Structural Characterization of the Aggregates of Gemtuzumab

Apr 8, 2019 - Data acquisition and processing were carried out using the ASTRA 6.1 software (Wyatt Technology). Samples were diluted to 1 mg/mL in 0.2...
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Article Cite This: ACS Omega 2019, 4, 6468−6475

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Structural Characterization of the Aggregates of Gemtuzumab Ozogamicin Qingping Jiang,*,† Bhumit Patel,†,∥ Xin Jin,†,⊥ Deanna Di Grandi,†,# Eric Bortell,¶,∇ Brooke Czapkowski,¶,○ Thomas F. Lerch,‡ Debra Meyer,‡ Shruti Patel,†,∥ Jodi Pegg,‡ Alan Arbuckle,‡ Julius Lagliva,† Verl Sriskanda,† Leo Letendre,¶ Heyi Li,† Elizabeth Thomas,§,◆ and Durgesh Nadkarni*,¶ Downloaded via 193.56.75.118 on April 8, 2019 at 22:36:25 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.



Analytical Research and Development, ¶Bioprocess Research and Development, and §Pfizer Global Supply, Pfizer Inc., 401 N. Middletown Road, Pearl River, New York 10965, United States ‡ Analytical Research and Development, Pfizer Inc., 700 Chesterfield Parkway West, Chesterfield, Missouri 63017, United States ABSTRACT: The preparation of antibody drug conjugates (ADCs), particularly those containing hydrophobic payloads, may promote aggregate formation. The aggregate (high molecular mass species, HMMS) is an impurity that must be controlled in the final drug substance and drug product formulation to meet product safety and efficacy. Although there are numerous methods designed to mitigate protein aggregation, some of the reasons for the formation of aggregates in ADCs are unique and specific to the synthetic preparation and structure of each conjugate. Proper structural characterization and identification of causes of aggregation are key to improving the process for preparation of ADCs for minimizing aggregate formation. In this article, we have characterized aggregates generated during the preparation of a lysine conjugate, gemtuzumab ozogamicin, prepared from an IgG4 antibody (hP67.6) and a calicheamicin-based linker-payload. Using analytical and biophysical techniques, structural details of the aggregates formed in the conjugation process are elucidated. The aggregates are predominantly composed of a mixture of dimers and multimers with high drug loaded species. The level of conjugation to the N-termini of the mAb was higher in aggregates than in the monomeric ADC. Studies performed on the biological activity revealed differences in binding affinity and cytotoxicity between aggregates and monomeric ADC species.



INTRODUCTION

that of gemtuzumab ozogamicin, which is indicated for the treatment of acute myeloid leukemia. It is composed of an antiCD33 humanized IgG4 monoclonal antibody (hP67.6) that is covalently conjugated to a toxic small molecule payload Nacetyl-γ-calicheamicin via a linker with an average drug to antibody ratio (DAR) of ∼2−3 (Figure 1).3 The conjugation process used for the preparation of ADCs can often lead to formation of minor amounts of aggregated species. Several factors may be involved in the formation of aggregates.4,5 The presence of aggregates in the final ADC formulation is undesirable due to the potential impact on efficacy and safety such as immunogenicity, toxicity, and pharmacokinetics. Because it is difficult to predict the effect of these aggregates on immunogenicity, regulatory agencies have required that these species be well controlled and monitored.6,7

Antibody drug conjugates (ADCs) have emerged as a new class of antitumor therapeutics combining the efficacy of cytotoxic small-molecule drugs with the targeting ability of an antibody. Since Mylotarg (gemtuzumab ozogamicin), the first ADC that won regulatory approvals in the USA (subsequently withdrawn in 2010) and Japan, the ADC field has made significant progress with two recent FDA approvals in 2011 for Adcetris (brentuximab vedotin, SGN-35) and in 2013 for Kadcyla (trastuzumab emtansine, T-DM1). Currently, more than 30 additional ADCs are in clinical trials.1,2 ADCs are complex molecules composed of three components: a monoclonal antibody, a linker, and a cytotoxic payload. There are also numerous chemistries used to conjugate the payload to an antibody such as conventional conjugation to lysine or cysteine residues, as well as more recent technology of site specific conjugation to natural or non-natural amino acids. The conventional conjugation reaction generally results in the formation of ADC species with a consistent range of drug loading. One such example is © 2019 American Chemical Society

Received: December 26, 2018 Accepted: March 4, 2019 Published: April 8, 2019 6468

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Figure 1. Preparation and purification of ADC 1 and aggregate enriched fractions.

