Pertuzumab variants and evaluation thereof.

VARIATIONS OF PERTUZUMAB AND ITS EVALUATION

This non-provisional application filed under 37 CFR § 1.53 (b), claims the benefit in accordance with 35 USC § 119 (e) of the US Provisional Application. Series No. 61 / 812,603, filed on April 16, 2013, incorporated herein in its entirety by reference.

Sequence Listing

This application contains a Sequence List submitted through EFS-Web and incorporated herein by reference in its entirety. Said ASCII copy, created on April 8, 2014, bears the name of P5584R1-WO_Seq_Listing.txt, and has a magnitude of 31,363 bytes.

Field of the Invention

The present invention relates to pertuzumab variants. In particular, it is a variant of unpaired cysteine ​​that comprises unpaired Cys23 / Cys88 cysteines in one or both light variable domains of pertuzumab, an afucosylated variant of pertuzumab, a low molecular weight species (LMWS, low molecular). pertuzumab weight-species), and a high-molecular-weight-species (HMWS) of pertuzumab. The invention further relates to isolated variants, compositions, pharmaceutical compositions, and articles of manufacture comprising the variants, as well as methods for preparing and characterizing their variants and compositions. Background of the invention

Pertuzumab (PERJETA®) (also called rhuMAb 2C4) is a monoclonal antibody (MAb), which is the first of its kind in a line of agents called "HER dimerization inhibitors." By binding to HER2, it inhibits the dimerization of HER2 with other HER receptors and therefore inhibits tumor growth. Pertuzumab has received approval from the U.S. Food and Drug Administration. (US FDA) for the treatment of HER2-positive metastatic breast cancer, June 8, 2012.

U.S. Pat. No. 7.62,817 (Adams et al.) Discloses a humanized variant of the 2C4 antibody called humanized 2C4 version 574 or recombinant humanized monoclonal antibody 2C4 (rhuMAb 2C4). The antibody bound to subdomain II in the extracellular domain (ECD) dominates Receptor 2 of human epidermal growth factor (HER2). The rhuMAb 2C4 antibody has been produced on a laboratory scale and shows binding to HER2 and inhibiting the growth of MDA-175 cells (expressing HER2 at a + 1 level) and of MCF7 xenografts implanted in mice. See, also, Adams et al. Immunol cancer. Immunother 55 (6): 717-727 (2006).

U.S. Pat. No. 6,339,142 (Blank and Basey) describes a HER2 antibody composition comprising a mixture of antiHER2 antibody and one or more of its acidic variants, in which the amount of the variant (s) is less than about 25% Variant 8 of humanized monoclonal antibody 4D5 (humMAb4D5-8 or trastuzumab) is the HER2 antibody given by way of example.

U.S. Pat. No. 7,560,111, U.S. Pat. No. 7,879,325, and U.S. Pat. No. 8,241,630 (Kao et al.) Describe a variant of pertuzumab (rhuMAb 2C4) comprising an amino terminal leader extension (HSV-) in one or both light chains of the antibody, the so-called "HSV variant" When, the material Reference (Phase I), Lot S9802A (Phase II), and scale process development material were tested for free thiol by Ellman analysis under native conditions, the free thiol level was below the detection limit in all the materials tested. From 1 to 2% pertuzumab in the compositions tested was afucosylated (G0-

F) as determined by capillary electrophoresis (CE capillary electrophoresis). See Table 5 of US Pat. No. 7,560,111 (Kao et al.).

WO 2009/099829 (Harris et al.) Describes acid variants of pertuzumab which include: deamidated variant, glycated variant, reduced disulfide variant, non-reducible variant, and sialylated variant. Variants were characterized as described as follows:

Table 1: Acid variants in WO 2009/099829 (Harris et al.)

<td colspan="3">Methods for characterizing acid claims</td>

<td>Method</td><td>Variants detected</td><td>Variant Name</td>

<td>treatment with CEX + / Sialidase</td><td>6% sialylated</td><td>Sialylated variant</td>

<td>CE-SDS reduced</td><td>1.5% Incompletely reduced</td><td>Non-reducible variant</td>

<td>CE-SDS not reduced</td><td>6% Reduced Disulfide</td><td>Reduced disulfide variant</td>

<td>Boronate Chromatography</td><td>Glycated at 3.5% (higher order)</td><td>Glycated variant</td>

<td>Peptide map</td><td>Discouraged</td><td>Deamidated variant</td>

CEX = cation Exchange (cation exchange). CE-SDS = Capillary Electrophoresis with Sodium Dodecyl Sulfate (Capillary electrophoresis with sodium dodecyl sulfate).

The experimental method used to characterize the disulfide variant in WO 2009/099829 (Harris et al.), Non-reduced CE-SDS of intact antibody, evaluated the inter-chain disulfide bonds, instead of the intra-chain disulfide bonds.

Zhang et al. Anal. Chem. 84 (16) 7112-7123 (2012) report a recombinant antibody (mAb A) that has unpaired cysteines (Cys22 and Cys96) in its heavy variable domain (VH domain). It was found that unpaired cysteines had no significant impact on antibody binding to CD20, and that mAb A with unpaired cysteines was fully active in a potency assay (complement dependent cytotoxicity, CDC, assay).

WO 2009/009523 (Kao et al.) Describes the prevention of the reduction of disulfide bonds between chains during the recombinant production of the creclizumab antibody (rhuMAb 2H7) that binds to CD20.

Harris, R. Dev. Biol. (Basel, Switzerland) 122: 117-127 (2005) revealed unpaired cysteines (Cys22 and Cys96) in the variable heavy domain (VH) of omalizumab, a humanized anti-IgE antibody. The unpaired cysteine ​​form had a significantly lower potency.

Synthesis of the invention

The experimental data herein refers to variant forms of pertuzumab, including an unpaired cysteine ​​variant, an afucosylated variant, a low tempo species (LMWS), and a high molecular weight species (HMWS). The means to identify, characterize, and quantify these variants are useful in manufacturing methods and quality control for the composition of the drug pertuzumab.

Therefore, in a first aspect, the invention relates to a composition comprising pertuzumab and its unpaired cysteine ​​variant, wherein the unpaired cysteine ​​variant comprises unpaired Cys23 / Cys88 cisterns in one or both light variable pertuzumab domains . The unpaired cysteine ​​variant includes a heterodimeric variant (which comprises unpaired Cys23 / Cys88 cysteines in only one light variable pertuzumab domain) and / or a homodimeric variant (comprising unpaired Cys23 / Cys88 cysteines in both light variable domains of pertuzumab).

The composition optionally further comprises one or more additional variants of pertuzumab such as an afucosylated variant, low molecular weight variant (LMWS), high molecular weight variant (HMWS), glycated variant, reduced disulfide variant, non-reducible variant, deamidated variant , sialylated variant, HSV variant, C-terminal lysine variant, oxidized methionine variant, G1 glycosylation variant, G2 glycosylation variant, and non-glycosylated heavy chain variant.

The invention also relates to a composition comprising pertuzumab and an afucosylated variant of pertuzumab, the amount of the afucosylated variant being 0.9 to 4.1% of the composition. In one embodiment, the invention relates to a composition comprising pertuzumab and an afucosylated variant of pertuzumab, in which the amount of the afucosylated variant is greater than 2% of the composition. In accordance with this embodiment, the amount of the afucosylated variant is greater than that reported in US Pat. N⁰ 7,560,111, U.S. Pat. No. 7,879,325, and U.S. Pat. No. 8,241,630 (Kao et al.).

In a further aspect, the invention relates to a composition comprising a mixture of pertuzumab, low molecular weight species (LMWS) of pertuzumab, and high molecular weight species (HMWS) of pertuzumab, wherein the amount of LMWS is &lt;1.6% and the amount of HMWS is £ 1.7%.

The invention also relates to a composition comprising a mixture of pertuzumab, Peak 1, and Peak 2, wherein the amount of Peak 1 is 0.5% and the amount of Peak 2 is 1.0% measured by the R.-CE-SDS test (reduced capilliary electrophoresis sodium dodecyl sulphate), reduced capillary electrophoresis with sodium dodecyl sulfate.

Additional aspects of the invention relate to pharmaceutical compositions, articles of manufacture, and methods for treating a cancer patient in what is used or comprising the compositions herein.

In a further aspect, the invention relates to a method for the evaluation of a pertuzumab composition comprising: (1) the measurement of the amount of unpaired cysteine ​​variant in the composition, in which the unpaired cysteine ​​variant comprises the sCys23 / Cys88 cysteines not paired in one or both of the light variable pertuzumab domains; and / or (2) the measurement of the amount of afucosylated pertuzumab in the composition; and / or (3) the measurement of the amount of low molecular weight species (LMWS) or high molecular weight species (HMWS) of pertuzumab in the composition.

And in another aspect, the invention relates to a method for evaluating the biological activity of a pertuzumab composition comprising measuring the amount of afucosylated pertuzumab variant in the composition to determine the cell-dependent cytotoxicity activity dependent on antibody (ADCC antibody-dependent cell-mediated cytotoxicity) of the composition, and confirmation that the amount of afucosylated pertuzumab is in the range of about 0.9 to about 4.1%.

In another aspect, the invention relates to a method for preparing a composition comprising: (1) producing a composition comprising pertuzumab and one or more of its variants; and (2) subject the composition thus produced to an analysis test to evaluate the amount of the variant (s) therein, the variants comprising: (i) unpaired cysteine ​​variant comprising Cys23 / Cys88 cysteines not paired in one or both light variable domains of pertuzumab; and / or (i) afucosylated pertuzumab variant; and / or (ii) high molecular weight species (HMWS) of pertuzumab; and / or (iv) low molecular weight species (LMWS) of pertuzumab, and / or fragment (s) of Peak 1 of pertuzumab, and / or (vi) fragment (s) of Peak 2 of pertuzumab.

In another aspect, the invention relates to an isolated variant of pertuzumab, wherein the isolated variant comprises: (1) an unpaired cysteine ​​variant of pertuzumab, wherein the variant is a heterodimeric variant comprising Cys23 / Cys88 tanks not paired in only a light variable domain of pertuzumab; and / or (b) an unpaired cysteine ​​variant of pertuzumab, the variant being a homodimeric variant comprising Cys23 / Cys88 cysteines not paired in both light variable pertuzumab domains; and / or (c) afucosylated variant of pertuzumab; and / or (d) high molecular weight species (HMWS) of pertuzumab; and / or (e) low molecular weight species of (LMW) of pertuzumab; and / or fragment (s) Peak 1 of pertuzumab, and / or fragment (s) Peak 2 of pertuzumab.

In a further aspect, the invention relates to a method for evaluating the fragmentation of a pertuzumab composition comprising measuring the amount of Peak 1 and Peak 2 in the composition by the reduced capillary electrophoresis assay with sodium dodecyl sulfate (R- CE-SDS) and confirmation that the amount of Peak 1 is £ 5% and the amount of Peak 2 is £ 1.0%.

Brief description of the drawings

Figure 1 provides a schematic representation of the structure of the HER2 protein, and the amino acid sequences for subdomains I-IV (SEQ ID Nos. 1-4, respectively) of its extracellular domain.

Figures 2A and 2B represent alignments of the amino acid sequences of the light (VL) (Fig. 2A) and heavy (VH) variable domains (fig.

2Β) 2C4 murine monoclonal antibody (SEQ ID No. 5 and 6, respectively); the VL and VH domains of the 574 / pertuzumab variant (SEQ ID Nos. 7 and 8, respectively), and the VL and VH human consensus frameworks (subgroup I moisture k1, light weight k; humlll, heavy subgroup III) (SEQ ID Nos. 9 and 10, respectively). Asterisks identify differences between the variable domains of pertuzumab and murine monoclonal antibody 2C4 or between the variable domains of pertuzumab and the human framework. The CDR (complementarity determining regions) are in parentheses.

Figures 3A and 3B show the light chain amino acid sequences of pertuzumab (Fig. 3A; SEQ ID No. 11) and heavy chain (Fig. 3B; SEQ ID No. 12). The CDRs are shown in bold. The calculated molecular mass of the light chain and heavy chain are 23,526.22 Da and 49,216.56 Da (cysteines in reduced form). The remaining carbohydrate is fixed to Asn 299 of the heavy chain.

Figures 4A and 4B show the amino acid sequences of the trastuzumab light chain (Fig. 4A; SEQ ID No. 13) and the heavy chain of trastuzumab (Fig. 4B; SEQ ID No. 14), respectively. The limits of the light variable and heavy variable domains are indicated by arrows.

Figures 5A and 5B represent the light chain sequence of a variant of pertuzumab (Fig. 5A; SEQ ID No. 15) and the heavy chain sequence of a variant of pertuzumab (Fig. 5B; SEQ ID No. 16 ), respectively.

Figure 6 represents the structure of pertuzumab (main species) including its 4 disulfide bonds between chains and its 12 intra-chain disulfide bonds, including the Cys23 / Cys88 intra-chain disulfide bonds in each of the light variable domains (VL). The domains illustrated are: VL = light variable domain; VH = heavy variable domain; CL = light chain constant domain; CH1 = constant heavy chain constant domain 1; CH2 = heavy chain constant domain 2; CH3 = heavy chain constant domain 3.

Figure 7 shows maps of non-reduced (native) tryptic peptides of pertuzumab.

Figure 8 depicts maps of tryptic peptides of reduced and non-reduced pertuzumab (full scale).

Figure 9 depicts maps of tryptic peptides of reduced and non-reduced pertuzumab (0-120 minutes).

Figure 10 depicts maps of tryptic peptides of reduced and non-reduced pertuzumab (120-204 minutes).

Figure 11 depicts hydrophobic interaction chromatography (HIC), analysis of pertuzumab digested by papain.

Figure 12 depicts the HIC analysis of pertuzumab digested by papain (enlarged view).

Figure 13 represents the HIC analysis of intact pertuzumab. The peaks are shown comprising: the free tillo-enriched homodimer (free thiols in both light chains), free thiol heterodimer (free thiol in a light chain), and wild type homodimer (main species antibody).

Figure 14 depicts the activity of pertuzumab (batch anti2C4907-2) in the ADDC assay (antibody-dependent cell-mediated cytotoxicity, antibody-dependent cell-mediated cytotoxicity).

