Quench

1.00

0.10

0.01 0

5

10 Diavolumes

15

20

25

Figure 3. Impact of quench agents on the clearance profile of the free drug related species during purification of 1 by UF-DF.


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The conjugate material 1 isolated using the process described above contained ~ 4 - 5 % aggregates as determined by SEC assay. Experiments designed to probe the origin of aggregate formation showed that the aggregate formation occurred only after the conjugation with the payload 2 and not during TCEP reduction step. No further change in the aggregation was observed during the quench and UF-DF steps. Though the level of aggregate content in the isolated 1 was acceptable for preclinical testing, further reduction in the aggregate level in the final isolated conjugate material was desirable for clinical trial evaluation. Minimizing Aggregate Formation by Optimization of Conjugation Conditions We sought to evaluate whether aggregate formation in the conjugation reaction could be minimized by optimization of the conjugation conditions. During initial experiments it was observed that the temperature of the conjugation step did not impact aggregate formation, however aggregate formation occurred only after the conjugation with 2. While complete absence of agitation during TCEP reduction step led to increase in the level of aggregation, the rate of agitation of the mixture during TCEP reduction step as well as during the conjugation step did not significantly impact the level of aggregation. A series of designed experiments were carried out to evaluate the conjugation parameters that were suspected to impact the level of aggregation in the conjugation mixture. Three parameters, concentration of the antibody in the conjugation mixture at TCEP reduction step, concentration of DMSO in the conjugation mixture, and temperature of reduction of antibody by TCEP were evaluated (Table 1, details in the supporting information). The conjugation reactions were executed by partial reduction of the antibody solution at varied concentrations with 2.2 equivalents of TCEP solution at defined temperature. The mixture was stirred at that temperature for 90 minutes. The solution was then cooled to 25 oC. A solution of 2 in DMSO was added to the conjugation mixture and the mixture was stirred at 25 oC for 60 minutes. At the end of the



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conjugation, an aliquot of the conjugation mixture was evaluated by SEC and HIC assays for aggregate content and DAR of the crude conjugate. Table 1 Experimental Design, Process Parameters, and Ranges Used in the DoE Process Parameters Antibody Concentration in the Conjugation Mixture (mg/mL) Level of DMSO in the Conjugation Mixture (%, v/v) Temperature of TCEP reduction Step (oC)

Low 3.8

Medium 16.7

High 29.6

5

15

25

5

22.5

40

Figure 4. Impact of the process parameters of DMSO and antibody concentrations at different temperatures on aggregation are depicted in contour plots. The process parameters of the antibody concentration and the temperature of TCEP reduction were found to have maximum impact on the aggregate content of the conjugate mixture. The level of


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aggregates varied within the narrow range (1.0 -5.4 %) for all experimental conditions within the DoE model. Lower aggregate levels were observed at lower reduction temperature and low antibody concentration. To further confirm the impact of reduction temperature on aggregate levels, conjugation experiments were carried out at two different reduction temperatures (5 oC and 37 oC) at the same antibody and DMSO concentrations. The conjugate material 1 prepared from an experiment with 5 oC TCEP reduction temperature (process A) contained lower aggregates (1 %) compared to the aggregate level (6 %) in the ADC 1 obtained from an experiment where the reduction temperature was 37 oC (process B) (Figure 4). The drug loadings (Drug to Antibody Ratio, DAR) for the conjugate materials prepared by both processes A and B were ~ 4 by the HIC assay (Figure 5). The identity of individual species in the HIC assay was confirmed by their separation and isolation by analytical HIC chromatography. The individual ADC species were further characterized by native size-exclusion chromatography coupled to mass spectrometry (SEC/MS) (Supporting information).




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Figure 5. a. SEC chromatograph of 1 (process A), b. SEC chromatograph of 1 (process B), c. HIC chromatograph of 1 (process A), DAR ~ 3.7, d. HIC chromatograph of 1 (process B), DAR ~ 4.2. Comparability Assessment of ADC 1 Prepared by Process A and Process B The ADC 1 products prepared by both processes A and B from above experiments were structurally characterized further by mass spectroscopy (MS) and other biophysical techniques to confirm structural identity and assess comparability. Organic Size-Exclusion Chromatography-Mass Spectrometry Accurate molecular masses of individually loaded species from samples of 1 prepared by processes A and B were determined under denaturing conditions by organic SEC-MS.10 The interchain disulfide bonds of 1 are partially reduced prior to conjugation with 2. As a result, many conjugated isoforms of 1 were detected in the dissociated form in this assay, as the secondary structure is only maintained under these conditions by those interchain disulfide bonds that remain intact. Two peaks were observed in the organic SEC chromatogram (Figure 6).