Figure 2. (a) Preparative SEC chromatogram from purification of the ADC mixture. (b) Further purification of the aggregate fraction into aggregate enriched fractions: A1 and A3. (c) Analytical SEC chromatogram of fraction A1. (d) Analytical SEC chromatogram of fraction A3.

two main aggregate enriched fractions (A1 and A3) (Figure 2b). The composition of each fraction was evaluated by analytical SEC to determine aggregate content. The aggregation levels of fractions A1 and A3 were 84 and 79%, respectively, by analytical SEC (Figure 2c,d). Molecular Weight Analysis of Aggregates by SECMALS and AUC. Size exclusion chromatography coupled with multiangle light scattering (SEC-MALS) demonstrated that fraction A3 consists of a mixture of dimeric and multimeric species as indicated by the molecular mass (Figure 3a). The black trace is primarily monomeric ADC 1, while the blue trace is the aggregate fraction A3. The average molecular weights determined via SEC-MALS are shown in Figure 3a for the region indicated by the dotted lines. The data show that fraction A3 is made up of monomeric (148 kDa) and aggregated species with molar mass distribution ranging from dimeric (300 kDa) through higher multimeric (446 kDa) species with some heterogeneity in size distribution. Due to the low concentration of fraction A1, we were unable to obtain SEC-MALS data. Orthogonal size distribution data were obtained for ADC 1 and fractions A1 and A3 using analytical ultracentrifugation-sedimentation velocity (AUC-SV) (Figure 3b). Fraction A1 is composed mainly of multimers, while fraction A3 is predominantly a dimer with some higher

Previous work from our laboratories focused on minimizing formation of the aggregates during preparation of the ADC.4 However, the underlying mechanism behind the formation of aggregates during this conjugation reaction is not well understood. Further characterization of the aggregated species would help enhance the knowledge of the process for preparation of lysine conjugates, such as gemtuzumab ozogamicin. With better understanding of the nature of product aggregation and formation of aggregated species, process parameters that impact aggregate formation can be controlled to minimize their formation. In the current study, we isolated and characterized the aggregates formed during conjugation of gemtuzumab ozogamicin. The results of these investigations are described in this paper.



RESULTS AND DISCUSSION Separation of Aggregates from Monomer of Gemtuzumab Ozogamicin. The crude ADC mixture was purified by preparative size exclusion column chromatography to separate aggregates from predominantly monomeric species ADC 1 formed during preparation of gemtuzumab ozogamicin (Figures 1 and 2a). The aggregate fraction was further fractionated on a preparative size exclusion chromatography column to produce 6469

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were observed in lower abundance in fraction A1, again consistent with SEC and AUC-SV results. Determination of Average Drug Load and Drug Distribution of Aggregates by SEC, UV, and Reduced RP-HPLC. After confirmation of size distribution of aggregates by analytical methods such as SEC-MALS and AUC-SV, experiments were performed to determine the drug loading and drug distribution of the aggregate samples. The average drug loading on the conjugated species was determined by analytical SEC and UV. By taking advantage of the unique absorbance of calicheamicin, the average drug loading of the conjugated species was calculated by the ratio of relative absorbances at 310 and 280 nm. The average drug loadings of the aggregate enriched fractions A1 and A3 were 72 μg of calicheamicin/mg of protein (DAR ∼6) and 58 μg of calicheamicin/mg of protein (DAR ∼5) by SEC (Figure 2), respectively. The drug loadings of fractions A1 and A3 were also confirmed by using a direct UV measurement method. The average drug loadings of the ADC 1 monomeric fraction were 27 μg of calicheamicin/mg of protein (DAR ∼2.4) by SEC and 29 μg of calicheamicin/mg of protein (DAR ∼2.6) by UV. These results indicate that the aggregates are primarily composed of ADC species with higher average drug loading. The presence of higher drug loaded species in the aggregate enriched fractions compared to ADC 1 was qualitatively assessed by using a reduced reversed-phase HPLC (RPHPLC) method. After reduction of the ADC species by DTT, the heavy chain components containing different numbers of linker species could be separated on the RP-HPLC column. The chromatograms (Figure 5) show that the heavy chains (HCs) of the aggregate enriched fractions A1 (cyan) and A3 (black) consist of significant percentage of species with higher drug loading compared to the HC of ADC 1 (red). Antibody hP67.6 does not contain any of the conjugated HC species (blue) The level of high drug loaded species, indicated by peaks labeled as “HC with linkers” in Figure 5, was greater in fractions A1 and A3 than in ADC 1. The control antibody (hP67.6) sample did not show any peaks at the same rentention area. The low resolution of the separation was due to the heterogeneity of the antibody and small differences in the drug loading of ADC speices. Under the acidic assay conditions, the conjugated calicheamicin drug is released