Figure 15 shows the impact of the GO-F level on ADCC activity. The samples tested were pertuzumab phase III (GO-F = 2.2%) and pertuzumab phase I (GO-F = 0.8%).

Figure 16 depicts the capillary electrophoresis analysis of N-linked oligosaccharides released from pertuzumab.

Figure 17 depicts a capillary electrophoresis analysis of N-linked oligosaccharides released from pertuzumab (enlarged view). Note: The oligosaccharide G1 has two isomeric forms (labeled G1 and GT) in which the terminal galactose residue is attached to either branch A1-6 or branch A1-3.

Figure 18 depicts the HP-HPLC (Reversed Phase-High Performance Liquid Chromatography) for pertuzumab Fab and Fe separation (limited digestion with LysC). They show pertuzumab digestion limited by Lys-C and pertuzumab digestion limited by Lys C and then treated with N-ethylmaleimide (NEM).

Figure 19 shows the peptide mapping confirming that pertuzumab-free thiol Fab contains free Cys23 and Cys88 in its light chain. Fab L2 peptide containing free thiols was labeled by NEM and therefore shifted in the peptide map analysis.

Figure 20A-20C schematically represents: lgG1 of the main or wild-type species (Fig. 20A), heterodimeric variant Cys23 / Cys88 (Fig. 20B), and homodimeric variant Cys23 / Cys88 (Fig. 20C).

Figure 21 represents% of GO-F versus ADCC activity for pertuzumab batches for which the test in Example 4 was used herein.

Figure 22 schematically represents pertuzumab binding at the heterodimeric binding site of HER2, thereby preventing heterodimerization with activated EGFR or HER3.

Figure 23 compares the activities of trastuzumab (which joins Subdomain IV near the juxtamembrane domain of HER2 ECD) and pertuzumab (which joins Subdomain II of HER2 ECD).

Figures 24A and 24B represent the oligosaccharide structures attached to an IgG antibody.

Figure 25 shows an analysis of SEC (Size Exclusion Chromatography, Chromatography of Size Exclusion) of pertuzumab (full scale).

Figure 26 represents the SEC analysis of pertuzumab (enlarged scale). Peaks include main peak (main species antibody), high molecular weight species (HMWS), and low molecular weight species (LMWS)

Figure 27 shows the SE-HPLC analysis (high performance liquid chromatography of size exclusion) of pertuzumab samples. Sample A is representative of a batch of pertuzumab drug product. Sample B is a batch of pertuzumab subjected to light exposure at a rate of 1.2 mlux hours. Sample C is a batch of pertuzumab subjected to light at a rate of 3.6 mlux hours. Sample D is a batch of pertuzumab subjected to an acid treatment with a pH of 3.2. Sample E represents purified basic variants of

HPLC ion exchange (IE-HPLC).

Figure 28 shows the graph of the agreement of the analytical ultracentrifugation sedimentation rate (AUC) and HPLC SE analysis. Error bars represent two standard deviations with a determination of n = 3. All other data points denote a single determination. Circles denote samples with HMWS levels below the AUC detection level.

Figure 29 depicts the analysis of CE-SDS (capillary electrophoresis with sodium dodecyl sulfate) with laser-induced fluorescence detection (LIF) of non-reduced pertuzumab.

Figure 30 represents CE-SDS-LIF of non-reduced pertuzumab (NR) (enlarged view).

Figures 31A and 31B illustrate SE-HPLC chromatograms for Example 6; full scale (Figure 31 A) and enlarged scale (Figure 31B).

Figures 32A and 32B represent electropherograms of non-reduced CE-SDS (NR-CE-SDS) for Example 6: full scale (Figure 32A) and expanded scale (Figure 32B).

Figures 33A and 33B illustrate reduced CE-SDS electropherograms (RCE-SDS) for Example 6: full scale (Figure 33A), and the expanded scale (Figure 33B).

Figure 34 provides a comparison of the NRCE-SDS and R-CE-SDS electropherogram for an acid treated sample (enlarged scale).

Figure 35 shows the correlation of Fab quantification between NR14

CE-SDS and SE-HPLC.

Detailed description of the preferred embodiments

I. Definitions

Here, the term "paired cysteines" refers to two cysteine ​​residues that form a disulfide bond in a protein, such as an antibody. Said disulfide bond can be an inter-chain disulfide bond (for example, a disulfide bond between the heavy and light chains of an antibody, or between two heavy chains of an antibody), or an intra-chain disulfide bond (for example, within a light chain of an antibody or within a heavy chain of an antibody). Most lgG1 antibodies comprise four disulfide bonds between chains and twelve intra-chain disulfide bonds. See Figure 6.

The term "unpaired cysteine ​​variant" refers to a variant of a protein (for example, an antibody such as pertuzumab) in the one or more paired cysteines are not in a disulfide bound state. Such unpaired cysteines may not have been paired so as to form a disulfide bond (for example, when the protein originally folded into its tertiary structure) or it may have formed a disulfide bond, but which has subsequently broken (for example during manufacturing or during storage). Unpaired cysteines are often referred to as free thiols or free sulfhydryls. In one embodiment, unpaired cysteines are from a disulfide bond within the chain. In one embodiment, unpaired tanks are in a light chain, for example, a variable light domain of the antibody. In one embodiment, the unpaired cysteine ​​variant is a Cys23 / Cys88 variant.

An unpaired "Cys23 / Cys88" cysteine ​​variant lacks an intramolecular disulfide bond in cysteine ​​residues 23 and 88 in one or both variable light domains of the antibody. See Figures 20B and 20C in this document.

A "homodimeric variant" lacks Cys23 / Cys88 disulfide bonds in both variable light domains of the antibody. See Figure 20C in this document.

A "heterodimeric variant" lacks only a Cys23 / Cys88 disulfide bond in a light variable domain of an antibody. See Figure 20B in this document.

An "afucosylated variant" is a glycosylation variant of an antibody in which one or both oligosaccharide structures linked to the Asn299 residue of one or both heavy chains lacks fucose, for example, lacks Fuco (1 -> 6), in the structure of nuclear oligosaccharide.

A "low molecular weight species" or "LMWS" of pertuzumab comprises a pertuzumab fragment having a molecular weight less than that of the main species or intact pertuzumab (for example, when the intact pertuzumab has a molecular weight of approximately 145,197 Da, measuring only its peptide chains). The LMWS can be detected by SE-HPLC chromatography (high performance liquid chromatography of size exclusion) and / or CE-SDS assay (capillary electroforesls not reduced with sodium dodecyl sulfate) for example as in Example 5. In One embodiment, the LMWS comprises or consists of "Peak 6 as obtained by CE-SDS (see, for example, Example 5).

A "low molecular weight species" or "HMWS" comprises a pertuzumab preparation having a molecular weight that is greater than that of the main species intact pertuzumab (for example, when intact pertuzumab has a molecular weight of approximately 145,197 Da measuring only its peptide chain). HMWS can be detected by SE-HPLC (high performance liquid chromatography of size exclusion) and / or by CE-SDS test (capillary electrophoresis not reduced with sodium dodecyl sulfate) for example, as in Example 5.

Here, the term "Peak 1" refers to one or more pertuzumab fragments that are smaller in size than the light chain (LC) of pertuzumab. The fragment or fragments of Peak 1 can be separated from the main pertuzumab species by the CE-SDS test, preferably by the reduced DE-SDS test (R-CE-SDS). See, for example, Figure 33B, Table 16, and Table 18 and herein. It is preferable that the amount of Peak 1 in a pertuzumab composition is £ 0.5%. Optionally, the R-CE-SDS assay is carried out as described in Example 6 and the corrected peak area (CPA) provides the% of peak 1 in a composition.

Hereby, the term "Peak 2" refers to one or more pertuzumab fragments that are larger than light chain pertuzumab (LC) and smaller than the non-glycosylated heavy chain (NGHC). of pertuzumab. Peak 2 can be separated from the main pertuzumab species by CE-SDS, preferably by reduced assay (R-CE-SDS). Peak 2 excludes peak 3 that may appear during the R-CE-SDS test as explained in Example 6 of this document. See, for example, Figure 33B, Table 16, and Table 18 and herein. It is preferable that the amount of peak 2 in a pertuzumab composition is £ 1.0%. Optionally, the R-CE-SDS assay is performed as described in Example 6 and the corrected peak area (CPA) provides the% of peak 2 in a composition.

The term "fragmentation" refers to the cleavage or cleavage of the polypeptide chain, for example, the cleavage of the heavy chain and / or light chain of pertuzumab. It does not include the dissociation of non-covalently associated polypeptide chains during NR-CE-SDS analysis, for example.

The term "analytical assay" refers to an assay that qualitatively evaluates and / or quantitatively measures the presence or amount of an analyte (eg, an antibody variant) in a composition. The composition tested may be a purified composition, including a pharmaceutical composition.

A "hydrophobic Fab interaction chromatography assay" or "HIC Fab assay" comprises generating fragments (eg, Fab fragments) of the antibodies in a composition (eg, using the papain enzyme) and subjecting the antibody fragments thus generated to HIC to separate unpaired cysteine ​​variants of the main pertuzumab species. An assay of this type is disclosed by way of example in Example 1 herein.

An "HER receptor" is a receptor tyrosine kinase protein that belongs to the HER family of receptors and includes the EGFR, HER2, HER3 and HER4 receptors. The HER receptor will generally comprise an extracellular domain, which can bind to an HER ligand and / or dimerize with another HER receptor molecule; a lipophilic transmembrane domain; a conserved intracellular tyrosine kinase domain; and a carboxyl terminal signaling domain that hosts several tyrosine residues that may be phosphorylated.

The term "HER2" refers to the human HER2 protein described, for example, in Semba et al., PNAS (USA) 82: 6497-6501 (1985) and in Yamamoto et al. Natura 319: 230-234 (1986) (Genebank accession number X03363).

Here, the term "extracellular domain of HER2" or "HER2 ECD" refers to a domain of HER2 that is outside a cell, either anchored to a cell membrane, or in the circulation, including its fragments. The amino acid sequence of HER2 is shown in Figure 1. In one embodiment, the extracellular domain of HER2 may comprise four domains: "Subdomain I" (amino acid residues of approximately 1-195; SEQ ID NO: 1), "Subdomain II" (amino acid residues of approximately 196 to 319; SEQ ID NO 2), "Subdomain III (amino acid residues of approximately 320-488; SEQ ID NO: 3)," Subdomain IV (amino acid residues of approximately 489-630; SEQ ID NO: 4) (residue numbering without signal peptide). See Garrett et al. Mol. Cell .. 11: 495-505 (2003), Cho et al. Nature 421: 756-760 (2003), Franklin et al. Cancer Cell 5: 317-328 (2004), and Plowman et al. Proc. Nati Acad. Sel. 90: 1746-1750 (1993), as well as Figure 1 in this document.

Hereby, the term "HER dimer" refers to a non-covalently associated dimer comprising at least two HER receptors. Such complexes can be formed when a cell expressing two or more HER receptors is exposed to an HER ligand and can be isolated by immunoprecipitation and can be analyzed by SDS-PAGE as described in Sliwkowski et al., J. Biol. Chem .., 269 (20): 14661-14665 (1994), for example. Other proteins, such as a cytokine receptor subunit (for example, gp130) may be associated with the dimer. It is preferable that the HER dimer comprises HER2.

Here, a "HER heterodimer" is a non-covalently associated heterodimer comprising at least two different HER receptors, such as the EGFR-HER2, HER2-HER3 or HER2-HER4 heterodimers.

The term "HER activation" refers to the activation or phosphorylation of one or more any HER receptors. Typically, activation of HER results in signal transduction (for example, that caused by an intracellular kinase domain of HER receptors that phosphorylates tyrosine residues in the HER receptor or in a substrate polypeptide). HER activation may be mediated by the HER ligand that binds to a dimer of

HER comprising the HER receptor of interest. Binding of the HER ligand to an HER dimer can activate a kinase domain of one or more of the HER receptors in the dimer and thereby result in phosphorylation of tyrosine residues in one or more of the HER receptors. and / or phosphorylation of tyrosine residues in one or more additional substrate polypeptides, such as the Akt or MAPK intracellular kinases.

The "humanized" forms of non-human antibodies (eg rodents) are chimeric antibodies that contain a minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (receptor antibody) in which the residues of a hypervariable region of the receptor are replaced by residues of a hypervariable region of a non-human species (donor antibody), such as mouse, rat, rabbit or non-human primate that has the desired specificity, affinity and capacity. In some cases, the residues of the framework region (FR, framework region) of the human immunoglobulin are replaced by the corresponding non-human residues. In addition, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine the antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a sequence. of human immunoglobulin. The humanized antibody will optionally also comprise at least a portion of a constant region of immunoglobulin (Fe), typically that of a human immunoglobulin. For more details, see Jones et al, Nature 321: 522-525 (1986); Riechmann et al., Nature 332: 323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2: 593-596 (1992). Humanized HER2 antibodies specifically include trastuzumab and humanized 2C4 antibodies such as pertuzumab as described and defined herein.

Here, the term "intact antibody" refers to an antibody comprising two antigen-binding regions, and a Fe region. It is preferable that the intact antibody has a functional Fe region. In one embodiment, the "intact pertuzumab" has a molecular weight of approximately 145,197 Da, measuring only its peptide chains.

Here, the term "hypervariable region" refers to the amino acid residues of an antibody that are responsible for antigen binding. The hypervariable region generally comprises amino acid residues of a "complementarity determining region" or "CDR" (eg, residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the domain light chain variable and 31 -35 (H1), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain; Kabat et al, et al., Sequences of Proteins of Immunological Interest, 5th Ed Public Health Service, National Institutes of Health, Bethesda, MD. (1991)) and / or those residues of a “hypervariable loop” (for example, residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the light chain variable domain and 26 -32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variable domain; Chothia and Lesk J.

Mol Biol. 196: 901-917 (1987)). The "Framework Region" or "FR" (region framework) residues are those residues of the variable domain other than the residues of the hypervariable region as defined herein.