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Figure 6. UV280 nm profile of ADC 1 using organic SEC/UV/MS From the mass analysis results, the earlier eluting peak (peak 1) contains isoforms that are larger than L chain, including various conjugated species of heavy and light chain (e.g. HHL, HL, HH, etc). The most abundant species detected by oSEC-ESI-MS is composed of disulfide linked L and H chains with two linker-payload 2 moieties and labeled as HL+2 x 2 (Figure 7). Additional minor and trace level species such H+3 x 2, HH+2 x 2, HHL+1 x 2, and mAb+4 x 2 were also observed and are consistent with the interchain disulfide bond conjugation chemistry. For the sample of ADC 1 prepared by process A, the levels of H+3 x 2 and HH+2 x 2 species are higher due to the increased relative level of species formed by conjugation to the cysteines involved in disulfide bonds between H and L. The later eluting peak (peak 2) in the SEC chromatogram contains one moiety of 2 conjugated to the interchain cysteine residue of the L chain involved in the disulfide bond to H chain (Figure 8).


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Figure 7. Zero-charge deconvoluted mass spectra of peak 1 in ADC 1 conjugated by process A and process B and analyzed by organic SEC/MS



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Figure 8. Zero-charge deconvoluted mass spectra of peak 2 in ADC 1 conjugated by process A and process B and analyzed by organic SEC/MS. Non-Reduced Peptide Mapping by Liquid Chromatography Mass Spectrometry (LC-MS) of ADC 1 The disulfide connectivity in the ADC samples prepared by processes A and B was established by alkylated non-reduced Lys-C peptide mapping.11 There are a total of 32 cysteine residues in each ADC sample. All intrachain cysteine residues are expected to be present as disulfide bonds. There are two intrachain disulfide bonds in each light chain and four in each heavy chain. Interchain cysteines are present as both intact disulfide bonds and as partially reduced that are conjugated to 2. There is one interchain disulfide bond between each light and heavy chain and two interchain disulfides between the heavy chains in the hinge region. All intrachain and interchain predicted


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disulfide bonds were detected as expected. Interchain cysteines were also detected as conjugated to 2. The non-reduced peptide mapping results for 1 prepared by both processes demonstrate that the disulfide linkages are consistent with the predicted structure. The similarity of profiles is seen for extracted mass chromatograms for expected disulfide bonded peptides produced by Lys-C digestion of process A and process B samples of ADC 1 (Figure 9). H15-ss-H20 RT: 59.52 - 95.96 H26-ss-H30 100

L8-ss-L14 Relative Abundance

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Process A

80 60 40

H6-ss-H7H8

H13H14-2x(ss)-H13H14 H6-ss-H7

L1-ss-L5

20

H13-2x(ss)-H13H14 H13-2x(ss)-H13

0 100 80

Process B

60 40 20 0 60

65

70

75

80 Time (min)

85

90

95

Figure 9. Extracted mass chromatograms for disulfide bonded peptides for ADC 1 prepared by process A and process B The extracted mass chromatograms for conjugated peptides of 1 prepared by process A and process B are shown in Figure 10. The L15 peptide containing the Cys214 and H12 peptide containing Cys200 are detected with one conjugated payload 2. The hinge region H13 and H13H14 peptides containing Cys residues 226 and 229 are observed as disulfide bonded using both Cys226 and



Cys229 (Figure 9) and as conjugated with one molecule of 2 to one of the cysteine residues and disulfide linked via the other cysteine residue with L15 peptide (Figure 10). Additionally, H13 and H13H14 peptides are observed with two moieties of 2 conjugated to both hinge region cysteines. However, the extracted mass chromatograms for the conjugated peptides show that the ADC material produced by process A has a lower level of hinge region conjugation (H13 + 2 x 2 and H13H14 + 2 x 2 peptides) and higher levels of L15 and H12 conjugated peptides than the level of the same conjugated peptides observed for 1 prepared by process B (Figure 10). The difference in the level of hinge region conjugation observed for the ADC materials prepared by the two processes was also confirmed by non-reduced capillary gel electrophoresis (CGE) and reduced reverse phase HPLC (supporting information). 100