Figure 3. Analysis of ADC 1 and aggregate fractions by (a) SECMALS and (b) AUC-SV.

multimeric species, consistent with SEC-MALS data. The sedimentation coefficient for the monomer present in these fractions is higher than that in ADC 1 (∼98% monomer), indicating that the monomeric species present in the aggregate fractions might be in a more compact conformation or undergo reversible association with aggregate species. The contents of monomer and aggregates determined by AUC-SV in all samples are consistent with those observed by analytical SEC. SEC-MALS demonstrates that fraction A3 is composed of predominantly dimers, with some monomer and larger multimer species. The analysis of aggregate fractions by AUC-SV confirmed that ADC 1 is composed primarily of monomer and fractions A1 and A3 predominently consist of multimer and dimer species, respectively. However, collectively the data suggests that A1 is primarily multimeric and A3 is primarily composed of dimers. Analysis of Aggregates and ADC 1 by Transmission Electron Microscopy (TEM). The sample of ADC 1 was imaged using transmission electron microscopy. A classical “Y” shape expected for monoclonal antibodies and ADC molecules was observed, and particles were found to be predominantly monomeric (Figure 4a). At the same concentration, fraction A3 was composed primarily of dimeric and small aggregate species, consistent with SEC and AUC-SV measurements (Figure 4b). Fraction A1 contains several large, amorphous aggregates, comprising multiple self-associated monomeric ADC molecules (Figure 4c). Monomer and smaller aggregates

Figure 4. Particle imaging analysis by TEM. (a) ADC 1, (b) fraction A3, (c) fraction A1. Inset images show expanded views of selected monomers in ADC 1 (a) and small aggregates in fraction A3 (b), while arrows in panel (c) point to some of the larger aggregates in fraction A1. Scale bars represent 50 nm. 6470

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Figure 5. RP-HPLC profiles of reduced heavy chain of anti-CD33 antibody (hP67.6), ADC 1, and fractions A1 and A3.

Figure 7. Size exclusion chromatograms of fraction A3 after dilution with acetonitrile containing mobile phase. The total peak areas of samples containing 0, 10, and 20% acetonitrile are the same.

during the denaturing and reduction steps leaving only the covalently conjugated linker on the antibody. The heavy chain species with variable loading of the attached linkers separate on the reversed-phase HPLC column. Characterization of Types of Aggregates. To determine if the aggregated species are covalently associated, the ADC 1 and aggregate fraction A3 were first characterized by SDS-PAGE (reducing and nonreducing) (Figure 6). Under

increasing content of acetonitrile in the sample, the aggregate species dissociate into monomeric ADC molecules. The conditions used in this study were relatively mild to avoid denaturing the ADC species. Under these conditions, only partial dissociation of the aggregates was observed. The results from the SDS-PAGE as well as from the dilution experiments with acetonitrile indicate that the aggregated species are predominanently noncovalent in nature. Acetonitrile may play a role in the disruption of the hydrophobic interactions between ADC molecules reducing the amount of aggregates. When the same experiment was carried out by increasing the salt concentration of the sample in sodium phosphate buffer, there was no change in the amount of aggregates (data not shown). Bioassays of Aggregated Species. CD33 Binding of ADC 1 and Aggregate Enriched Fractions A1 and A3. The binding of ADC 1 and fractions A1 and A3 to the CD33 antigen was evaluated by surface plasmon resonance (SPR) analysis using CD33 mFc covalently immobilized on a biosensor chip. The results of kinetic analyses of the binding of ADC 1 to CD33 are shown in Table 1.