The term "region of Fe" herein is used to define a C-terminal region of an immunoglobulin heavy chain, which includes the native sequence Fe regions and the variant Fe regions. Although the limits of the Fe region of an immunoglobulin heavy chain may vary, the human IgG heavy chain Ig region is generally defined by stretching from an amino acid residue at position Cys226, or from Pro230, to its terminal. carboxyl The C-terminal lysine (residue 449 according to the EU numbering system) of the Fe region can be removed, for example, during the production or purification of the antibody, or by recombinant engineering of the nucleic acid encoding a heavy chain of the antibody. Accordingly, an intact antibody composition may comprise antibody populations with all K449 residues removed, antibody populations without K449 residues removed, and antibody populations having a mixture of antibodies with and without the K449 residue.

Unless otherwise indicated, the numbering of residues in an immunoglobulin heavy chain in this document is that of the EU index as in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991), which is expressly incorporated herein by reference. The "EU index as in Kabat" refers to the numbering of the EU antibody residues of human lgG1.

A "functional Fe region has an" effector function "of a native sequence Fe region. Examples of "effector functions" include C1q with a binding effect: complement dependent cytotoxicity; Fe receptor binding; antibody-dependent cell mediated cytotoxicity (ADCC); phagocytosis; downstream regulation of cell surface receptors (eg, B cell receptor; BCR), etc. Such effector functions generally require that the Fe region be combined with a binding domain (eg, an antibody variable domain) and can be evaluated using various assays.

A "native Fe region" comprises an amino acid sequence identical to the amino acid sequence of a Fe region found in nature. Native human sequence Fe regions include an FC region of native sequence human IgG1 (oA and A allotypes); Fe region of human IgG1 of native sequence; Fe region of human IgG3 of native sequence; and Fe region of human lgG4 of native sequence as well as its natural variants of natural presentation.

A "naked antibody" is an antibody that is not conjugated to a heterologous molecule, such as a cytotoxic or radiomarker moiety.

Here, the term "main species antibody" or "wild type antibody" refers to the structure of the amino acid sequence of the antibody in a composition that is the quantitatively predominant antibody molecule in the composition. It is preferable that the main species antibody is an HER2 antibody, such as an antibody that binds to subdomain II of HER2, an antibody that inhibits HER dimerization more effectively than trastuzumab, and / or binds to a site of heterodimeric binding in HER2. In one embodiment, the main species antibody is one comprising CDR-H1 (SEQ ID NO: 17 or 23), CDR-H2 (SEQ ID NO: 18), and CDR H3 (SEQ ID NO: 19), CDR - L1 (SEQ ID NO: 20), CDR - L2 (SEQ ID NO: twenty-one or 24) and CDR-L3 (SEQ ID NO: 22), the amino acid sequences of VL and VH in SEQ ID NO: 7 and 8, respectively (see Figures 2A-2B), and, optionally, the sequences of light chain amino acids in SEQ ID NO 11 or 15 and heavy chain amino acid sequences in SEQ ID NO 12 or 16 (see Figures 3A-3B and 5A-5B). In one embodiment, the antibody of main species is pertuzumab.

An antibody that "inhibits the dimerization of HER" is an antibody that inhibits or interferes with the formation of a dimer or heterodimer of HER. In one embodiment, such an antibody binds to HER2 at its heterodimeric binding site. Here, the most preferred antibody to inhibit dimerization is pertuzumab.

The term "heterodimeric binding site" in HER2 refers to a region in the extracellular domain of HER2 that comes into contact or has an interface with a region in the extracellular domain of EGFR, HER3 or HER4 when the formation of A dimer with him. The region is located in Subdomain II of HER2 (SEQ ID No.: 2). Franklin et al. Cancer Cell 5: 317-328 (2004).

An HER2 antibody that "binds to a heterodimeric binding site" of HER2, binds to residues in Subdomain II (SEQ ID NO: 2) and, optionally, also binds to residues in another of the domains of the extracellular domain of HER2, such as Subdomains I and III (SEQ ID NO: 1 and 3), and can sterically impede, at least to some extent, the formation of a heterodimer HER2-EGFR, HER2-HER3, HER2-HER4. Franklin et al. Cancer Cell 5: 317-328 (2004) characterize the crystal structure of HER2 pertuzumab, registered in the RCSB Protein Data Bank (Identification Code IS78), which illustrates an antibody given by way of example that binds to the binding site HER2 heterodimeric.

An antibody that "binds to Subdomain II" of HER2 binds to residues in Subdomain II (SEQ ID NO: 2) and, optionally, to residues in another Subdomain (s) of HER2, such as Subdomains I and III (SEQ ID NO: 1 and 3, respectively).

For the purposes of this document, the terms "pertuzumab" and "rhuMAb 2C4", which are used interchangeably, refer to an antibody comprising the light variable (VL) and heavy variable (VH) amino acid sequences in SEQ ID NO: 7 and 8, respectively. Figures 22 and 23 in this document illustrate the exemplary biological functions of pertuzumab. When pertuzumab is an intact antibody, it is preferable that it comprises comprising a lgG1 antibody; in an embodiment comprising the amino acid sequence of the light chain in SEQ ID NO 11 or 15, and the amino acid sequence of the heavy chain in SEQ ID NO 12 or 16. The antibody is optionally produced by recombinant cells of Chinese hamster ovary (CHO, Chínese Hamster Ovary). The terms "pertuzumab" and "rhuMAb 2C4" in this document cover biosimilar or intended copies of the drug with the name adopted in the United States (USAN, United Status Adopted Ñame) or International Common Denomination (INN): pertuzumab.

For the purposes of the present, the terms "trastuzumab" and rhuMAb4D5 ", which are used interchangeably, refer to an antibody comprising the light chain (VL) and heavy chain (VH) amino acid sequences from within the SEQ ID Nos: 13 and 14, respectively (see Figures 4A4B). When trastuzumab is an intact antibody, it is preferable that it comprises a lgG1 antibody; in an embodiment comprising the amino acid sequence of the light chain of SEQ ID NO: 13 and the amino acid sequence of the heavy chain of SEQ ID NO: 14 The antibody is optionally produced by Chinese hamster ovary cells (CHO) The terms “trastuzumab” and “rhuMAb4D5” in this document cover biosimilar or intended copies of the drug with the name adopted in the United States (USAN, United States Adopted Ñame) or International Common Denomination (INN, International Nonproprietary Ñame): trastuzumab.

An amino acid sequence variant antibody herein is an antibody with an amino acid sequence that differs from the antibody of the main species. Typically, the amino acid sequence variants will possess a homology of at least about 70% with the antibody of the main species, and preferably, said homology will be at least about 80%, and more preferably at least about 90% with the antibody of the main species. The amino acid sequence variants have substitutions, deletions and / or additions at certain positions within or adjacent to the amino acid sequence of the antibody of the main species. Examples of amino acid sequence variants herein include deamidated antibody variant, antibody with an amino terminal leader extension (eg HSV) in one or two of its light chains, antibody with a C-terminal lysine residue in one or two of its heavy chains, etc., and includes combinations of variations in the amino acid sequences of heavy and / or light chains.

An acid variant is a variant of the antibody of the main species that is more acidic than the antibody of the main species. An acid variant has acquired a negative charge or has lost a positive charge in relation to the antibody of the main species. Such acidic variants can be resolved using a separation methodology, such as ion exchange chromatography, which separates the proteins according to the charge. Acid variants of a main species antibody elute before the main peak upon separation by cation exchange chromatography.

A reduced disulfide variant has one or more inter-chain disulfide bridges reduced to the free thiol form. This variant can be monitored by CE-SDS (capillary electrophoresis not reduced with sodium dodecyl sulfate), for example, as described in WO 2009/099829 (Harris et al.).

Here, a non-reducible variant "or" incompletely reduced variant "is a variant of the main species antibody that cannot be chemically reduced to the heavy chain and light chain by treatment with a reducing agent such as dithiothreitol. Such variants can be evaluated by treating the composition with a reducing agent and evaluating the resulting composition by a methodology that evaluates the size of the protein, such as capillary electrophoresis with sodium dodecyl sulfate (SDSCE), for example by techniques described in WO 2009 / 099.829 (Harris et al.).

Hereby, a "glycosylation variant" is an antibody with one or more carbohydrate moieties attached to them that differ from one or more carbohydrate moieties attached to the antibody of the main species. In one embodiment, the glycosylation variant has oligosaccharide structures attached to one or both heavy chains of an antibody, for example, in residue 299 of the heavy chain. In one embodiment, the antibody of the main species (for example, pertuzumab) comprises oligosaccharide G0 as the predominant oligosaccharide attached to its region of Fe. Examples of oligosaccharide structures attached to lgG1 are shown in Figures 24A-24B. Examples of glycosylation variants herein include afucosylated variant, antibody with an oligosaccharide structure G1 or G2, instead of an oligosaccharide structure G0, fixed to one of its Fe regions ("glycosylation variant G1" or "variant of G2 glycosylation ”), carbohydrate-free antibody bound to one or two heavy chains of the antibody (“ non-glycosylated heavy chain variant ”), sialylated variants, etc., as well as combinations of such glycosylation alterations. See, for example, US Pat. 7,560,111 (Kao et al.).

When the antibody has a region of Fe, there may be an oligosaccharide structure attached to one or two heavy chains of the antibody, for example, in residue 299. In one embodiment, GO is the predominant oligosaccharide structure, having other oligosaccharide structures such as GO-F, G-1, Man5, Man6, G1-1, G1 (1-6), G1 (1-3) and G2 that are present in smaller amounts in the composition.

Unless otherwise indicated, an oligosaccharide structure AG1 herein includes structures G1 (1-6) and G1 (1-3).

For the purposes of the present, a "sialidated variant" is a variant of the main species antibody comprising one or more sialylated carbohydrate moieties attached to one or two of its heavy chains. A sialylated variant can be identified by evaluating a composition (for example by ion exchange chromatography) with or without sialidase treatment, for example, as described in WO 2009/099829

A "glycated variant" is an antibody to which a sugar, such as glucose, has been covalently bound, for example, to one or both of its light chains. This addition can be produced by reacting glucose with a lysine residue in the protein (for example, in a cell culture medium). A glycated variant can be identified by mass spectrometry analysis of the reduced antibody that evaluates the increase in the mass of the heavy or light chains. A glycated variant can also be quantified by boronate chromatography as explained in WO 2009/099829 (Harris et al.).

A "deamidated antibody" is an antibody in which one or more asparagine residues have been derived, for example, to an aspartic acid, a succinimide, or an isoaspartic acid. An example of a discouraged antibody is the variant of pertuzumab, in which Asn-386 and / or Asn-391 in one or two heavy chains of pertuzumab are discouraged. See WO 2009/099829 (Harris et al.), For example.

Here, the term "amino-terminal leader extension variant" refers to an antibody of the main species with one or more amino acid residues of the amino terminal leader sequence at the amino-terminal end of one or more any heavy chains. or light antibody of the main species. An example of amino terminal leader extension comprises or consists of three amino acid residues, HSV, present in one or both light chains of an antibody variant, designated as "HSV-variant" herein. See US Pat. 7,560,111 (Kao et al.).

The term "" C-terminal lysine variant "refers to a variant comprising a lysine (K) in the C-terminal residue of its heavy chain. See US Pat. 7,560,111 (Kaoetal.).

The term "oxidized methionine variant" refers to a variant comprising one or more oxidized methionine residues therein. For example, oxidized Met254. See US Pat. 7,560,111 (Kao et aL).

The term "cancer" refers to the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer of the present invention include breast cancer (eg metastatic breast cancer), gastric (or stomach) cancer, ovarian cancer, primary peritoneal cancer, and fallopian tube cancer. Examples of cancer herein include HER2-positive cancer and low HER3 cancer.

A cancer sample or biological sample that "shows expression, amplification, or activation of HER" is one that, in a diagnostic test, expresses (including overexpressed) an HER receptor, has an amplified HER gene, and / or in some other way demonstrate the activation or phosphorylation of an HER receptor.

A "HER2 positive" cancer comprises cancer cells that have higher levels of HER2 than normal. Examples of HER2 positive cancer include HER2-positive breast cancer and HER2 positive gastric cancer. Methods to identify HER2 positive cancer include: assays that measure HER2 protein such as the immunohistochemistry assay (IHC immunohistochemistry), assays that measure nucleic acid encoding HER2 such as in situ hybridization (ISH) in situ hybridization ), including fluorescent in situ hybridization (FISH, fluorescent in situ hybridization; see WO98 / 45479 published in October 1998) and chromogenic in situ hybridization (CISH), see, for example Tanner et al, Am. J. Pathol 157 (5): 1467-1472 (2000); Bella et al, J. Clin Oncol 26: (May 20 suppl; abstr 22147) (2008)), southern blotting, or PCR techniques (polymerase chain reaction) such as real-time quantitative PCR (QRT-PCR); antigen shed assays (eg, HER2 ECD) (see, for example, U.S. Patent No. 4,933,294 issued June 12, 1990; and U.S. Patent 5,401. 638 issued on March 28, 1995); and rehearsals in vivo. Optionally, HER2 positive cancer has an immunohistochemical score (IHC) of 2 + or 3 + and / or an in situ hybridization amplification ratio (ISH) of £ 2.0.

A "low HER3" cancer comprises cancer cells that have much lower levels of HER3 than normal. Examples of low HER3 cancers include ovarian, primary peritoneal cancer and carcinoma of the fallopian tubes. See, for example, US Pat. No. 7,981,418 (Amler et al.). In one embodiment, the low HER3 is determined on the basis of HER3 mRNA expression levels (concentration ratio equal to or less than 2.81, as evaluated by qRT-PCR in a COBAS z480® instrument) .

The "epitope 2C4" is the region in the extracellular domain of HER2 to which the 2C4 antibody binds. In order to systematically select antibodies that bind essentially to the 2C4 epitope, a routine cross-block assay such as that described in Antibodies, A Laboratory Manual, Coid Spring Harbor Laboratory, Ed Harlow and David Lañe (1988) can be carried out. . It is preferable that the 2C4 antibody blocks bind to HER2 in about 50% or more. Alternatively, epitope mapping can be carried out to assess whether the antibody essentially binds to the 2C4 epitope of HER2. Epitope 2C4 comprises residues of Subdomain II (SEQ ID NO: 2) in the extracellular domain of HER2. 2C4 and pertuzumab join the extracellular domain of HER2 in the meeting of Subdomains I, II and III (SEQ ID NO: 1, 2, and 3, respectively). Franklin etal. Cancer Cali 5: 317-328 (2004).