Relative Abundance

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Process A

H12 + 1 x 2 L15 + 1 x 2

80 60

H13 + 2 x 2 40

H13H14 + 2 x 2

20

H13H14 + 1 x 2-ss-L15 H13 + 1 x 2-ss-L15

0 100

Process B

80 60 40 20 0 85

90

95

100

105 110 Time (min)

115


120

125

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Figure 10. Extracted mass chromatograms for conjugated peptides of 1 prepared by process A and process B Trace levels of mispaired interchain cysteine-containing peptides were observed for L15 and H12. These include mispairing to the nearby cysteine-containing peptides such as L14 for L15 and H6, H7, and H15 for H12 peptide. In addition, trace levels of mispairing of the hinge region peptide H13H14 conjugated to one molecule of 2 were observed for both L15 and H12 peptides. The levels of mispairing of the hinge region peptide H13H14 is higher for the sample of ADC 1 produced by the process B. The non-bonded (free thiol) cysteine containing peptides were also observed at very low levels. Stability Comparison of Samples of 1 Prepared by Processes A and B by Differential Scanning Calorimetry The stability of the ADC samples prepared by processes A and B were evaluated by differential Scanning calorimetry (DSC) (Figure 11).12 In the thermogram of the ADC sample prepared by process B an additional peak was observed at approximately 62°C that was not present in the similar trace of the ADC sample prepared by process A. This indicates potentially slightly lower conformational stability of the ADC 1 prepared by process B compared to the sample that was prepared by process A.

100 Process A Process B 80

Cp (kcal/mole/°C)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


60

40

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0

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40

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60

Temperature (°C)

70

80

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Figure 11. DSC thermograms of samples of ADC 1 from process A and process B Assessment of Stability of 1 Prepared by Processes A and B by Extrinsic Fluorescence Spectroscopy The stability of 1 prepared by processes A and B was further evaluated by extrinsic fluorescence spectroscopy. Fluoresence dye Sypro Orange and 4-(dicyanovinyl)Julolidine (DCVJ) were used in differential scanning fluorimetry (DSF) experiments to monitor thermal unfolding and formation of aggregates.13 ADC samples from the solutions of 1 were prepared in BioRad multiplate® PCR plate. The sample plates were subjected to thermal stress from 5 oC to 95 oC at an increment of 1 o

C. At each temperature, the plate was equilibrated for 1 minute prior to measurement.

SYPRO

®

Orange binds to hydrophobic residues or patches of a protein and spectral changes reflect

changes in the environment around the probe. In an aqueous solution, the fluorescence emission intensity from the probe is negligible. However in the presence of an unfolded protein the fluorescence intensity increases significantly due to the increased exposure to hydrophobic regions to which the probe can bind.13 During SYPRO® Orange fluorescence study, two transitions were observed for both the ADC samples of 1 with similar Tm1 at 47 oC, indicating thermal unfolding of CH2 domain occurs at similar temperatures. However, Tm2 for the ADC 1 at 62°C prepared by the process A is approximately 2°C higher (62 oC) compared to that prepared by the process B. The data from this study under heating conditions showed that the ADC 1 prepared by process A was less prone to unfolding than the sample that was prepared by the process B (Figure 12).


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200 150 - First derivative of F.I.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


100 50 0 0

20

40

60

80

100

-50 -100

Temperature (◦C) Process A

Process B

Figure 12. Sypro® orange fluorescence intensity profile as a function of temperature. DCVJ is one of a class of viscosity-sensitive fluorescent dyes, commonly referred to as molecular rotors. When DCVJ binds to aggregates, the dye becomes partially immobilized, accompanied by an increase in quantum yield.14, 15 Studies have shown that DCVJ responds to the early stages of aggregation and, thus, has a strong preference for oligomeric aggregates. The decrease in fluorescence intensity upon heating up to 40 °C is observed in all the solutions including DCVJ buffer solution which was not plotted in the figure, thus could be attributed to the inherent property of the fluorophor.16 The transitions detected by DCVJ are at 53 oC for 1 prepared by process A and 50 oC for 1 prepared by process B, indicating that the ADC 1 produced by the process A is less prone to aggregation (Figure 13).