Figure 6. SDS-PAGE of ADC 1 and fraction A3 under reducing and nonreducing conditions. Lane M (MW marker), lane A (nonreducing, ADC 1), lane B (nonreducing, fraction A3) (black box is shown around the faint aggregate species band), lane C (reducing, ADC 1), lane D (reducing, fraction A3).

Table 1. Binding and Cytotoxicity Data for ADC 1 and Aggregate Enriched Fractions A1 and A3

reducing conditions (lanes C and D), all species from ADC 1 and aggregate enriched fraction A3 dissociated into light and heavy chains as expected. Under nonreducing conditions, for fraction A3 (lane B in Figure 6), a very faint band consistent with the molecular weight of the aggregate species was observed (indicated by the black box); however, the abundance was much lower than expected compared to the ∼80% aggregate content found in fraction A3. This indicates that the aggregate species are predominantly noncovalent and dissociate in the presence of SDS (denaturing agent) to the monomeric ADC species. Next, a sample of fraction A3 was diluted with the mobile phase containing phosphate buffer (pH 7) with varying levels of acetonitrile. The resulting solutions were analyzed by size exclusion chromatography (Figure 7). As the concentration of acetonitrile in the sample was increased to 20%, the aggregate content of the sample decreased from ∼78 to ∼44% with a concomitant increase in the monomer content of the sample eluting between 7.8 and 9.0 min (Figure 7). A relative quantification of aggregate and monomer species is shown in the table (Figure 7 inset). With

sample ID ADC 1 aggregate enriched fraction A1 aggregate enriched fraction A3

KD for SPR (pM)

CD33 relative binding by flow cytometry (%)

relative cytotoxicity (%)

8 29

103 16

95 152

29

33

140

The data were fitted globally to a 1:1 Langmuir binding model with compensation for mass transfer. Both the aggregate fractions (A1 and A3) exhibited ∼3.5-fold lower affinity (Ka = 1.1 × 105 M−1 s−1, Kd = 3.1 × 10−5 s−1, KD = 29 pM for A1 and Ka = 9.5 × 104 M−1 s−1, Kd = 2.8 × 10−5 s−1, KD = 29 pM for A3) than the ADC 1 (Ka = 1.5 × 105 M−1 s−1, Kd = 1.2 × 10−5 s−1, KD = 8 pM). The binding of ADC 1 and aggregate samples to CD33 expressed on HL60 cells was also evaluated by flow cytometry. The % relative CD33 binding of the aggregate samples (A1 and A3) and ADC 1 control was determined based on the EC50 comparison to gemtuzumab 6471

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This is in contrast to the observation in the cytotoxicity assay that showed samples with more aggregation led to higher cytotoxicity. The aggregate enriched fraction A1 contained more multimers compared to mainly dimeric aggregate fraction A3. Though the aggregate samples enriched in dimer and higher order aggregates show decreased antigen binding compared to monomeric ADC 1, the average drug loading in aggregate enriched fractions is also higher than that in ADC 1. This may result in the apparent increase in cytotoxicity of the aggregate enriched fractions despite exhibiting relatively lower binding affinity in the binding assays compared to the reference material. Comparison of Conjugation Sites on Aggregates and ADC 1. The samples of aggregate A3 and ADC 1 were analyzed by peptide mapping and LC−MS. Four primary sites of conjugation on the antibody were identified in both molecules as denoted by a black asterisk in the peptide mapping data (Figure 10).8 The sites are identified as Lys 242,

ozogamicin reference material. As shown in Figure 8, the aggregate fractions A1 and A3 had significantly lower relative CD33 binding (16 and 33%, respectively) than the ADC 1 (103%).

Figure 8. CD33 binding of ADC 1 and aggregate enriched fractions A1 and A3 to HL60 cells by flow cytometry.