"Treatment" refers to both therapeutic treatment and prophylactic or preventive measures. Those in need of treatment include those who already have cancer, as well as those in whom cancer is to be prevented. Therefore, the patient to be treated herein may have been diagnosed with cancer or may be predisposed to get cancer or be susceptible to getting it.

The term "effective amount" refers to an amount of an effective drug to treat cancer in the patient. The effective amount of the drug can reduce the number of cancer cells; reduce tumor size; inhibit (ie, slow down to some extent and preferably stop) the infiltration of cancer cells in peripheral organs; inhibit (ie, slow to some extent and preferably stop) tumor metastasis; inhibit, to some extent, tumor growth; and / or relieve to some extent one or more of the symptoms associated with cancer. To the extent that the drug can prevent growth and / or kill existing cancer cells, it can be cytostatic and / or cytotoxic. The effective amount can prolong progression-free survival (for example, as measured based on the evaluation criteria for solid tumor response, RECIST (Response Evaluation Criteria for Solid Tumors) or CA-125 changes), gives place to an objective response (which includes a partial response, PR (partial response), or complete response, CR (complete response), increase in overall survival time, and / or cause an improvement in one or more cancer symptoms (for example, according to the FOSI evaluation).

A "fixed" or "flat" dose of a therapeutic agent herein refers to a dose that is administered to a human patient regardless of the patient's weight (WT) or body surface area (BSA). Therefore, the fixed dose or not is given as a dose in mg / kg or as a dose of mg / m², but rather as an absolute amount of the therapeutic agent.

A "container" refers to an object that can be used to support a pharmaceutical composition or composition. Examples of containers in this document include vial, ampoule, syringe, IV bag, etc.

An "IV bag" or "IV bag" is a bag that can contain a solution that can be given through a patient's vein. In one embodiment, the solution is a saline solution (for example, about 0.9% or about 0.45% NaCl). Optionally, the IV bag is made of polyolefin or polyvinyl chloride.

A "bottle" is a suitable container for containing a liquid or lyophilized preparation. In one embodiment, the bottle is a single-use bottle, for example, a 20 cc single-use bottle provided with a cap.

A “leaflet” is a booklet that, by order of the Food and Drug Administration (FDA) or other regulatory authority, must be placed inside the package or package of each prescription drug. The brochure generally includes the drug's trademark, its generic name, and its mechanism of action; establishes its indications, contraindications, warnings, precautions, adverse effects, and dosage forms; and includes instructions about recommended dose, time and route of administration.

A "pharmaceutical composition" is a composition comprising a pharmaceutically active drug (for example, pertuzumab and its variant forms such as those described herein) and one or more "pharmaceutically active excipients" (eg, buffer, stabilizer, modifier of tonicity, preservatives, surfactant, etc.) that can be safely administered to a human patient. Such compositions may be liquid or lyophilized, for example.

A "recombinant" protein is one that has been produced by a genetically modified host cell, such as a Chinese hamster ovarian host cell (CHO, Chínese Hamster Ovary).

The term "manufacturing scale" refers to the production of a protein drug (eg, an antibody) on a commercial scale, for example, at 12,000 liters (L) or more, using a commercial process approved by the FDA or other Regulation organism.

The term "purification" refers to one or more purification steps, such as protein A chromatography, ion exchange chromatography, etc.

The term "isolated variant" refers to the variant that has been separated from the main species or wild-type antibody by one or more purification or analytical procedures. Said isolated variant can be evaluated to determine its biological activity and / or potency.

II. Antibody compositions (i) Main species antibody

The antibody compositions herein comprise an antibody that binds to HER2 (an HER2 antibody), optionally a humanized HER2 antibody. The humanized antibodies herein may, for example, comprise non-human hypervariable region residues incorporated into a human variable heavy domain and may further comprise a framework framework region (FR) substitution in a selected position of the group consisting of 69H, 71H and 73H using the variable domain numbering system set forth in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991). In one embodiment, the humanized antibody comprises substitutions of FR in two or all positions 69H, 71H and 73H.

An example of a humanized antibody of interest herein comprises the following VH CDR residues:

- GFTFTDYTMX (SEQ ID NO: 17), where X is preferably D or S, for example, GFTFTDYTMD (SEQ ID NO: 23) for CDR-H1;

- DVNPNSGGSIYNQRFKG (SEQ ID NO: 18) for CDR-H2; I

- NLGPSFYFDY (SEQ ID NO: 19) for CDR-H3, which optionally comprises amino acid modifications of those CDR residues, for example, where the modifications essentially maintain or improve the affinity of the antibody. For example, an antibody variant for use in the methods of the present invention may have from about one to about seven or about five amino acid substitutions in the variable heavy CDR sequences mentioned above. Such antibody variants can be prepared by affinity maturation, for example, as described below.

The humanized antibody may comprise VK CDR residues:

- KASQDVSIGVA (SEQ ID NO: 20) for CDR-L1;

- SASYX¹X²X³where X¹ it is preferably R or L, X² it is preferably Y or E, and X³ it is preferably T or S (SEQ ID NO: 21), for example, SASYRYT (SEQ ID NO: 24) for CDR-L2; I

- QQYYIYPYT (SEQ ID NO: 22) for CDR-L3, for example, in addition to those variable heavy domain CDR residues in the preceding paragraph.

Such humanized antibodies optionally comprise amino acid modifications of the above-mentioned CDR residues, for example, where the modifications essentially maintain or improve the affinity of the antibody. For example, the antibody variant of interest may have from about one to about seven or about five amino acid substitutions in the light CVDR sequences mentioned above. Such antibody variants can be prepared by affinity maturation.

The present application also considers affinity matured antibodies that bind to HER2. The genitor antibody may be a human antibody or a humanized antibody, for example, one comprising light variable and / or heavy variable sequences of SEQ ID NO. 7 and 8, respectively (that is, comprising the VL and / or VH of pertuzumab). A mature affinity variant of pertuzumab binds preferentially to the HER2 receptor with an affinity greater than that of murine 2C4 or pertuzumab (for example, about two or about four times, about 100 times or about 1,000 times the improved affinity, for example, as assessed by an HER2 ECD ELISA test). Examples of variable heavy CDR residues for substitution include H28, H30, H34, H35, H64, H96, H99, or combinations of two or more (eg, two, three, four, five, seism seven of these residues) . Examples of light variable CDR residues for alteration include L28, L50, L53, L56, L91, L92, L93, L94, L96, L97 or combinations of two or more (for example, two to three, four, five or even ten of this waste).

Various forms of the humanized antibody or the mature affinity antibody are considered. For example, the humanized antibody or the affinity matured antibody, as an alternative. The humanized antibody or the affinity matured antibody can be an intact antibody, such as an intact lgG1 antibody.

It is preferable that the HER2 antibody (both the HER2 antibody of the main species and its variant antibody) is one that binds to HER2 Subdomain II, inhibits HER dimerization more effectively than trastuzumab, and / or binds to a heterodimeric binding site of HER2. The preferred embodiment herein of the main species antibody antibody is one that comprises the light variable and heavy variable amino acid sequences in SEQ ID NO 3 and 4, and more preferably comprises the light chain amino acid sequences in SEQ ID NO 11 or 15 and heavy chain amino acid sequence in SEQ ID NO 12 or 16.

(ii) Unpaired cysteine ​​variants

Examples 1 and 3 herein describe unpaired cysteine ​​variants of pertuzumab. Analytical assays to isolate, characterize, and quantify such variants include assays that specifically assess intra-chain disulfide bonds (as opposed to disulfide bonds within chains), for example, hydrophobic interaction chromatography (HIC) analysis of antibody fragments. (for example of Fab fragments) as in Example 1, the HIC of an intact antibody as in Example 1, the analysis of differentially labeled antibody peptide mapping as in Example 3, and / or reverse phase high performance liquid chromatography (RP-HPLC) as in Example 3 herein and in Zhang et al. Anal. Chem. 84 (16): 7112-7123 (2012).

In general terms, the predominant form of pertuzumab comprises a disulfide bond between Cys23 and Cys88 in both VL domains of its two domains Fab. See Figure 6.

A cysteine ​​variant unpaired herein, a heterodimeric variant, lacks the Cys23 / Cys88 disulfide bond in the light variable domain (VL) of only one of its Fab regions. See Figure 20B. This was determined to be the predominant unpaired cysteine ​​variant.

An additional unpaired cysteine ​​variant herein, a homodimeric variant, lacks the Cys23 / Cys88 disulfide bonds in its two Fab regions. See Figure 20C.

In one embodiment, the amount of the unpaired cysteine ​​variant in the composition (including homodimer and heterodimeric variant) is &lt;about 25%, for example, determined by Fab hydrophobic interaction chromatography (HIC).

In one embodiment, the amount of the homodimeric variant in the composition is £ 4.9% as determined by intact antibody HIC.

In one embodiment, the amount of heterodimeric variant in the composition is from about 13% to about 18%, for example, determined by intact antibody HIC.

The composition optionally further comprises one or more additional variants as described below.

The invention also relates to an unpaired cysteine ​​variant isolated from pertuzumab, where the unpaired cysteine ​​variant comprises unpaired Cys23 / Cys88 cysteines in one or both of the light variable pertuzumab domains. Such isolated unpaired cysteine ​​variant may comprise or consist of a heterodimeric variant and / or a homodimeric variant. Such variants can be isolated using HIC methods or by other purification methods, and can be subjected to a biological assay such as the potency assay (using HER2-positive breast cancer cells) as shown in the Example 1 segment.

(iii) Afucosilated variant

Examples 2 and 4 herein describe afucosylated variants of pertuzumab and demonstrate how to determine ADCC activity based on the percentage of afucosylated pertuzumab in a composition.

In one embodiment, the invention relates to a composition comprising pertuzumab and an afucosylated variant of pertuzumab, in which the amount of the afucosylated variant is greater than 2% of the composition. See, for example, ant2C4907 2, and Pass 1 in the following Table 9.

In an alternative embodiment, the invention relates to a composition comprising pertuzumab and an afucosiladsa variant of pertuzumab, in which the amount of the afucosylated variant is 0.9 to 4.1% of the composition. This quantity of afucosylated variant can, for example, be quantified using the CE-LIF test validated in Example 4.

Opclonally, the composition further comprises unpaired cysteine ​​variants (heterodimer and / or homodimer as described in the previous chapter) and / or additional variants described in the following.

(iv) LMWS and HMWS

The invention further relates to a low molecular weight species (LMWS) of pertuzumab and / or a high molecular weight species (HMWS) of pertuzumab, either in isolation or in compositions comprising the main species antibody or variants . LMW and HMWS can be isolated, characterized and quantified by various techniques, including, without limitation, high performance liquid chromatography of size exclusion (SE HPLC), and / or capillary electrophoresis with sodium dodecyl sulfate (CE-SDS ).

If the SE-HPLC assay is used (for example, as in Example 5), the amount of the main pertuzumab species and HMWS or LMW in a composition may be:

Main peak: z. about 96%, for example,> about 96.7%, 2: about 97.3%, for example,> about 97.4%. HMWS: £ approximately 2%, for example, approximately 1.7%, for example, <approximately 15%, for example <approximately 1.4%, for example s approximately 0.8%.

LMWS: £ approximately 2%, for example, £ approximately 1.6%, for example, £ approximately 1.2%, for example s approximately 0.6%.

If the NR-CE-SDS test is used (for example as in Example 5), the amount of pertuzumab and HMWS or LMWS main species in a composition can be:

Main Peak: £ approximately 95%, for example, £ approximately 96.0%, for example, 2 approximately 97.8%

HMWS: £ about 1%, for example about 0.6%.

LMWS: &lt;approximately 4%, for example &lt;approximately 3.4%.

For example, the amount of Main Peak or the main pertuzumab species (excluding LMWS and HMWS) determined by CE-SDS can be from about 95% to about 99%, for example, from about 96.0% to about 97.8 %, for example from about 95.3% to about 97.3% of the Main Peak.

Optionally, the LMWS comprises or consists of "Peak 6" obtained by NR-CE-SDS (see, for example Example 5). Said Peak 6 can be determined with a value of about 0.9% to about 2.3%, for example from about 2% to about 2.3% of the composition.

(v) Fragments of Peak 1 and Peak 2 of pertuzumab

The invention further relates to Pico 1 fragments and / or pertuzumab Pico 2 fragments either separately or in isolation or in compositions comprising the fragment (s) and the antibody of the main species. Peak 1 and Peak 2 can be isolated, characterized and quantified by the use of various techniques, including, without limitation, high performance liquid size exclusion chromatography (HPLC SE), and / or sodium dodecyl sulfate capillary electrophoresis (EC -SDS), including R-CE-SDS and NR-CE-SDS. In one embodiment, Peak 1 and Peak 2 are separated and / or analyzed by RCE-SDS, for example as described in Examples 5 and 6 and the corrected peak area (CPA) provides the% Peak 1 of Pico 2 in the composition.

By using the R-CE-SDS test (for example, as in Examples 5 and 6), the amount of Peak 1 in a composition is &lt;5% (for example, from 0.13% to 0.41% of CPA) and the amount of Peak 2 in a composition is &lt;1.0% (for example, 0.47% to 0.74% CPA) (vi) Additional variants

The compositions of the present invention optionally comprise additional variants of pertuzumab such as those described in US Pat. 7,560,111 (Kao et al.) And / or in WO 2009/099829 (Harris et al.).

Examples of such additional variants include, without limitation, any one or more of the following: glycated variant, reduced disulfide variant, non-reducible variant, deamidated variant, sialylated variant, HSV variant, C-terminal lysine variant, oxidized methionine variant , afucosylated variant, glycosylation variant G1, glycosylation variant G2, and non-glycosylated heavy chain variant.

For example, the composition may comprise acidic variants (see WO 2009/099829, Harris et al.), Wherein the acidic variants in the composition may include one, two, three, four, or five of the following: glycated variant , deamidated variant, reduced disulfide variant, sialylated variant, and non-reducible variant. It is preferable that the total amount of all acidic variants in the composition be less than about 25%. In one embodiment, the glycated variant, deamidated variant, reduced disulfide variant, sialylated variant, and non-reducible variant constitute at least about 75-80% of the acidic variants present in the composition.