1800 1600 - First Derivative of F.I

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1400 1200 1000 800 600 400 200 0 -200 0

20

40 60 Temperature (◦C) Process A

80

100

Process B

Figure 13. DCVJ fluorescence intensity profile as a function of temperature. Reduction in the Aggregate Level by Chromatographic Purification of 1 Prepared by Process B As described above, the level of aggregates formed during the conjugation reaction could be minimized by the conjugation protocol used for the process A (5 oC TCEP reduction). However this protocol also led to changes in the drug distribution profile of the ADC 1 compared to the conjugate mixture produced by the process B (37 oC TCEP reduction) (Figure 10). Since in-vivo properties of 1 prepared by process B were more thoroughly studied during early development work, we decided to develop a chromatographic purification procedure for the removal of aggregates from 1. Several resins were screened for the selective separation of aggregates from monomeric 1. Hydroxyapatite resin yielded the best results leading to the removal of a major portion of the aggregates without significantly impacting the average DAR of the conjugate mixture. The predominantly monomeric ADC material was obtained by elution with the phosphate buffer at pH 7 using a linear gradient (Figure 14). The solvent DMSO and the residual free drug species were separated from the ADC in the flow-through fraction during this purification.


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% Mobile Phase B

Aggregates

Figure 14. Purification profile of 1 on the hydroxyapatite resin column with phosphate buffer as an eluent (UV detection at 280 nm). The solution of purified conjugate was concentrated and diafiltered in 20 mM histidine buffer (pH 5.8) using regenerated cellulose membrane cassette. The isolated ADC material contained primarily monomeric ADC species (~ 98 %) with the average DAR ~ 3.6 – 4.0. Characterization of Aggregates Produced During Preparation of 1 The aggregates isolated during purification of 1 by the column chromatography were further characterized by SEC, RP- HPLC and SEC/MS assays (Figure 15). The SEC result confirmed that the isolated aggregate material consisted of 68 % aggregates and 32 % monomer species. (Figure 15a). The average loading of the aggregate sample was determined to be ~ 7 by RP-HPLC assay (Figure 15b) and

SEC/MS assay (Figure 15c). The SEC/MS assay was performed under



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denaturing conditions where the ADC species of the light and heavy chains without covalent interchain disulfide linkages separated on the SEC column into two main peaks (peak 1 and peak 2). From the mass analyses results, the peak 1 contained ADC species with various combinations of heavy and light chain (e.g. HHL, HL, HH, etc) attached to the payload 2. The most abundant species detected was composed of the heavy chain with three molecules 2. The peak 2 contained one molecule of payload 2 conjugated to the light chain. These data from RP-HPLC and organic SEC/MS together showed that the majority of isolated aggregates were non-covalent in nature and primarily composed of 8-loaded ADC species. Comparison of the analytical HIC chromatograms of the purified ADC 1 and isolated aggregates (Figure 16) did not show presence of monomeric 8loaded species in the sample of ADC 1. Evidently either a majority of the 8-loaded species formed in the reaction mixture during conjugation lead to the formation of aggregates, or the monomeric 8loaded ADC species co-elute with the aggregates in the HIC chromatogram.


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Figure 15. Aggregate characterizations by a. SEC assay, b. reduced reverse phase HPLC assay c. SEC/MS assay.



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Figure 16. Analytical HIC chromatograms of 1 purified by column chromatography (blue) and isolated aggregates (black).

■ CONCLUSION A novel Anti-Notch 3 cysteine conjugated ADC indicated for triple negative breast cancer and ovarian cancers is currently in phase I clinical trials. The conjugation process for the preparation of this ADC led to the formation of higher aggregate levels in the conjugation reaction mixture. The aggregates were formed in the conjugate mixture only after conjugation with the payload 2 and not during the TCEP reduction step. The formation of aggregates is likely due to a combination of several factors such as the nature of the antibody, hydrophobicity of the payload, average drug loading (DAR) on the conjugate, and the sites of drug conjugation. Optimization studies on the reduction/conjugation steps led to identification of conjugation conditions that resulted in the formation of the ADC mixture with low level of aggregates. At lower TCEP reduction temperature (0 – 5 oC) followed by the conjugation, the ADC 1 was obtained with lower aggregate level. Differences in the TCEP reduction temperatures also caused changes in the distribution of ADC species. At higher temperature (~ 37 oC) of the TCEP reduction step, the resulting ADC contained more species conjugated at the hinge region of the mAb. Stability studies by DSC and extrinsic