Cytotoxicity of ADC 1 and Aggregate Enriched Fractions A1 and A3. A cytotoxicity assay was performed on the samples of aggregate enriched fractions, A1 and A3, along with ADC 1 using CD33 expressing HL60 cells. At the end of the assay, a luminescence reagent to measure viable cells, Cell Titer Glo, was used to determine the cytotoxic activity of the samples where the relative luminescence units (RLU) are directly proportional to the number of viable cells in the culture. Each sample was assayed twice, and the biological activities of the samples were determined by comparing each sample curve to the gemtuzumab ozogamicin reference material. As shown in Figure 9, the predominantly dimeric fraction A3 and multimeric fraction A1 show a shift to the left in the dose Figure 10. Results from peptide mapping of ADC 1 and aggregate enriched fraction A3. The retention time window where there were differences between peaks between unconjugated mAb hP67.6, ADC 1, and fraction 3 aggregate is shown here. There were no differences in the peptide map of the three samples shown here at other retention times in the chromatogram. The peaks with a black asterisk indicate sites of conjugation on the antibody. The peak indicated by a red asterisk in fraction A3 indicates the conjugation site on the N-terminal peptide observed at significantly higher levels in aggregates than in ADC 1.

Lys 286, Lys 330, and Lys 380. In the aggregate sample A3, the same four sites on the antibody were found to be predominantly conjugated. The relative peak areas of these four conjugation sites were higher in the aggregate sample than the peak areas observed for ADC 1. In addition to these four sites, several minor sites of conjugation were also observed for both samples. However, for the aggregate sample A3, additional main peptide (designated by the red asterisk in Figure 10) was found to be present at significantly higher levels (7- to 10-fold) than that in ADC 1. The relative abundance of isotope peaks (m/z 745. 89 and m/z 1490.77) extracted from this peptide peak was also much higher in aggregate fraction A3 than in ADC 1 (Figure 11a,b). The mass matches with the N-terminal peptide H1 (amino acids 1−12) conjugated with one linker-payload. The relative abundance of a peptide (m/z 1052.07) with no conjugated drug was comparable between aggregate A3 and ADC 1 (Figure 11a,b). Using this peptide (m/z 1052.07) as an internal control, a qualitative comparison of the average drug loading between aggregate fraction A3 and

Figure 9. Representative cytotoxicity assay of ADC 1 and aggregate enriched fractions of A1 and A3.

response curve and greater maximal cytotoxicity (lower viable cells at the maximal concentration of ADC tested) compared to the reference material indicating increased cytotoxicity. In contrast, the ADC 1 control showed comparable cytotoxicity to the reference material. In order to determine relative biological activity, the IC50 for each sample curve was determined and compared to the IC50 of the reference material. The mean relative biological activities for the dimeric fraction A3 and multimeric fraction A1 are 152 and 140%, respectively, relative to the reference material indicating increased relative cytotoxicity, whereas the mean relative activity for the ADC 1 control was 95% demonstrating activity comparable to the reference material (Table 1). Based on CD33 binding data by SPR and flow cytometry, it appears that increased aggregation leads to a decrease in CD33 binding. 6472

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Figure 11. Mass spectra extracted from the peptide peak labeled with a red asterisk in Figure 10 for (a) ADC 1 and (b) aggregate fraction A3, and (c,d) fragmentation data obtained on drug-attached peptide H1 of the parent ion m/z 1490.77. The m/z values for y/b annotations allow us to assign the modification site.

ozogamicin. The aggregate enriched fractions A1 and A3 and monomeric fraction ADC 1 were characterized by analytical and biophysical techniques. The aggregate species were found to be a mixture of dimers and multimers. The aggregates were composed mainly of ADC molecules with higher average loading of the drug. The higher average drug loading and the hydrophobic nature of the calicheamicin drug (linker-payload) may partly contribute to the formation of aggregates.3 The aggregates and monomeric ADC 1 also contained species with conjugation to the N-termini of the mAb. The level of conjugation to the N-termini of the mAb was 7- to 10-fold higher in aggregate enriched fraction A3 than in monomeric ADC 1. Compared to the other four main sites, the Nterminus is more exposed and the linker-payload conjugated to the N-termini may potentially increase the aggregation propensity due to much higher hydrophobic interaction between molecules. The binding affinities of the aggregate enriched fractions A1 and A3 to the CD33 antigen were found to be lower than that of the monomeric ADC 1. However, the aggregates exhibited higher potency than ADC 1 in the cytotoxicity assay. This is likely due to the higher average drug loading on the aggregates compared to ADC 1 despite the lower CD33 binding. The studies presented here have helped reveal the composition of aggregates formed in this lysine conjugation process. The ADC 1 is composed of a mixture of unconjugated and conjugated species.8 The conjugation conditions that would minimize formation of higher drug loaded species while maintaining the same average drug loading of the ADC by changing the drug distribution profile will likely lead to a lower aggregate level in the conjugate product.12,13 Efforts are underway in our laboratory to optimize the conjugation conditions toward that objective.