Acid variants can be evaluated by a variety of methods, but preferably such methods include one, two, three, four, or five of the following: ion exchange chromatography (IEC) in which the composition is treated with sialidase before, after, and / or during the IEC (for example, to evaluate the sialylated variant), reduced CE-SDS (for example, to evaluate the variant of reduced disulfide), non-reduced CE-SDS (for example, to evaluate the non-reducible variant), boranato chromatography (for example, to evaluate the glycated variant), and peptide mapping (for example, to evaluate deamidated variant).

The composition optionally includes an amino terminal leader extension variant. It is preferable that the amino terminal leader extension is in a light chain of the antibody variant (for example, in one or both light chains of the antibody variant). The antibody variant herein may comprise an amino-terminal leader extension in any one or more of its heavy or light chains. It is preferable that the amino terminal leader extension be in one or more light chains of the antibody. The amino-terminal leader extension preferably comprises or consists of HSV (ie, HSV variant). The presence of the amino-terminal leader extension in the composition can be detected by various analytical techniques, which include, without limitation: Nterminal sequence analysis, assay to establish the heterogeneity of the charge (eg, cation exchange chromatography or electrophoresis capillary zone), mass spectrometry, etc. The amount of the antibody variant in the composition generally varies between an amount that constitutes the lower limit of detection of any assay (preferably cation exchange analysis) that is used to detect the variant at an amount less than the amount of the species antibody. principal. Generally, from about 20% or less (for example, from about 1% to about 15%, for example from about 5% to about 15%, and preferably from about 8% to about 12%) of the antibody molecules in the composition they comprise an amino-terminal leader extension. Such percentage amounts are preferably determined by cation exchange analysis.

Other alterations of the amino acid sequence of the main and / or variant species antibody are considered, including, without limitation, an antibody comprising a C-terminal lysine residue in one or both of its heavy chains (a variant of said antibody it may be present in an amount of about 1% to about 20%), antibody with one or more oxidized methionine residues (eg, pertuzumab comprising oxidized Met-254), etc.

In addition, apart from the afucosylated variant of the slalilated variant described above, the antibody or variant of the main species may comprise additional glycosylation variations, non-limiting examples of which include antibody comprising an oligosaccharide structure G1 or G2 fixed to its FC region , antibody comprising one or two non-glycosylated heavy chains, etc.

III. Manufacturing and analytical methods

According to an embodiment of the invention, there is provided a method for evaluating a pertuzumab composition comprising one, two, three, or four of the following: (1) the measurement of the amount of unpaired cysteine ​​variant in the composition, wherein the unpaired cysteine ​​variant comprises unpaired Cys23 / Cys88 cysteines in one or both light variable domains of pertuzumab, and / or (2) the measurement of the amount of afucosylated pertuzumab in the composition, and / or (3) the measurement of the quantity of low molecular weight species (LMWS) of pertuzumab in the composition, and / or (4) the measurement of the amount of high molecular weight species (HMWS) of pertuzumab in the composition. Optionally, all four analytical tests are performed in a composition comprising pertuzumab and its variants.

The invention also relates to a method for preparing a composition comprising: (1) the production of a composition comprising pertuzumab and one or more of its variants; and (2) subject the composition thus produced to one or more tests to evaluate the amount of the variant or variant in it. The analytical test (s) allow to evaluate and quantify the amount of any one or more of the following: (i) the unpaired cysteine ​​variant comprising the Cys23 / Cys88 cysteines paired in one or both light variable domains of pertuzumab and / or (ii) a heterodimeric variant comprising the Cys23 / Cys88 cysteines paired in a single light variable domain of pertuzumab and / or (iii) a homodimeric variant comprising Cys23 / Cys88 cysteines not paired in both light variable domains of pertuzumab and / or (iv) the afucosylated variant of pertuzumab and / or (v) the high species molecular weight (HMWS) of pertuzumab and / or (vi) the low molecular weight species (LMWS) of pertuzumab, and / or (vü) the fragment or fragments of Peak 1 of pertuzumab and / or the or fragments of Peak 2 of pertuzumab. Therefore, it is possible to analyze one, two, three, four, five, six, seven or eight of these variants.

Optionally, the analytical test evaluates, quantifies, or isolates the unpaired cysteine ​​variant, including heterodimeric and / or homodimeric variants. For example, the analytical assay may comprise hydrophobic interaction chromatography (HIC) of an antibody fragment (eg, a Fab fragment) or an intact antibody (see, for example, Example 1), peptide mapping analysis (see, for example, Example 3), or reverse phase high performance liquid chromatography (HPLC) (see, for example, Example 3).

In one embodiment, the amount of the unpaired cysteine ​​variant (heterodimeric and / or homodimeric variant) in the composition is about 25% as determined by Fab hydrophobic interaction chromatography (HIC)

In one embodiment, the amount of the homodimeric variant in the composition is &lt;4.9% determined by hydrophobic interaction chromatography (HIC) of intact antibody.

In one embodiment, the amount of the heterodimeric variant in the composition is from about 13% to about 18% as determined by intact antibody hydrophobic interaction chromatography (HIC).

Optionally, the analytical test evaluates, quantifies or isolates afucosylated variant. The amount of afucosylation can be used to determine or quantify the biological activity, for example the ADCC, of ​​the composition.

In addition, the method comprises evaluating the biological activity of a pertuzumab composition comprising measuring the amount of afucosylated pertuzumab variant in the composition to determine the activity of the antibody-dependent cell-mediated cytotoxicity (ADCC) of the composition. , and to confirm that the amount of afucosylated pertuzumab is in the range of 0.9% to about 4.1%. For example, the method comprises measuring the amount of afucosylated pertuzumab by capillary electrophoresis - laser induced fluorescence (CELIF).

Optionally, the analytical test for the evaluation of afucosylation is capillary electrophoresis (EC), including capillary electrophoresis - laser induced fluorescence (CE-LIF), see Examples 2 and 4 below. The amount of afucosylated variant is optionally from about 0.9 to about 4.1% of the composition (for example, measured by CE-LIF in Example 4). In one embodiment, the amount of afucosylated variant is greater than 2% of the composition (for example, as measured by CE-LIF in Example 4).

Optionally, the analytical test evaluates, quantifies, or isolates species of low molecular weight (LMW) and / or species of high molecular weight (HMWS) of pertuzumab. The tests given as examples include SE-HPLC and / or CE-SDS (see, for example, the following Example 5).

In one embodiment, the analytical assay comprises SE-HPLC (for example, as in Example 5) and the amount of pertuzumab, HMWS or LMW main species in a composition thus analyzed and determined is:

Main Peak:> about 96%, for example,> about 96.7%, £ about 97.3%, for example, S: at about 97.4%.

HMWS: £ approximately 2%, for example, £ approximately 1.7%, for example, approximately 1.5%, for example &lt;approximately 1.4%, for example £ approximately 0.8%.

LMWS: approximately 2%, for example, £ approximately 1.6%, for example, &lt;approximately 1.2%, for example approximately 0.6%.

In one embodiment, the analytical assay comprises CE-SDS (for example, as in Example 5), and the amount of pertuzumab and pertuzumab main species of HMWS or LMW in a composition thus analyzed and determined is:

Main Peak:> approximately 95%, for example, £ approximately 96.0%, for example, £ approximately 97.8%

HMWS: s approximately 1%, for example &lt;approximately 0.6%.

LMWS: &lt;approximately 4%, for example &lt;approximately 3.4%.

In one embodiment, a composition is evaluated by NR-CE-SDS, and the amount of Main Peak or pertuzumab main species (excluding LMW and HMWS) proves to be from about 95% to about 99%, for example, of about 96.0 to about 97.8%, for example from about 95.3% to about 97.3% of the composition thus analyzed.

In one embodiment, the amount of "Peak 6" in a composition is evaluated by CE-SDS (see, for example Example 5), and the amount of LMWS of Peak 6 determined is from about 0.9% to about 2.3%, for example, from about 2% to about 2.3% of a composition thus analyzed.

In one embodiment, the amount of Peak 1 and / or Peak 2 in a composition is evaluated by R-CE-SDS (see, for example, Examples 5 and 6), and the amount of Peak 1 determined is 5 % (for example, 0.13% to 0.41% CPA) and the amount of Peak 2 determined is £ 1.0% (for example, 0.47% to 0.74% CPA).

The methods optionally further comprise combining the purified composition with one or more pharmaceutically acceptable excipients to prepare a pharmaceutical composition. In addition, the pharmaceutical composition can be placed in a container that is packaged together with a package or booklet insert (for example, with the prescription information that instructing the user how to use the pharmaceutical composition to treat cancer) so as to obtain a manufacturing item.

IV. Pharmaceutical compositions

Pharmaceutical compositions comprising pertuzumab and its variants are prepared for storage by mixing the composition having the desired degree of purity with optional pharmaceutically acceptable excipients (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980), generally in the form of lyophilized formulations or aqueous solutions are also considered antibody crystals (see US patent application 2002/0136719). Pharmaceutically acceptable excipients are non-toxic to the receptors at the doses and concentrations employed, and include buffers such as histidine acetate; ant ioxidantes including ascorbic acid and methionine; low molecular weight polypeptides (less than about 10 residues); proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt forming counterions such as sodium; metal complexes (for example, Zn-protein complexes); and / or non-ionic surfactants such as polysorbates (eg, polysorbate 20 or 80), PLURONICS ™ or polyethylene glycol (PEG).

Lyophilized antibody formulations are described in US Pat. No. 6,267,958, U.S. Pat. No. 6,685,940 and U.S. Pat. No. 6,821,515, which are expressly incorporated herein by reference. An example trastuzumab pharmaceutical composition is a sterile lyophilized powder, white to pale yellow, without preservatives, for intravenous (IV) administration, comprising 440 mg of trastuzumab, 400 mg of a, dehydrated trehalose, 9.9 mg of L-histidine-HCI, 6.4 mg of L -histidine, and 1.8 mg of polysorbate 20. The reconstitution of 20 ml of bacteriostatic water for injections (BWFI), which contains 1.1% benzyl alcohol as a preservative, allows a multi-dose solution containing 21 mg / ml of trastuzumab to be obtained at pH of approximately 6.0.

An example of a pharmaceutical composition of pertuzumab for therapeutic use comprises 30 mg / ml of pertuzumab in 20 mM histidine acetate, 120 mM sucrose, 0.02% polysorbate 20, at pH 6.0. An alternative pertuzumab formulation comprises 25 mg / ml pertuzumab, histidine buffer — HC110 mM, 240 mM sucrose, 0.02% polysorbate 20, pH 6.0.

Pharmaceutical compositions to be used for in vivo administration must be sterile. This is easily achieved by filtration through sterile filtration membranes.

V. Therapeutic applications and uses

The compositions of this invention can be used to treat cancer, such as HER2-positive breast cancer, for example, metastatic or locally recurrent, non-resectable breast cancer, or de novo Stage IV disease, which herein defined as immunohistochemistry (IHC) 3+ and / or in situ hybridization of FIS fluorescence) with a £ 2.0 amplification. Optionally, patients in the population have not received prior treatment or have had a relapse after adjuvant therapy, have a left ventricular ejection fraction (LVEF, left ventricular ejection fraction,> 50% at baseline, and / or have a performance status of the Eastern Cooperative Ontology Group (ECOG PS Eastern Cooperative Oncology Group performance status) of 0 or 1.

In an alternative embodiment, it is possible to use the composition to treat early-stage HER2-positive breast cancer, for example, in combination with trastuzumab and chemotherapy, in which chemotherapy comprises anthracycline-based chemotherapy or carboplatin-based chemotherapy. . In one embodiment, the chemotherapy comprises anthracycline-based chemotherapy, for example comprising 5 -FU, epirubicin and cyclophosphamide (FEC). In an alternative embodiment, the chemotherapy comprises carboplatin-based chemotherapy, for example comprising taxane (for example, Docetaxel), carboplatin, in addition to HERCEPTIB® / trastuzumab (for example, a HRCT regime). In one embodiment, the composition is administered simultaneously with anthracycline-based chemotherapy or with carboplatin-based chemotherapy, for example, in which pertuzumab, trastuzumab and chemotherapy are administered in 3-week cycles, and pertuzumab, trastuzumab and chemotherapy are given on day -1 of each cycle. The treatment of early-stage HER2-positive breast cancer considered herein includes neoadjuvant adjuvant and adjuvant therapy.

In yet another embodiment, the composition can be used to treat HER2-positive gastric cancer, optionally in combination with trastuzumab and chemotherapy, such as a platinum (for example, cisplatin) and / or a fluoropurimidine (for example, capecitabine and / or 5-fluorouracil (5-FU))

In an alternative embodiment, the composition can be used to treat HER2-positive breast cancer optionally in combination with trastuzumab and vinorelbine. Breast cancer according to this embodiment is optionally metastatic or is locally advanced. Optionally, the patient has not previously received non-hormonal systemic therapy in metastatic situations.

In another aspect, the composition is used to treat HER2-positive breast cancer in a patient, which comprises administering the composition, trastuzumab, and the aromatase inhibitor (eg, anastrozole or letrozole) to the patient. According to this embodiment, breast cancer is an advanced breast cancer, including hormone receptor positive breast cancer such as estrogen receptor positive (ER) and / or progesterone receptor positive ( PgR). Optionally, the patient has not previously received non-hormonal systemic therapy against cancer in the metastatic situation. This method of treatment optionally further comprises the administration of induction chemotherapy (for example comprising taxane) to the patient.

In a further aspect, the composition is used to treat low HER3 cancer, such as ovarian cancer, primary peritoneal cancer or fallopian tube cancer. See, for example, the U.S. patent. 7,981,418 (Amler et al.) And U.S. Patent Publication US-2006-0013819-A1 (Kelsey, S.).

According to a particular embodiment of the invention, approximately 840 mg (loading dose) of pertuzumab is administered, followed by one or more doses of approximately 420 mg (one or more maintenance doses of pertuzumab). The maintenance doses are preferably administered approximately every 3 weeks, for a total of at least two doses, to progressive clinical disease, or unmanageable toxicity, for example, from 6 to 20 doses. Longer treatment periods are also considered, including a greater number of cycles.

According to another particular embodiment in which the cancer is gastric cancer, pertuzumab is administered at a dose of 840 mg for all treatment cycles.