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florescence spectroscopy indicated that 1 prepared by the process B may be more prone to aggregation at higher temperature compared to the ADC material produced by the process A, though at ambient temperature no discernible difference in the stability was observed. Separation of aggregates in the crude mixture of 1 (prepared by process B) was accomplished by purification on the hydroxyapatite column under mild conditions without significantly impacting the average DAR of the purified ADC. In addition to aggregates, solvent DMSO, and residual free drug species were successfully removed under these purification conditions.

■ EXPERIMENTAL SECTION General Procedure for the Preparation of ADC 1 by Processes A and B Process A The pH of a stock solution of anti-Notch 3 antibody was adjusted to 6.5 – 7 with a histidine buffer solution. The solution of the antibody was cooled to 0-5 oC. An aqueous solution of TCEP (tris(2carboxyethyl)phosphine, 2.4 equivalents./1 equivalent of antibody) was added to the antibody solution. The resulting mixture was stirred for 90 minutes at 0-5 oC. The solution was then cooled to ambient temperature. To this antibody solution was added a solution of 2 (6 equivalents) in DMSO (11% v/v). The reaction mixture was stirred for 1 hour. The mixture was quenched by addition of a solution of L-cysteine. The resulting unpurified product consisted of an ADC with an average drug to antibody ratio (DAR) of ~ 4 by analytical HIC assay and with aggregate levels 1-2 % by SEC assay. Process B The ADC 1 was prepared using the same process as in process A except the temperature of the TCEP reduction step was 37 oC. Purification of 1 Prepared by Process B Using Column Chromatography



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The ADC mixture prepared by the process B was purified by column chromatography over hydroxyapatite resin (type 1, 40 µm). The following purification parameters were used for separation of aggregates from the monomer species, Mobile phase A: 5 mM sodium phosphate, pH 7; Mobile phase B: 200 mM sodium phosphate, pH 7; buffer gradient = 10-70 % mobile phase B over 20 column volumes, protein loading = 15 mg/mL, flow rate = 1 mL/min, column bed height = 22 cm. Most of the desired monomer product eluted between the beginning of the peak through to the next 6.9 column volume affording yield of 85-88 % (based on the protein yield) and the average DAR of 3.6 (HIC assay) and aggregates ~ 1 % (SEC assay). Evaluation of Different Quench Reagents The crude ADC mixture of 1 was prepared by the process B described above except the quench step was omitted. The reaction mixture was split into four equal size portions. The reaction mixtures were quenched with appropriate quench reagent; L-Cysteine, N-acetyl cysteine, and 2(dimethylamino) ethane thiol. Each mixture was stirred for 30 minutes at 25 oC. No quench reagent was added to one of the portion of the reaction mixture. All four portions of the reaction mixture were purified by ultrafiltration-diafiltration (UF-DF) process using regenerated cellulose membrane (30 kD) and histidine buffer as diafiltration buffer at protein concentration of 40 mg/mL and 500 LMH feed flux. General Procedure for the Execution of DoE The pH of a stock solution of anti-Notch 3 antibody was adjusted to 6.5 – 7 with a histidine buffer solution. The solution of the antibody (100 mg, 1 equivalent) was adjusted to the desired concentration with the histidine buffer. The temperature of the antibody solution was set to the predetermined temperature based on the requirement of each experiment. The aqueous solution of