ADC 1 shows higher drug loading on the fraction A3 compared to ADC 1. To further confirm the conjugation locations in aggregate fraction A3, we searched the fragment ions within the mass spectrum of this peptide in A3 following collision induced dissociation. The fragmention data showed good coverage of a series of y- and b-ions. The diagnostic ions y-ions and b-ions (Figure 11c,d) conclusively identified the drug conjugation site of glutamine 1 for the H1 peptide. For example, the difference between the b4 ion of the modified peptide (m/z 674.34) and the b4 ion of the unmodified peptide (m/z 469.23) correlates well with the theoretical m/z of the linker (m/z 204.08). Based on the LC−MS data of the aggregate enriched fraction A3, the conjugation to the amino acid glutamine of the N-terminal peptide was confirmed. The pKa of the N-terminus α-amino group of the protein is lower (pKa ∼8) than the pKa of ε-amino group of the lysine side chain (pKa ∼10−11).9,10 At the conjugation pH 7.5−8, the α-amino groups of the N-termini of the mAb molecules are more likely to exist as free base compared to the ε-amino groups of the lysine side chains. The higher reactivity of the Nterminal amines at the conjugation pH along with other factors such as better accessibility to the reactive linker-payload may lead to some conjugation at the N-termini of the mAb molecules.11 The peptide mapping data revealed that the ADC species containing the linker-payload at the N-termini are more prevalent in the aggregate enriched fraction A3 compared to the mainly monomeric ADC 1. It is unclear whether conjugation of the hydrophobic calicheamicin containing linker-payload to the N-termini of the mAb preferentially leads to the formation of aggregates. In summary, in the present study, we isolated the aggregate species formed during the preparation of gemtuzumab 6473

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EXPERIMENTAL SECTION Purification and Isolation of Antibody Drug Conjugate 1 and Aggregate Enriched Fractions. The sample of ADC 1 was prepared by the procedure reported previously.3 The crude conjugate mixture of ADC 1 was purified on a Superdex 200 PG resin size-exclusion chromatography (SEC) column using 5 mM sodium phosphate/100 mM sodium chloride buffer (pH ∼7). The isolated aggregate fraction was first concentrated using a Millipore 50 kD MWCO spin concentrator to a protein concentration of 7−10 mg/mL. The concentrated aggregate material was further fractionated on the same SEC column using the same mobile phase. Individual fractions that are enriched in aggregates (≥75% aggregates) were collected. Size Exclusion Chromatography for DAR Determination. The SEC system consisted of an HPLC system (HP1100; Agilent), an SEC column (TSKgel G3000SWXL; Tosoh Bioscience), and a diode-array detector (Agilent, USA). Samples were diluted to 1 mg/mL in 0.2 M sodium phosphate at pH 7 and run on SEC chromatography monitoring 280 and 310 nm wavelengths. The DAR values were determined based on the peak areas by using correction factors for the calicheamicin at 280 nm and the antibody at 310 nm. Evaluation of ADC 1 and Aggregate Fractions by SDS-PAGE. Four micrograms each of aggregated fraction A3 and ADC 1 was loaded onto an Invitrogen NuPage 4−12% Bis-Tris Gel under both reducing and nonreducing conditions following the manufacturer’s protocol. For the reduced sample, the proteins were diluted in a 10× reducing agent (Invitrogen) and heated at 70 °C for 5 min. The Mark12 Protein Ladder was loaded for comparison (20 μL) of the marker. The gel was run at 200 V with MES running buffer for 45 min and stained using GelCode Blue Safe Protein Stain by Thermo Scientific according to the manufacturer’s protocol. Size Exclusion Chromatography and Multiangle Light Scattering (SEC-MALS). The SEC system consisted of an HPLC system (HP1100; Agilent), an SEC column (TSKgel G3000SWXL; Tosoh Bioscience), an MALS detector (DAWN HELEOS II, λ 658 nm, 25 °C; Wyatt Technology, USA), and a refractive index detector (Optilab rEX, λ 685 nm, 35 °C; Wyatt Technology, USA). Data acquisition and processing were carried out using the ASTRA 6.1 software (Wyatt Technology). Samples were diluted to 1 mg/mL in 0.2 M sodium phosphate at pH 7 and run on SEC chromatography monitoring 280 and 310 nm wavelengths. Evaluation of Aggregate in the Presence of Varying Levels of Acetonitrile. Fraction A3 was run on SEC using mobile phase containing 0.2 M sodium phosphate at pH 7 containing 0, 10, or 20% acetonitrile. The diluent used for sample preparation was the same as the mobile phase. For example, the sample was diluted in 0.2 M sodium phosphate at pH 7 with 10% acetonitrile and run on SEC using 0.2 M sodium phosphate at pH 7 with 10% acetonitrile as mobile phase. Once diluted, the samples were incubated at room temperature for 5 min and placed in the autosampler at 4 °C. Reduce Reversed-Phase HPLC. The sample was denatured in 2.5 M guanidine hydrochloride and subsequently reduced in 200 mM DTT at 37 °C for about 30 min. The reduced materials were separated and monitored at 280 nm by using an Agilent HPLC with an Agilent Zorbax 300SB-CN reversed-phase column (150 × 4.6 mm, 3.5 μm). Mobile phase