SAW. Manufacturing items

An embodiment of an article of manufacture of the present comprises a container, such as a vial or ampoule, syringe, or intravenous (IV) bag containing the pharmaceutical composition or composition of the present. Optionally, the article of manufacture also includes a leaflet or brochure with information about the prescription that describes how to use the composition according to the previous chapter in this document.

Vile. Biological Materials Registry

The following hybridoma cell lines have been registered in the

Amerlcan Type Culture Collection, 10801 University Boulevard, Manassas, VA

20110-2209, USA (ATCC)

ATCC antibody designation No.

Registration Date

4D5

ATCC CRL 10463 May 1990

2C4

ATCC HB-12697 April 1999

Other details of the invention are illustrated by the following non-limiting examples. The disclosures of all references in the specification are expressly incorporated herein by reference.

EXAMPLE 1

Cys23 / Cvs88 unpaired cysteine ​​variant of pertuzumab and its characterization

Pertuzumab is a humanized monoclonal antibody (MAb) based on a human lgG1 (K) framework. The recombinant antibody is produced by Chinese hamster ovary (CHO) cells and comprises two heavy chains (449 amino acid residues each) and two light chains (214 amino acid residues each) with interchain and intra-chain disulfide bonds. The light chain and heavy chain sequences of pertuzumab are shown in Figures 3A and 3B, respectively. The calculated molecular mass of intact pertuzumab is 145,197 Da (peptide chains only, without the heavy chain C-terminal lysine residue).

The CH2 domain of each heavy chain also has a single glycosylation site conserved in Asn299.

Pertuzumab differs from trastuzumab (HERCEPTIN®) in the complementarity determining regions (CDRs) of the light chain (differences of 12 amino acids) and the heavy chain (29 differences of amino acids), and the fact that it binds to an epitope different at human epidermal growth factor receptor 2 (p185^HER2). The binding of pertuzumab to the HER2 receptor on human epithelial cells prevents HER2 from complexing with other members of the HER receptor family (including EGFR, HER3, HER4) and forming HER2 homodimers. By blocking the formation of complexes, pertuzumab inhibits intracellular signaling initiated by ligand through two important signal pathways, the mitogen-activated protein (MAP) kinase (MAP) and the phosphoinositide 3 kinase (PI3K), resulting in inhibition of cell proliferation and survival, respectively.

This example refers to the identification and characterization of an unpaired cysteine ​​variant of pertuzumab: the unpaired cysteine ​​variant Cys23 / Cys88 comprising unpaired cysteines in one or both light chains of the antibody.

Free sulfhydryls were measured using Ellman's reagent, and showed a reactive free sulfhydryl content of 0.1 to 0.3 moles per mole of protein. Hydrophobic interaction chromatography (HIC) analysis and peptide map analysis revealed unpaired cysteine ​​residues in Cys23 and Cys88 in one or both light chains. Through the use of papain HIC, it was found that the levels of the Fab variant containing free sulfhydryls at these sites were 12.7% -13.5% in pertuzumab materials produced using the commercial manufacturing process. The HIC analysis of the intact antibody indicated that the two main forms are 78% -85% wild-type pertuzumab and 13.4% -18.4% heterodimeric pertuzumab (pair of unpaired cysteines in one arm)

MATERIALS AND METHODS

Compositions tested: This example describes the characterization of the current standard reference batch of pertuzumab ant2C4907 2 and Pass 1, which represents phase III clinical material, and five commercial phase III batches (Passes 3-7), all produced at a scale of 12,000 liters (L) using the commercial process. The comparison is also made with the previous batch of previous standard reference ant¡2C4 900 1, which is representative of the clinical material Phase l / ll.

The compositions tested were batches of drug substance formulated in the commercial formulation at 30 mg / ml in 20 mM Lhistidine acetate, 120 mM sucrose, and 0.02% (w / v) polysorbate 20 at pH 6.0. The ant2C4 900 1 lot was previously formulated in clinical development at 25 mg / ml in 10 mM L-histidine chloride, 240 mM sucrose, and 0.02% (w / v) polysorbate 20 at pH 6.0.

Analysis of the disulfide bond by non-reduced peptide Map and mass spectrometry: To denature pertuzumab under non-reducing conditions and to rent any buried free sulfhydryl groups, approximately 0.5 mg of pertuzumab in formulation buffer was mixed with denaturation buffer (consistent in 8 M GdHCI, 10 mM Netylmaleimide (NEM), 0.1 M sodium acetate, pH 5.0) and then incubated at 37 ° C for 3 hours. The solution buffer was exchanged in 600 μΙ of 0.1 M TRis, 1 mM CaCh, pH 7.0 using -5 columns. Acetonitrile (ACN) was added to each sample to achieve a concentration of 10%. Trypsin digestion was carried out at a ratio between enzyme and substrate of 1:10 (weight / weight) at 37 ° C for 16 hours. The resulting peptides were separated by RP-HPLC using the methods described below for tryptic sulfitolysis maps.

Map of tryptic sulfitolysis peptides: To generate pertuzumab peptide maps, the protein was digested with trypsin after reduction and sulfitolysis of cysteine ​​residues. Aliquots (1 mg) of pertuzumab were added to Tris HCI 360 mM pH 8.6, 6 M guanidine hydrochloride (GdHCI), ethylenediaminetetraacetic acid (EDTA2 mM), 13 mM sodium sulphite, and sodium tetrathionate 38 mM for the reduction and sulfitolysis of cysteine ​​residues. The samples were incubated at 37 ° C for 20 minutes. Sulfitolized samples were loaded on PD-10 columns and eluted with 10 mM Tris,

0.1 mM CaCh, pH 8.3. After buffer exchange, 20 μl of a 10% octyl glycoside B solution was added and 20 μΙ of 1 mg / ml trypsin was loaded. The samples were incubated at 37 ° C for 5 hours. The digestion reaction was quenched with 25 μΙ of 10% trifluoroacetic acid (TFA). The resulting peptides were separated by RP-HPLC using a Zorbax 300SB-C8 column (4.6 mm x 150 mm). The peptides were separated after a 5 minute wait under the initial conditions with a linear gradient of 0% to 17% of solvent B in 57 minutes, to 32% of solvent B in 149 minutes, to 45% of solvent B in 162 minutes, and 95% of solvent B in 173 minutes. At 179 minutes, the column was reconditioned to 100% solvent A for 25 minutes, for a total run time of 204 minutes. Solvent A consisted of 0.1% TFA in water and solvent B consisted of 0.08% TFA in acetonitrile. The column was maintained at 37 ° C and eluted at a flow rate of 0.5 ml / min. The elution profile was monitored at 214 nm and 280 nm. The masses of the tryptic peptides were determined by liquid chromatography analysis of mass spectrometry (LC-MS) of the separated digest mixture for which an ORBITRAP ™ mass spectrometer was used.

Free sulfhydryl content by Ellman analysis: The buffer of pertuzumab samples was exchanged for reaction buffer (100 mM potassium phosphate, 1 mM EDTA, 8 M urea, pH 8) and adjusted to a concentration that resulted Free thiol concentrations within the range of the standard curve. A solution of ditionitrobenzene (DTNB) (10 mM) and a standard cysteine ​​curve (eight points between 0 and 100 mM) were prepared in reaction buffer. In a 96-well plate, 165 pl of sample or standard in cavities were added, in triplicate. The reaction was initiated by the addition of 10 μΙ of DTNB and then incubated for 30 minutes. After incubation, absorbance at 412 nm was measured using a SPECTRAMAX M plate reader²®. The concentration of free thiol was calculated by the linear equation obtained from the standard curve. The protein concentration was determined by absorbance at 280 nm obtained from a spectrophotometer. Free thiol is reported as moles of free thiol per mole of pertuzumab.

HIC by Papain: For samples of pertuzumab digested by papain, samples were digested with papain after removing lysine from terminal C with carboxypeptidase B (CpB). The Fab and Fe domains were separated by HIC for which a PopIyPropyl Aspartamide column (4.6 mm * 100 mm, 1,500 A, 3 pm) was used. Solvent A consisted of 1.6 M ammonium sulfate, 20 mM potassium phosphate, pH 6.05 and solvent B consisted of 20 mM potassium phosphate pH 6.05. The analytes were separated with a gradient of 0% to 18% of solvent B from 3 to 6 minutes, to 24% of solvent B in 21 minutes. The column was maintained at 25 ° C with a flow rate of 0.8 ml / min. The elution profile was monitored at 280 nm.

HIC of intact antibody: Intact pertuzumab samples were separated by HIC using a PolyPropyl Aspartamide column (9.4 mm χ 100 mm, 1500 A, 3 pm). Solvent A consisted of 1.0 M ammonium sulfate, 20 mM potassium phosphate, pH 6.05 and solvent B consisted of mM potassium phosphate, pH 6.05. The analytes were separated socratically with 12% solvent B for 25 minutes. The column was maintained at 30 ° C with a flow rate of 2 ml / min. The elution profile was monitored at 280 nm.

SDS-PAGE with digital peptide mass printing: Both reduced and non-reduced pertuzumab samples were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Samples (5 pg) were denatured by heating in the presence of SDS PAGE sample buffer for 5-10 minutes at 60 ± 2 ° C with iodoacetamide for non-reduced samples. Samples were reduced for 15-20 minutes to 60⁰ C ± 2 ° C in the presence of 80 m dithiothreitol; (DTT). Denatured samples were separated on 4% polyacrylamide gels and 20% gradient polyacrylamide gels and stained with SYPRO ™ Ruby dye so as to obtain the protein band pattern. Together with pertuzumab samples, molecular weight standards and SYPRO ™ patterns of Ruby dye sensitivity (2 ng / lane and 8 ng / bovine serum albumin lane (BSA)) were included in the gels.

Digital mass impressions of peptides is an analytical technique for protein identification. The gels were loaded with 10 pg of standard commercial reference lot ant2C4907 2 and Pass 5. All bands separated by SDS-PAGE were cleaved into peptides by trypsin. The absolute masses of the peptides are accurately measured with the laser-assisted matrix BRUKER ™ instrument for the desorption / ionization time of flight mass spectrometry (MALDI-TOF MS). Peptide mass lists were used to identify proteins by searching for protein sequences. All bands observed in both non-reduced pertuzumab and reduced pertuzumab were identified by digital peptide printing.

Potency by Bioassay: The pertuzumab potency method evaluates the potency of pertuzumab by measuring its ability to inhibit the proliferation of a breast cancer cell line that expresses human HER2. In a typical assay, one or more 96-cavity microtiter plates were seeded with breast cancer cells and incubated in a humidified incubator. After incubation, the medium was removed and variable concentrations of standard reference pertuzumab, assay control, and sample (s) were added to the plate (s). The plate (s) were then incubated, and the relative number of viable cells was indirectly quantified using a redox dye, ALAMARBLUE®. Fluorescence was measured using excitation at 530 nm and emission at 590 nm. ALAMARBLUE® is blue and non-fluorescent in its oxidized state, but is reduced by the intracellular environment of the cell by acquiring a pink form that is highly fluorescent (Page et al. Int. J. Oncol 3: 473-476 (1993) ). Changes in color and fluorescence are proportional to the number of viable cells. The results, expressed in relative fluorescence units (RFU) were plotted against pertuzumab concentrations and a parallel line program was used to estimate the anti-proliferative activity of pertuzumab samples in relation to the Reference Standard.

RESULTS

Assignment of disulfide bonds: there are 32 cysteines in pertuzumab, which form 16 disulfide bonds, of which four bonds are between chains and 12 are links within chains. However, due to the multimeric nature of the molecule, there are only nine different disulfide bonds. The native protein was digested with trypsin to achieve the release of all peptides with disulfide bonds. Chromatographic profiles for pertuzumab batches are shown in Figure. 7. The reverse phase LC-MS analysis of the digest of the standard commercial reference lot ant2C4907 2 allowed to obtain all the pairs of 10 peptide bound peptides provided (Table 2).

Table 2 - Disulfide-linked peptide pairs identified by LC-MS

<td>Provided³</td><td>Disulfide bond</td><td>Found</td><td>Mass previst a (Da)^b</td><td>Observed mass (Da)^b</td>

<td>T2H = T10H</td><td>Cys22 = Cys96</td><td>T2H = T10H</td><td> 3429,4</td><td> 3429,4</td>

<td></td><td></td><td></td><td> 8</td><td> 8</td>

<td>T13H = T14H</td><td>Cys146 = Cys202</td><td>T13H = T14H</td><td> 7917,9</td><td> 7917,9</td>

<td></td><td></td><td></td><td> 2</td><td> 2</td>

<td>T19H = T19H</td><td>Cys228 = Cys228</td><td>T19H = T19H</td><td> 5455,7</td><td> 5455,7</td>

<td></td><td>Cys231 = Cys231^c</td><td></td><td> 8</td><td> 9</td>

<td>T21H = T27H</td><td>Cys263 = Cys323</td><td>T21H = T27H</td><td> 2329,1</td><td> 2329,1</td>

<td></td><td></td><td></td><td> 0</td><td> 0</td>

<td>T35H = T40H</td><td>Cys369 = Cys427</td><td>T35H = T40H</td><td> 3845,8</td><td> 3845,8</td>

<td></td><td></td><td></td><td> 2</td><td> 2</td>

<td>T18H = T20L</td><td>Cys222 = Cys214</td><td>T18H = T20L</td><td> 757,24</td><td> 757,24</td>

<td>T18H = T20L</td><td>Cys222 = Cys214</td><td>T18H = T19L-</td><td> 1261,4</td><td> 1261,4</td>

<td></td><td></td><td>T20L^d</td><td> 9</td><td> 9</td>

<td>T2L = T7L</td><td>Cys23 = Cys88</td><td>T2L = T7L</td><td> 5393,4</td><td> 5393,4</td>

<td></td><td></td><td></td><td> 8</td><td> 8</td>

<td>T11L = T18L</td><td>Cys134 = Cys194</td><td>T11L = T18L</td><td> 3556,7</td><td> 3556,7</td>

<td></td><td></td><td></td><td> 5</td><td> 5</td>

Note: an equal sign (=) represents a disulfide bond.

Η = heavy chain; L = light chain; LC-MS = high performance liquid chromatography mass spectrometry; T = tryptic peptide.

^to Refer to Figures 9 and 10. ^b Monoisotopic masses (MH⁺).