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TCEP (2.21 equivalents) was added to the antibody solution with stirring. The mixture was stirred with a magnetic stir bar for 90 minutes. The mixture was cooled to 25 oC and a solution of linkerpayload 2 in DMSO (containing required amount of DMSO for each experiment) was added to the reaction mixture. The mixture was stirred at 25 oC for 1 h. L-cysteine (4 equivalents) was added to the reaction and the mixture was stirred for 30 minutes. An aliquot from each reaction mixture was taken for evaluation by the analytical HIC and SEC assays. Determination of Average Drug Loading (DAR) and Drug Distribution by Analytical HIC The analytical HIC method was used to determine the drug loading and the drug load distribution of the ADC. The reference material (25 µL) and test samples were diluted to 2 mg/mL with diluent (half strength of the mobile phase A) and injected onto a Thermo Propac HIC10 column (4.6 mm x 10 cm, 5 µm). The mobile phase A was 1.5 M ammonium sulfate, 50 mM potassium phosphate dibasic, pH 7 and mobile phase B was 10% IPA, 50 mM potassium phosphate dibasic, pH 7. The samples were eluted with the flow rate of 0.8 mL/minute and at the column temperature of 30 °C. Species of different drug to antibody ratio (DAR) were separated using a salt gradient and detected by UV absorbance at 280 nm. The average DAR and distribution profile were determined by peak area percentage of each species. Determination of Aggregate Content by Analytical SEC The SEC method was used to determine product purity and aggregate content. The test samples were diluted with the mobile phase (20 mM sodium phosphate, 400 mM sodium chloride, pH 7.2) at the concentration of 2 mg/mL and injected onto the YMC-Pack Diol-200 (300 x 8 mm ID) column at 0.75 mL/minute for 20 minutes. The aggregate and monomer species in the crude ADC preparation were reported as the percent of the total area for all protein-related peaks. Determination of Average Drug Loading (DAR) by Reduced Reverse Phase HPLC



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The sample was denatured in 4 M Gdn-HCl, 50 mM Tris, pH 7.8 and 20 mM DTT at 37 ºC for about 30 minutes. The reduced samples (10 µL) were separated and monitored at 214 nm by using an Agilent HPLC with a Agilent Zorbax 300SB-CN column (150 x 4.6 mm, 3.5 um) at the column temperature of 75 ºC. The samples were eluted using the mobile phase A (0.1% TFA in water) and mobile phase B (80% acetonitrile, 20% isopropanol and 0.1% TFA) with the flow rate of 0.75 ml/minute. Characterization of ADC 1 by LC-MS and Peptide Mapping Intact Mass Analysis by Organic Size-Exclusion Chromatography - Electrospray Ionization Mass Spectrometry (oSEC/MS) The ADC 1 samples and HIC fractions were analyzed by oSEC liquid chromatography interfaced with mass spectrometry. Samples were transferred into HPLC vials and placed in a 6 °C autosampler. Approximately 20 µg of sample was applied to a Waters Acquity UPLC® BEH200 SEC column (4.6 mm x 150 mm, 1.7 µm) at a column temperature of 25 °C. The samples were eluted at a flow rate of 0.2 mL/min using an isocratic separation of 70% water and 30% acetonitrile with 0.1% trifluroacetic acid (TFA) and monitored at 280 nm with a TUV detector. The Waters Acquity UPLC was coupled to a Waters Xevo Q-Tof mass spectrometer (Waters, Milford, MA) that was calibrated by infusing 5 µL/min of 2 mg/mL of NaI solution. Liquid Chromatography – Mass Spectrometry (LC-MS) of Non-Reduced and Alkylated LysC Peptides For the non-reduced and alkylated sample preparation, 100 µg of protein sample was denatured by addition of 100 µL of the buffer containing 7 M GndHCl, 0.1 M Tris, pH 7.5 and 80 µL of water. The alkylation step was performed at room temperature for 1 hour by addition of 3 µL of 50 mM iodoacetic acid (IAA). The Lys-C digestion reaction was prepared by addition of 10 µL of 1 mg/mL


