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Bioconjugate Chem. 2008, 19, 358–361
Selection of Reaction Additives Used in the Preparation of Monomeric Antibody-Calicheamicin Conjugates Irwin Hollander, Arthur Kunz, and Philip R. Hamann* Wyeth Research, 401 N. Middletown Road, Pearl River, New York 10965. Received August 24, 2007; Revised Manuscript Received September 24, 2007
The formation of protein aggregates can be a major problem during the preparation of antibody-drug conjugates. Herein is described the methods by which reaction additives were selected, which reduce the tendency of antibodies to aggregate during the attachment of the cytotoxic agent calicheamicin to form an immunoconjugate. Reaction conditions were delineated that produced optimized yields of monomeric conjugates. These conditions were used in the preclinical preparations of gemtuzumab ozogamicin (Mylotarg), the first commercially available chemotherapeutic immunoconjugate.
INTRODUCTION In the more than thirty years since the development of methods for preparing monoclonal antibodies, there have been numerous attempts to use these proteins as effective vehicles for targeting cytotoxic agents to human cancers. Although there are now six antibodies and two radioimmunoconjugates approved for cancer treatment in the United States, the only conjugate of a cytotoxic agent, such as a phytotoxin, a cytotoxic oncology agent, or a natural product analogue, is gemtuzumab ozogamicin (gem-ozo1 , Mylotarg, 2 (Figure 1), antibody ) hP67.6) (1), in spite of numerous agents being examined in the clinic (2). Gem-ozo is a conjugate of the anti-CD33 antibody hP67.6 and NAc-gamma calicheamicin DMH, a derivative of the naturally occurring gamma calicheamicin. The calicheamicins are members of the enediyne family of antitumor antibiotics that, even at subpicomolar concentrations, produce doublestranded DNA cleavage (3, 4). In gem-ozo, the antibody and calicheamicin derivative are covalently attached by a bifunctional linker referred to as the AcBut linker (5). This linker allows for attachment of the calicheamicin to the lysines on the antibody and forms an acyl hydrazone with the calicheamicin derivative that supplies a site of hydrolytic release in the lysosomes of the CD33-positive target cells. In preclinical models of acute myeloid leukemia, for which gem-ozo is approved, a hydrazone linkage was required for potent, selective cytotoxicity and gave conjugates superior to the amide conjugate (4), which does not contain that linkage (6). This is not required for all calicheamicin conjugates (7). Unfortunately, the introduction of the AcBut linker made conjugation to antibodies problematic, in that the maximum usable amount of the cosolvent DMF produced only modest yields of low-loaded, monomeric conjugates because of aggregation (Figure 2A). Although this material was used in the initial biological characterization of gem-ozo, scale up for development activities required a dramatic improvement in the loading and yield of monomeric conjugates. This article details * To whom correspondence should be addressed. Phone: (845) 6023423. Fax: (845) 602-5561. E-mail:
[email protected]. 1 Abbreviations: gem-ozo, gemtuzumab ozogamicin (Mylotarg); NAc, N-acetyl; DMF, N,N-dimethylformamide; DMH, dimethyl hydrazide; gamma, calicheamicin γ1I; OSu (ester), ester of N-hydroxysuccinimide; PBS,phosphate-bufferedsaline;SDS-PAGE,sodiumdodecylsulfate-polyacrylamide gel electrophoresis; SEC, size-exclusion chromatography.
Figure 1. Structures of calicheamicin derivatives and conjugates. In both conjugates, the natural product, gamma calicheamicin, is modified to contain an NAc group and a disulfide stabilized by two methyl groups (DM ) dimethyl). The conjugates differ in the presence of the hydrolyzable hydrazone bond in the AcBut conjugates.
how the use of various unique additives in the conjugation reaction solved this problem.
EXPERIMENTAL PROCEDURES Reagents. Antibody hP67.6 (CDP771/2, anti-CD33 humanized IgG4) was produced by cell culture in CHO cells and purified with affinity and ion exchange column chromatography by Lonza Biologics. The purity of the antibody was greater than 95%. The synthesis of NAc-gamma calicheamicin DMH AcBut OSu (1; see Figure 1 for structures) and the original procedures for making conjugates have been published (5).