^c The disulfides are inferred. The allocation of the T19H dimer did not include the verification of the disulfides Cys228 = Cys228 and Cys231 = Cys23.

^d The presence of this disulfide bound pair has been confirmed by the use of an additional enzyme, Lys-C, which does not divate T19L or T20L.

The identified peptides were further confirmed by the identification of peptides predicted from the peptide pairs after the reduction of the disulfides (Figure 8 with the expanded views in Figures 9 and 10). A dimer of the T19H heavy chain peptide (T19H = T19H) was identified as containing two disulfide bonds; the identification of the pairs Cys228 = Cys228 and Cys231 = Cys231 is inferred. A pair of disulfides, T18H = T20L, was detected by LC-MS but eluted near the void volume and was not dentifiable as a peak in the chromatograms (UV). The presence of this pair of disulfides was further confirmed by LC-MS analysis of a Lys C digest, in which the peptide T18H = T19L-T20L was observed. No unexpected links were found. A disulfide bond is partially unpaired, as set forth below.

Suifhydryl free analysis: all cysteine ​​residues in pertuzumab properly folded should participate in disulfide bonds. The Ellman assay (Ellman, G. Arch Biochem Biophys 82: 70-77 (1959)), a method for measuring the content of sulfhydryl-free peptides and proteins, was used to determine whether non-modified (free) reactive thiols are present. in pertuzumab. All materials were evaluated to determine free thiol (unpaired cysteine ​​residue), and the results have been summarized in Table 3.

Table 3: Free thiol content by Ellman test

<td></td><td>Moles of free thiols per mole of</td>

<td>Lot name</td><td>pertuzumab</td>

<td>Ant2C4-900-1</td><td> 0,06</td>

<td>ant2C4907-2</td><td> 0,15</td>

<td>Past 1</td><td> 0,28</td>

<td>Past 3</td><td> 0,16</td>

<td>Past 4</td><td> 0,17</td>

<td>Past 5</td><td> 0,16</td>

<td>Past 6</td><td> 0,16</td>

<td>Past 7</td><td> 0,14</td>

Note: Free thiol levels were determined by the Ellman assay in the presence of 8 M urea.

Approximately 0.1-0.3 moles of free thiols per mole of pertuzumab were observed in all batches analyzed. In the absence of 8 M urea, the free thiol levels were below the quantification limit (QL; approximately 0.1 mol of free thiol per mole of protein) in all the materials tested, indicating that the free thiols (is that is, unpaired cysteines) present in pertuzumab molecules were buried and were inaccessible to Ellman reagent 5 under the condition of non-denaturation

Analysis of pertuzumab materials by HIC after CpB and papain digestion revealed an additional peak between the Fe and Fab peaks that was identified as a Fab variant that contains unpaired cysteine ​​residues in Cys23 and Cys88 (Figures 11 and 12, marked as thiol-free Fab). This identification was confirmed by mapping MS LC tryptic peptides, in which the sample was subjected to denaturation in the presence of NEM before reduction and tryptic digestion. The amplitude of the free thiol variant was measured using the HIC method with papain through the pertuzumab batches and found to be consistent with the current process (Table 4).

Table 4: Relative amount of unpaired Cys23 / Cys88 cysteine ​​Fab variant determined by HIC with papain or calculated intact antibody variant

<td></td><td colspan="2">Percentage</td><td>from</td><td>Percentage</td><td>from</td>

<td> 5</td><td>Name of</td><td>Fab variant</td><td>from</td><td>variant</td><td>from</td>

<td></td><td>lot</td><td colspan="2">unpaired cysteine</td><td>intact antibody</td><td> ★</td>

<td></td><td>Ant2C4-900-</td><td></td><td></td><td> 17,9</td><td></td>

<td></td><td></td><td> 9,4</td><td></td><td></td><td></td>

<td></td><td> 1</td><td></td><td></td><td></td><td></td>

<td></td><td>ant2C4907-2</td><td> 12,7</td><td></td><td> 23,8</td><td></td>

<td> 10</td><td>Past 1</td><td> 13,2</td><td></td><td> 24,6</td><td></td>

<td></td><td>Past 3</td><td> 13,3</td><td></td><td> 24,9</td><td></td>

<td></td><td>Past 4</td><td> 13,5</td><td></td><td> 25,2</td><td></td>

<td></td><td>Past 5</td><td> 13,3</td><td></td><td> 24,9</td><td></td>

<td></td><td>Past 6</td><td> 12,9</td><td></td><td> 24,2</td><td></td>

<td> 15</td><td>Past 7</td><td> 13,2</td><td></td><td> 24,6</td><td></td>

Note: The percentage of unpaired cysteine ​​Fab peak was obtained by dividing unpaired cysteine ​​Fab peak by unpaired cysteine ​​+ Fab peak areas.

* Calculated as described below.

Through the papain HIC test, the values ​​for the material produced by the commercial process ranged from 12.7% to 13.5%, while the value for the standard reference lot 2C4 900 1 (Phase l / ll ) was slightly lower than 9.4%.

Conversion of% of HIC FAb variant with papain Estimated% of intact antibody variant: The relative amount of Fab fragments containing unpaired cysteines can be used to calculate the relative distribution of heterodimeric or homodimeric forms of unpaired cysteine ​​variants . If the HIC test with papain shows that 10% (or X%) of Fauz fragments of pertuzumab contain unpaired cysteines in Cys23 / Cys88, then there must be 10 Fab fragments containing unpaired cysteines released from every 50 molecules of pertuzumab because the digestion of the 50 antibodies by papain should produce 100 Fab fragments. Assuming that these 10 Fab fragments are from 10 different pertuzumab molecules, the relative amount of pertuzumab q containing one Fab with mismatched Cys23 / Cys88 cysteines is approximately 20%, that is, 10 out of 50 pertuzumab molecules, (or 2x%) . Stated more precisely, if the probability of a pertuzumab with two Fab containing unpaired Cys23 / Cys88 cysteines is taken into account, then the relative amount of perturbed cysteine ​​heterodimer variants of pertuzumab should be 2 χ 10% χ 90% = 18 % (or 2 ^x x% χ [100-x]%). In addition, the relative amount of unpaired cysteine ​​variants of pertuzumab homodimer should be 10% χ 10% = 1% (ox% χ x%). In this case, the ratio between pertuzumab containing 2 Fab without unpaired cysteines in any Fab should be 90% χ 90% = 81% (or [100x]% χ [100-x]%).

In addition, the wild-type homodimer (without unpaired cysteines) and the heterodimer (with unpaired cysteines in a single Fab) can be quantified directly by HIC. The HIC of the intact antibody separates pertuzumab into two main peaks (Figure 13), which were identified as wild-type homodimer (without free thiols) and heterodimer (with a free thiol pair in a Fab) by mapping LC MS tryptic peptides. The lower frontal shoulder peak was also collected and characterized by papain HIC as predominantly homodimer (thiol free pair in both Fab, approximately 40%) and pertuzumab with Fe oxidation. Using the intact antibody HIC, it was estimated that the pertuzumab contained approximately 17% -18% heterodimer for the materials produced using the current process, and 13% using the phase l / ll process (Table 5). Without being limited by any theory, it is possible that the increase in the amount of the unpaired cysteine ​​variant produced by the commercial process (in relation to the Phase 1/11 process) may result from the fact that protein folding (that is, the VL domain) thiol oxidation (disulfide formation) occurs more rapidly, whereby free cysteines are trapped in the variant.

Table 5- Relative amount of unpaired cysteine ​​variants determined by intact pertuzumab HIC:

Peak

Cysteine ​​Homodimer No

Paired heterodimer homodimer

Name of the wild type of cysteine ​​not partially batch (%) paired (%) enriched (%) ant¡2C484.7 13.4 1.9

900-1 Ant2C4907-

<td> 2</td><td> 78,9</td><td> 18,2</td><td> 2,9</td>

<td>Cumshot 1</td><td> 78,4</td><td> 18,4</td><td> 3,2</td>

<td>Cumshot 3</td><td> 79,1</td><td> 17,6</td><td> 3,2</td>

<td>Cumshot 4</td><td> 79,3</td><td> 17,3</td><td> 3,4</td>

<td>Cumshot 5</td><td> 79,1</td><td> 17,4</td><td> 3,5</td>

<td>Cumshot 6</td><td> 79,7</td><td> 17,2</td><td> 3,1</td>

<td>Cumshot 7</td><td> 79,3</td><td> 17,3</td><td> 3,4</td>

Note: The relative peak percentage was obtained by dividing the area of ​​the individual peak by the total peak area of ​​all three peaks.

Since a pair of unpaired cys23 / Cys88 cysteines in the pertuzumab light chain was observed by HIC, purified fractions of each of the unpaired cysteine ​​variants were subjected to the test in the anti-proliferation assay. Unpaired cysteine ​​containing Fab was purified and estimated to have reduced potency (estimated potency ~ 50% relative to native Fab) (Table 6).

Table 6: Antiproliferation of the unpaired cysteine ​​Fab variant

Samples of pertuzumab and conditions% average activity (n = 2)

100 native Fab

Unpaired cysteine-Fab 50^to

% Difference

Ñ ​​/ A

Note: percentage activity referred to native Fab ^to Estimated value of the power. The dose response curves are not parallel, and the lower plateau does not converge.

In addition, three intact forms (wild-type homodimer, free thiol-containing heterodimer, and free cysteine-containing homodimer) were isolated by HIC and tested with the antiproliferation potency test, see Table 7.

Table 7: Anti-proliferation activities of fractions of unpaired cysteine ​​variants of full-length pertuzumab

<td rowspan="2">Samples of pertuzumab and conditions</td><td colspan="2">antiproliferation</td>

<td>Average activity in% (n = 3)</td><td>CV (%)</td>

<td>Starting material</td><td> 110</td><td> 11</td>

<td>heterodimer</td><td> 112</td><td> 7</td>

<td>wild type homodimer</td><td> 104</td><td> 15</td>

<td>homodimer containing unpaired cysteine</td><td> 90</td><td> 15</td>

Note: percentage of activity reported with respect to pertuzumab Standard Reference (lot ant2C4907 2).

^to the fraction contains approximately 40% homodimer containing unpaired cysteine ​​and 60% heterodimer or mixture of wild type homodimers.

These data demonstrate that an unpaired cysteine ​​variant of pertuzumab is present in the commercially manufactured composition. The HIC (evaluation of the Fab fragment or intact antibody) methods in this example or peptide mapping in the following Example 3 are assays that can be used to evaluate the presence and amount of the unpaired cysteine ​​variant in a pertuzumab composition.

EXAMPLE 2

Afucosylated composition of pertuzumab and its characterization

ADDC (Antibody-Dependent Cell-Mediated Cytotoxity, Cell-Mediated Antibody Dependent Cytotoxicity) is an aspect of cell-mediated immunity by which a cell actively smooths an object cell that has bound to specific antigen antibodies. Pertuzumab had an ADCC activity when tested with 3 + HER2 cells, but very little activity was observed with 1 + HER2 cells (Figure 14).

The afucosylation levels in the Phase I and Phase II reference standards of pertuzumab were measured by capillary electrophoresis. The higher level of afucosylated material (G0 -F = 2.2%) in the pertuzumab Phase III reference standard correlates with the higher ADCC activity observed compared to the reference standard in Phase I (Figure 15), which had a lower Go-F (0.8%). An enzymatically deglycosylated pertuzumab was also prepared and tested and showed no binding to FcyRIIIa or any ADCC activity (Table 8).

Table 8: Biological activities of deglycosylated pertuzumab

Samples of average activity in% (n = 3)

<td>Pertuzumab and AntiProliferation</td><td>Union</td><td>to Union</td><td>to</td><td>Union to</td>

<td>conditions on</td><td>HER2</td><td>FcyRIIIa</td><td>ADCC</td><td>FcRn</td>

<td>Control 90</td><td> 105</td><td> 106</td><td> 101</td><td> 85</td>

<td>Deglycosylated</td><td></td><td></td><td>Without</td><td></td>

<td> 87</td><td> 94</td><td>Without activity</td><td></td><td> 72</td>

<td></td><td></td><td></td><td>exercise</td><td></td>

<td>Note; percentage activity</td><td>informed</td><td>Referring to</td><td>reference</td><td>Standard of</td>

pertuzumab (lot anti2C4907-2).

ADDC = antibody-dependent cell-mediated cytotoxicity, antibody-dependent cell-mediated cytotoxicity

These data show that the measurement of pertuzumab (afucosylated) GO-F is an effective means for the quantification of ADCC activity of pertuzumab. Experiments to quantify afucosylation are as follows.

Analysis of oligosaccharides by capillary electrophoresis (EC): samples of pertuzumab (250-500 pg) were purified by affinity tips for solid phase extraction of Protein A (PHYTIPS ™) and an automated liquid handling system. The pertuzumab samples were eluted from Protein A resin using 12 mM hydrochloric acid, pH 2.0 and neutralized with 10 µΙ of 50 mM sodium succinate. The resulting sample was incubated with 2.5 U / ml PNGase F for 15 hours at 37 ° C. The protein was precipitated by heating the solution at 95 ° C for 5 minutes and removed by centrifugation. Solutions of supernatant material containing released oligosaccharides were dried under vacuum.

The released glycans were derived with 8-aminopyrene-1,2,6 trisulfonic acid (APTS, 8-aminopyrene-1,2,6-trisulfonic acid) in a 15% acetic acid solution containing 55 ° sodium cyanoborohydride C for two hours. Derivative glycan analyzes were performed with a capillary electrophoresis system (EC, capillary electrophoresis) equipped with a fluorescence detection module using an argon ion laser (488 nm excitation, 520 nm emission) and a coated capillary N-CHO (50 pm χ 50 cm). The execution buffer was 40 mM amino epsilon.amino-n-caproic acid / acetic acid, pH 4.5, 0.2% hydroxypropyl methylcellulose (HPMC, hydroxypropyl methylcellulose). Samples were injected into the capillary by pressure at 0.5 psi. The separation was carried out at 20 kV, and the capillary temperature was maintained at 20 ° C.

Pertuzumab contains an N-linked oligosaccharide site in the Ch2 domain of the Fe portion of the Asn299 molecule. The relative distribution of neutral oligosaccharides found at this site for each batch was determined by CE after treatment with PNGase F and labeled with APTS.