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(10 µg) of endoproteinase Lys-C (Wako Chemicals) [enzyme: substrate = 1:10 (w: w)] and then incubating at 37 °C for 5 hours. Prior to analyses, samples were diluted with 90 µL of 0.1% TFA in water for a final concentration of 0.33 mg/mL. The digested sample was applied to a Vydac 214TP C4 column (5 µm particle size, 300 Å pore size, and 2.1 × 250 mm) set at 60 ºC and eluted using an Agilent 1200 HPLC. A linear gradient from 0 to 45% B in 116 minutes at a flow rate of 0.2 mL/min was used using mobile phase A [0.1% trifluoroacetic acid (TFA)] in H2O while mobile phase B consisted of [0.1% TFA in acetonitrile] to elute the peptides. Peptides were monitored at 214 nm. The Agilent 1200 HPLC was coupled to a Thermo LTQ Orbitrap XL mass spectrometer (Thermo Scientific, San Jose, CA) which utilized an internal lock mass ion of hexakis(1H,1H,3H-perfluoropropoxy)phosphazene at m/z 922.009798 for [M+H+]1+ for dynamic calibration. The observed multiply-charged peptides were converted to neutral molecular masses using Xtract algorithm of Xcalibur software. Neutral, monoisotopic masses were reported for all peptides observed in the LC-MS data. Stability Evaluation of 1 by Extrinsic Fluorescence Spectroscopy SYPRO® Orange and DCVJ were used to monitor thermal unfolding and formation of aggregates. SYPRO® Orange dye was supplied in 5000 fold concentrated stock solutions. The dye was diluted 2000-fold in the assay samples. With excitation at 560-590 nm, the fluorescence emission was collected using a ROX filter (600-630 nm). DCVJ stock solutions (4 mM) were prepared in DMSO. Prior to use, the stock solutions were diluted 200 fold to 20 µM in the assay samples. After excitation at 450-490 nm, the fluorescence emission was collected using a FAM filter (515-530nm). The ADC solutions were prepared in BioRad multiplate® PCR plate. Each cell was filled with 25 µL of sample solution. The plate was sealed with optically clear adhesive PCR Microseal® ‘B’ film. The fluorescence was monitored by Bio-Rad CFX Real-Time System, C1000 Thermal



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Cycler. The above plate, plate sealer and plate reader were purchased from BioRad Laboratories (Hercules, CA).The sample plates were subject to thermal stress from 5 to 95 °C at an increment of 1°C. At each temperature, the plate was equilibrated for 1 minute prior to measurement. The fluorescence data and the second derivatives obtained from the built-in software were exported as a CSV file into Microsoft Excel (Microsoft Corporation, Redmond, WA) for further data analysis. Stability Assessment of 1 by Differential Scanning Calorimetry Samples were diluted with the degassed buffer to a final protein concentration of 0.5 mg/mL for all runs. The same degassed buffer was also used as a reference in the reference cell. Samples were injected into a MicroCal VP capillary DSC (Malvern Instruments, Worcestershire, United Kingdom). DSC scans were performed from 25 °C to 95 °C at a rate of 60 °C/hour. The data was corrected for buffer and the raw signal was converted to molar heat capacity with protein concentrations and theoretical molecular weight. ■ AUTHOR INFORMATION Corresponding Author *Phone: 845-602-4505, Fax: 845-474-3359 E-mail: [email protected] Present Addresses ⊥Sanofi Genzyme, 1 The Mountain Road, Framingham, Massachusetts 01701, United States. ǁ 4110 Olive, St. Louis, Missouri 63108, United States. Notes The authors declare the following competing financial interest(s): All authors are or were employees of Pfizer Inc. when the research was conducted.


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■ ACKNOWLEDGMENTS The authors thank Manoj Charati, Nadira Prashad, Frank Loganzo, Elwira Muszynska, Kiran Khandke, and Ken Geles for providing details of early development work. The authors also thank Heyi Li, Leo Letendre, Bo Arve, Aparna Deora, Margaret Ruesch, and Brad Evans for supporting this work and providing valuable comments and suggestions. ■ ABBREVIATIONS ADC, antibody-drug conjugate; AUC-SV, analytical unltracentrifugation-sendimentation velocity; CGE, capillary gel electrophoresis; LC/MS, liquid chromatography/mass spectroscopy; MW, molecular weight; MWCO, molecular weight cutoff; SDS-PAGE, sodium dodecylsulfatepolyacrylamide gel electrophoresis; SEC, size-exclusion chromatography; TEM, transmission electron microscopy; TFA, trifluoroacetic acid; TCEP, (tris(2-carboxyethyl)phosphine; UF-DF, ultrafiltration-diafiltration ■ ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at http://pubs.acs.org Details of experimental design and results from DoE evaluation, anova summary for the level of aggregates, data for the characterization of individual ADC species by native SEC/MS, characterization data for ADC 1 by non-reduced CGE and reduced reverse phase HPLC.



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Process Development and Structural Characterization of an Anti-Notch

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