A was 0.1% TFA in water, and mobile phase B was 80% acetonitrile, 20% isopropanol, and 0.1% TFA. Analytical Ultracentrifugation-Sedimentation Velocity (AUC-SV). The aggregate samples were diluted to 0.5 mg/ mL with 5 mM sodium phosphate and 100 mM sodium chloride at pH 7.5 to obtain a UV absorbance (280 nm) signal of ∼1 AU in 1.2 cm centerpieces. Samples were run in triplicate using a Beckman Coulter Analytical Ultracentrifuge Proteome XL/I at 45000 rpm at 20 °C. One hundred scans were collected at a wavelength of 280 nm. Data was analyzed using sedfit (version14.1)14 to generate c(s) size distribution plots and to determine the relative abundance of the various species. Sednterp (version 20130813 BETA) was used to determine the partial specific volume of the mAb and solvent viscosity and density. A partial specific volume of 0.729 mL/g was used based on the amino acid composition of the antibody hP67.6 that was used for preparation of ADC 1. A solvent density of 1.0031 g/mL and a solvent viscosity of 1.013 cP were used for data analysis. Transmission Electron Microscopy (TEM). The aggregate samples were diluted in water to 0.01 mg/mL and adsorbed onto glow discharged 200 mesh copper grids with copper formvar coating (Ted Pella, Inc.). Grids were stained for 1 min with a 1% solution of filtered uranyl formate. Grids were imaged at the Nano Research Facility at Washington University (www.nano.wustl.edu) using a Tecnai G2 transmission electron microscope operated at 120 kV. Peptide Mapping and LC−MS Analysis. The ADC 1 (300 μg) was reduced with 5 mM tris(2-carboxyethyl)phosphine hydrochloride (TCEP) in 6.2 M guanidine HCl at pH 8.2, followed by alkylation with 6 mM sodium iodoacetate in the dark. The reaction mixture was then diluted with water and digested with Lys-C proteinase (enzyme μg/ protein μg ratio is 1:20) at 37 °C for 16 h. The digested material was quenched with trifluoroacetic acid (TFA) and stored at −70 °C until analysis. The digested peptides were separated using an Agilent HPLC with a Kinetex C18 reversedphase column (150 × 4.6 mm, 2.6 μm) obtained from Phenomenex (Torrance, CA, USA). The eluted peptides were monitored by UV absorbance at 214 nm and an Agilent 6220 TOF mass spectrometer (Santa Clara, CA, USA) in positive ion mode. Each peptide was identified by the molecular weight determined from its MS data. Surface Plasma Resonance (SPR). Biosensor analyses were carried out using a BIAcore 3000 (BIAcore, Uppsala, Sweden). CD33 mFc was covalently immobilized on the Nhydroxysuccinimide-activated carboxymethyl dextran-coated biosensor chip (CM 5) using a standard amine-coupling chemistry at a protein density of approximately 1000 resonance units. Samples of ADC 1 or aggregate fractions were diluted in the HBS buffer (10 mM HEPES, pH 7.4, containing 150 mM NaCl, 3 mM EDTA [ethylenediaminetetraacetic acid], and 0.005% polysorbate 20 [v/v]) and injected in the concentration range of 0 to 600 nM over the CD33 mFc-coated biosensor chip surface at a flow rate of 30 μL/min for 8 min to allow binding. After the binding phase, dissociation of the bound antibody was monitored by washing the chip with the HBS buffer over a 60 min period. The antigenic surface was regenerated by washing the biosensor chip with 15 μL of the regeneration buffer (10 mM NaOH and 200 mM NaCl) for 30 s, followed by a stabilization time of 2 min before the next cycle. Kinetic constants were calculated by nonlinear least square regression analysis using a 1:1 Langmuir 6474