10.1021/bc700321z CCC: $40.75 2008 American Chemical Society Published on Web 11/10/2007
Monomeric Antibody Conjugates
Bioconjugate Chem., Vol. 19, No. 1, 2008 359 Table 1. Additives Examined and Their Concentrations
Figure 2. Size-exclusion chromatography trace of conjugation reactions. Representative profiles of conjugation reactions performed on 30 mg of antibody hP67.6 and 6 equivalents of the AcBut OSu (1) with 20% DMF and no additives (A) or with 5% DMF, 20% propylene glycol, and 80 mM octanoic acid (B). Pooled monomer for the conjugation shown in A was about 1 mol/mol, while in B, the loading was 2.5 mol/mol.
Tween-80, glycine, maltose, histidine, Pluronic F-68, octanoic acid, N-acetyl tryptophan, benzyl alcohol, propylene glycol, glycerol, benzoic acid, isopropanol, and t-butanol were all reagent grade (Sigma Chemical). The PBS used is 50 mM sodium phosphate and 100 mM NaCl at pH 7.4, prepared using reagent grade mono and dibasic sodium phosphate salts and sodium chloride (Sigma Chemical). Aggregated Conjugate. Aggregated antibody-calicheamicin conjugate with the AcBut linker (2) was prepared using the published procedure as follows (5). To a 10 mg/mL solution of antibody hP67.6 in PBS was added additional buffer and the cosolvent DMF to give a solution of 5 mg/mL antibody and 20% DMF. To this solution was added 6 mol equiv of NAc-gamma calicheamicin DMH AcBut OSu (1) in DMF at 10 mg/mL. The mixture was shaken gently at room temperature for 16 h and then purified by sizeexclusion chromatography on a Sephacryl S-200 (GE Healthcare) column eluted with PBS. The eluted protein was fractionated on the basis of its 280 nm absorbance. Fractions were further analyzed by high-performance liquid chromatography on a Zorbax GF-250 column (Dupont) in 0.2 M sodium phosphate and 25% propylene glycol at pH 7.0. Fractions containing aggregate (Figure 2A) were selected to give conjugate (2) having approximately 25% aggregated protein as measured by peak areas at 280 nm. This material was concentrated to ∼1 mg/mL using Amicon Centriprep 30 ultrafiltration devices. Aggregate Dissociation Assay. Each additive was aliquoted as a 10X concentrate to a 50 µL aliquot of the aggregate fraction prepared as described above, to give the desired percent of additive as listed in Table 1. The mixtures were incubated overnight at room temperature and then analyzed by sizeexclusion chromatography (SEC). SEC was performed both on a Zorbax (Dupont) GF-250 column eluted with 0.2 M sodium phosphate containing 25% propylene glycol and on a Superose
additive
concentration
Tween-80 glycine glycine maltose glycine histidine Pluronic F-68 octanoic acid octanoic acid N-acetyl tryptophan benzyl alcohol sodium benzoate propylene glycol glycerol
1% 300 mM 200 mM/2% 100 mM/100 mM 1% 80 mM 40 mM/6 mM 1% 0.5% 33% 25%
12 HR (Pharmacia) column eluted with PBS. Both columns were monitored by UV absorbance at 280 and 333 nm to determine protein and calicheamicin profiles. Conjugation Experiments. To a 10 mg/mL solution of antibody hP67.6 in PBS was added additional buffer and one or more of the additives to give a solution of 3.25 mg/mL antibody (∼250 µL). To this solution was added 6 mol equiv of NAc-gamma calicheamicin DMH AcBut OSu (1) in DMF at 10 mg/mL (5% DMF final concentration). The mixture was shaken gently at room temperature for 16 h and then purified by size-exclusion chromatography on a Superose 200 (Pharmacia) column eluted with PBS. The eluted protein was fractionated on the basis of its 280 nm absorbance. Fractions were further analyzed by high-performance liquid chromatography on a Zorbax GF-250 column (Dupont) in 0.2 M sodium phosphate and 25% propylene glycol at pH 7.0. Fractions containing predominantly monomer were pooled to give the final conjugate (2). Optimized Large-Scale Conjugation Conditions. To 30 mg of hP67.6 antibody at 6.5 mg/mL in PBS was added propylene glycol (30% final concentration) and 1 M octanoic acid (final concentration 60 mM). The pH was then adjusted to ∼7.75 with 1 M NaOH. Six molar equivalents of NAc-gamma calicheamicin DMH AcBut OSu (1) in propylene glycol at 6 mg/mL was then added. The mixture was shaken gently at 25 °C for 3 h before filtering through a Millex HV filter to remove particulates and purifying by SEC on a Sephacryl S-200 column eluted with PBS (Figure 2B). Monomeric fractions were then pooled to give conjugate (2) with