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binding curve fitting model and BIA evaluation program (version 3.0, BIAcore). CD33 Binding by Flow Cytometry. HL60 cells (ATCC, Manassas, VA) at 5 × 104 per well were incubated on ice for 30 min with gemtuzumab ozogamicin reference material and samples in duplicate. After incubation, cells were stained with phycoerythrin-labeled goat anti-human IgG F(ab′)2 (Jackson ImmunoResearch, catalog # 109-116-088, West Grove, PA) on ice for 30 min. Flow cytometry analysis was carried out on a BD FACSCanto II Instrument (BD Biosciences, Franklin Lakes, NJ). The EC50 concentration for each curve was calculated, and the % relative CD33 binding was determined by comparing the sample EC50 to the reference material EC50. Cytotoxicity Assay. HL60 cells (ATCC, CCL-240), which constitutively express CD33, were incubated with gemtuzumab ozogamicin reference material, ADC 1, fraction A1, and fraction A3 for approximately 72 h. After the incubation, Cell Titer Glo (Promega, G7571) was added that generates a luminescence signal proportional to the amount of ATP present in each well and directly relates to the number of viable cells. The relative luminescence units were measured using a plate reader (Molecular Devices, M5), and a four parameter dose response curve was plotted for the control and samples. The IC50 concentration for each curve was calculated, and the % relative biological activity was determined by comparing the sample IC50 to the gemtuzumab ozogamicin reference material. Each sample was tested in triplicate in two separate assays.



Article

ABBREVIATIONS ADC, antibody drug conjugate; ACN, acetonitrile; SEC, size exclusion chromatography; TEM, transmission electron microscopy; AUC-SV, analytical ultracentrifugation-sedimentation velocity; LC/MS, liquid chromatography/mass spectroscopy; SDS-PAGE, sodium dodecylsulfate-polyacrylamide gel electrophoresis; HBS, 4-(2-hydroxyethyl)piperazine-1ethanesulfonic acid buffer saline; MW, molecular weight; MWCO, molecular weight cutoff



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AUTHOR INFORMATION

Corresponding Authors

*E-mail: qingping.jiang@pfizer.com. Phone: 845-602-4602. Fax: 845-474-3181 (Q.J.). *E-mail: durgesh.nadkarni@pfizer.com. Phone: 845-602-4505. Fax: 845-474-3359 (D.N.). ORCID

Durgesh Nadkarni: 0000-0003-0733-4380 Present Addresses ∥

Merck & Co., Inc., Process Research and Development, 2000 Galloping Hill Road, Kenilworth, New Jersey 07033, United States ⊥ Bristol-Myers Squibb, Molecular and Analytical Development, 1 Squibb Drive, New Brunswick, New Jersey 08903, United States # Regeneron Pharmaceuticals, Inc., 777 Old Saw Mill River Road, Tarrytown, New York 10591, United States ∇ Pfizer Inc., GTS Biomanufacturing Sciences, 4300 Oak Park, Sanford, North Carolina 27330, United States ○ Pfizer Inc., WSR Regulatory Operations, 219 East 42nd Street, New York, New York 10017, United States ◆ Lexicon Pharmaceuticals, 8800 Technology Forest Place, The Woodlands, Texas 77381, United States Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS The authors thank David Merkoolof and Robby Mohammed for providing sample of the aggregate fraction. The authors also thank April Xu, Aparna Deora, Bo Arve, and Margaret Ruesch for supporting this work. 6475

DOI: 10.1021/acsomega.8b03627 ACS Omega 2019, 4, 6468−6475