The EC analysis electropherograms of the released derived oligosaccharides are shown in Figure 16 with enlarged view profiles in Figure 17. The relative amounts of oligosaccharides in pertuzumab for the materials analyzed are summarized in Table 9.

Table 9: Distribution of oligosaccharide structures in pertuzumab (percentage peak area)

<td rowspan="2">Lot name</td><td colspan="2">G0-</td><td rowspan="2">Man5</td><td rowspan="2">G0</td><td rowspan="2">G1^to</td><td rowspan="2">G2</td>

<td>G0-F</td><td>GIcNAc</td>

<td>Ant2C4-900-</td><td> 0,8</td><td> 2,7</td><td> 1,2</td><td> 72,1</td><td> 20,4</td><td> 2,0</td>

<td> 1</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>ant2C4907-2</td><td> 2,2</td><td> 0,8</td><td> 0,3</td><td> 63,6</td><td> 27,6</td><td> 3,0</td>

<td>Cumshot 1</td><td> 2,5</td><td> 1,6</td><td> 0,2</td><td> 62,4</td><td> 29,1</td><td> 3,4</td>

<td>Cumshot 3</td><td> 1,7</td><td> 1,2</td><td> 0,3</td><td> 70,3</td><td> 23,4</td><td> 2,2</td>

<td>Cumshot 4</td><td> 1,8</td><td> 1,8</td><td> 0,3</td><td> 75,4</td><td> 18,3</td><td> 1,4</td>

<td>Cumshot 5</td><td> 1,4</td><td> 1,0</td><td> 0,4</td><td> 71,8</td><td> 22,4</td><td> 2,1</td>

<td>Cumshot 6</td><td> 1,1</td><td> 1,0</td><td> 0,2</td><td> 73,3</td><td> 21,3</td><td> 1,9</td>

<td>Cumshot 7</td><td> 1,2</td><td> 0,8</td><td> 0,2</td><td> 69,7</td><td> 24,5</td><td> 2,5</td>

Note: The total% may not add up to 100% due to rounding. In addition, smaller species (&lt;0.5%) may have been included in the total percentage area of ​​the peak, but this table is not reported.

^to Sum of the two G1 isomers (refer to Figure 17).

The oligosaccharide with structure G0 is the predominant species in all materials (62% -75%). The G0 glycoform was slightly more abundant in Runs 3-7 and in previous reference of the standard lot 2C4 900 1 (70% -75%) compared to the standard standard reference lot anti2C4907 2 and the Run (62% -64% ). G1 glycoform was observed as two peaks corresponding to the two isomers with the terminal galactose in the two branches of the biantennial structure. The areas of these two peaks were combined in order to determine the relative amount of G1 glycoform. G1 and G2 glycoforms represent approximately 18% -29% and 1% -3%, respectively, of the oligosaccharides released for all materials. Peaks that arise from other oligosaccharide structures were also observed in the electropherograms (all present at 3% or less). These structures include G0-F (G0 lacking nucleus fucose), GO-GIcNAc (G0 lacking a GIcNAc), Man5, and other minor glycoforms (Ma and Nashabeh Anal Chem 71: 5185-92 (1999)). The oligosaccharide structures in pertuzumab were compatible with those found in CHO-derived MAbs (Ma and Nashabeh, supra) and naturally occurring human immunoglobulins (Flynn et al. Mol Immunol 47: 2074-82 (2010)).

EXAMPLE 3

RP-HPLC v peptide mapping to evaluate unpaired cysteine ​​variant

Materials: Materials and devices used in the experiments include: 3- [N-morpholino] propanosulfonic acid (MOPS, Sigma-Aldrich), Netylmaleimide (dO-NEM: Thermo Scientific, Rockford, IL), N-ethylmaleimide (d5NEM; Cambridge IsotopeLaboratories, Andover, MA), L-cysteine ​​(Sigma-Aldrich), trypsin (Promega, Madison, Wl), trifluoroacetic acid (TFA; Fisher, Fair Lawn, NJ), acetonitrile (ACN, Burdick &amp; Jackson, Muskegon, MI) . All chemicals and reagents received were used without further purification.

Differential labeling of antibodies with N-ethylmaleimide (NEM): The differential labeling method with NEM allowed free thiols already present in the antibodies to be labeled with dO-NEM and the remaining disulfide bridges to be reduced and labeled with d5-NEM. For initial labeling with dO-NEM, 100 μΙ of antibody (3 mg / ml) was gently mixed with 400 μΙ of denaturation buffer (7.5 M GdnHCI, pH 5) containing 6.25 mM of dO-NEM and It was incubated at 37 ° C for 2 h. twenty μΙ of cysteine ​​(125 mM) was added to the sample and incubated at 37 ° C for 15 minutes to deactivate the remaining DONEMATODE. To reduce the remaining disulfide bridges in the antibody, 10 μΙ of TCEP (0.5 M) was added to the sample and incubated at 37 ° C for 30 minutes. Then 70μΙ of d5-NEM (171 mM) was added to the sample and incubated at 37 ° C for 2 h to label the free thiols created by the reduced disulfide bridges. At 0.5 ml of the sample labeled with differential NEM, the buffer was changed using NAP-5 columns and eluted with 0.6 ml of MOPS buffer (20 mM MOPS, 0.5 mM TCEP, pH 7).

Antibody peptide map analysis: Samples labeled with differential NEM were digested with trypsin in a 1:50 ratio (weight / weight) of trypsin: anti-body at 37 ° C for 2 h. Digestions were inactivated with 10% TFA. The samples labeled with differential NEM digested by trypsin were separated using an Agilent 1200 HPLC system (Agilent, Palo Alto, CA). A Jupiter C18 column (250 x 2 mm, 5 pm) (Phenomenex, Torrance, CA) with a pore size 300 ansgtrom was used for chromatographic separation of the samples. The injection volume was 95 pL, and the column temperature was 55 C. Mobile phase A was 0.1% TFA in water and mobile phase B was 0.08% TFA in ACN at 90% (volume / volume). The initial conditions were set at 100% of mobile phase A and were maintained for the first 3 minutes after the injection of the sample. The mobile phase B was increased to 10% in the next 20 minutes and then increased again to 40%, for up to 160 minutes and 100%, for up to 162 minutes, along all linear gradients. The mobile phase B was maintained at 100%, for up to 170 minutes. The column was rebalanced at 100% of the mobile phase A for up to 195 minutes. The flow rate was maintained at 0.28 ml / min.

The HPLC effluent was directly connected to the electrospray ionization source of the LTQ ORBITRAP ™ mass spectrometer operating in a positive ion mode. The spraying voltage was 4.5KV, and the capillary temperature was 300 ° C. The mass spectrometer was operated in the data dependent mode to automatically switch between the MS and MS / MS modes. The full scan MS spectra for monitoring were acquired from m / z 300 am / z 2000 at the FT-Orbitrap with a resolution set for R = 60000 am / z 400. The five most intense ions were fragmented in the trap of linear ions using collision induced dissociation (CID) with a normalized collision energy of 35% with an activation time of 30 ms and an insulation width of 2.5 m / z units. The dynamic exclusion (DE) function was enabled in order to reduce data redundancy and allow low intensity ions to be selected for data-dependent MS / MS scans. The dynamic exclusion parameters were as follows: a repetition duration of 30 seconds, a magnitude of the exclusion list of 500, an exclusion duration of 90 seconds, a low exclusion mass width of 0.76, a high exclusion mass width of 1.56, and a repetition number of 2. Data analyzes were performed using the XCALIBUR ™ software.

The standard reference lot ant¡2C4907-2 1 of current pertuzumab was analyzed by the method described above. It was found that 10.9% of the T2L peptides (produced by digestion with triposine and containing Cys23) and 8.3% of the T7L peptides (produced by digestion of trypsin and containing Cys88) were labeled with dO-NEM. Because only unpaired cysteines were labeled with dO-NEM in this experiment, these results suggest that approximately 10% of Cys23 and Cys88 in ant2C4907 -2 are not bound by a disulfide bond (i.e. 10% of unpaired cysteine ​​variants). By using a calculation method similar to that described above to convert the percent of unpaired cysteine ​​Fab variant into percent of intact unpaired cysteine ​​variant, it was estimated that 18% of pertuzumab molecules in the ant¡ 2C4907-2 are unpaired heterodimeric cysteine ​​variants, 1% of pertuzumab molecules are homodimeric unpaired cysteine ​​variants, and 81% are the homodimeric form of wild type (without unpaired cysteines). These results agree in general terms with the results of the HIC analysis of any of the Fab fragments or the intact pertuzumab fragments.

Limited digestion with Lys-C endoproteinase to generate the Fab: The Fab fragment of MAb A was generated by means of a limited digestion process by Lys-C. Briefly, an amount of MAb A (1 mg / ml) was mixed with Lys-C enzyme in a 1: 400 ratio in 100 mM Tris, pH 7.6, and then the mixture was incubated at 37 ° C for 30 minutes . The reaction mixture was labeled with NEMin pH 5.5, 350 mM sodium acetate and 8M guanidine HCI. The digested were analyzed by an RP-HPLC method described below.

RP-HPLC conditions: RP-HPLC analysis was carried out in an AGILENT 1200 ™ HPLC system (Palo Alto, CA, USA) equipped with a binary gradient pump, automatic injector, temperature column compartment controlled, and a diode array detector. The system included a Pursuit 3 diphenyl reverse phase column (150 x 4.6 mm, 3 pm, Vary, Lake Forest, CA, USA), which was operated at 75 C and at 1 ml / min. The separation was monitored by absorbance at 280 nm. The mobile phase consisted of 0.1% TFA in water (mobile phase A) and 0.09% TFA in ACN (mobile phase B). The 38-minute method began with a three-minute linear gradient from 32% to 36% of mobile phase B, followed by an 18-minute linear gradient to 42% of mobile phase B. The column was washed at 95% of mobile phase. B for 5 minutes and equilibrated at 32% of mobile phase B for 10 minutes.

RP-HPLC analysis of Fab of free thiol generated by limited digestion with Lys-C (Figure 18) indicated that Fab of free thiol is about 13%, which is compatible with HIC in Example 1. Free labeled thiol becomes more hydrophobic, and therefore eludes later compared to the free thiol Fab (Figure 18) and the presence of free thiol was further confirmed. See also Figure 19 in which peptide mapping confirms the free thiol Fab.

EXAMPLE 4

Afucosylation and quantification by CE-LIF

This example describes an EC-LIF (capillary electrophoresis-laser-induced fluorescence, capillary electrophoresis fluorescence) assay, fully validated for the quantification of the afucosylated pertuzumab variant. Modifications to the methods described in Example 2 above include: no robotic preparation of the sample, no purification step of protein A, which ensures consistent protein concentrations between the samples, and changes in electrophoretic parameters (excipient concentration tampon).

In the assay, pertuzumab samples are diluted to 10 mg / ml using formulation buffer, and the buffer is exchanged with digester peptide N-glycanase F buffer (PNGase F). Then the asparagine-linked ollgosaccharides are enzymatically released with PNGase F. The released glycans are subsequently derived with 8 aminopyrene-1,3,6-trisulfonic acid (APTS), a fluorophore with negative charge. The APTS provides all glycans with three negative charges, allowing for rapid electrophoretic analysis. The mixture containing the excess bypass agent and the glycan-APTS conjugates is analyzed by CE using a coated capillary that reduces electroosmotic flow. The separation is monitored with a laser-induced fluorescence system using an argon ion laser with an excitation wavelength of 488 nm and an emission bandpass filter of 520 nm.

A correlation graph is shown in the test in Figure 21. Through the correlation graph (ADCC% = 30,133 + 12,439x, where x =% G0-

F) and an ADCC range of 40-135%, the final specification for pertuzumab corresponds to 0.9-4.1% G0-F.

Therefore, using this validated CE-LIF test, it is possible to evaluate pertuzumab compositions to confirm that biological activity in terms of ADCC is within the desired range (ADCC activity 40-135% = 0.9 to 4 , 1% G0-F).

EXAMPLE 5

High molecular weight species of pertuzumab (HMWS), low molecular weight species of pertuzumab (LMWS), and its characterization

Pertuzumab was analyzed by SE-HPLC and CE-SDS to determine the amount of high molecular weight species (HMWS), generally dimer, and low molecular weight species (LMWS). There was no difference in HMWS after dilution, which suggests that the aggregates are not dissociable. There was a good agreement between analytical ultracentrifugation (AUC) and the results of SE HPLC in terms of quantification of HMWS, with no evidence of exclusion chromatography in which a significant amount of HMWS is missing or overestimated.

MATERIALS AND METHODS

Tested pertuzumab compositions: This example describes the characterization of the current standard reference batch of pertuzumab ant¡2C4907 2 and Corrida 1, which represents the Phase III clinical material, and five commercial lots in Phase III (Runs 3-7) , all of them produced on a scale of 12,000 liters (L) through the commercial process. Pertuzumab compositions tested:

Isolated HMWS: To prepare representative HMWS used for biological characterization, a batch of pertuzumab of the runs was injected into a preparative HPLC system using a preparative SE-HPLC column (TSK G3000SW, 21.5 mm x 600 mm) and the Same Socratic mobile phase as described above in 4.5 ml / min. The high molecular weight species were collected from fractions and subsequently the buffer was exchanged for formulation buffer. The HMWS showed to be 70% pure by subsequent analysis by SE-HPLC, the rest being predominantly Pico Principal. The Main Peak was also collected and found to be 100% pure.

Isolated LMWS: To prepare isolated LMWS, a batch number of Corrida 3 was digested with papain and subjected to fraction collection by preparative HPLC, as before. The predominant forms were verified as Fe and Fab by intact ESI-MS analysis. The LMWS were shown to be 99% pure by subsequent analytical SE-HPLC analysis. Isolated Fab variants were also prepared by papain treatment and collected by preparative IE HPLC. The Fab variant was shown to have a purity of 100% by subsequent analytical SE-HPLC.

SE-HPLC: aliquots of pertuzumab were diluted to 10 mg / ml with mobile phase (0.2 M potassium phosphate, pH 6.2, 0.25 M potassium chloride). The samples were separated on a TSK G3000SW column_XL (7.8 mm x 300 mm) which was eluted socratic. The flow rate was 0.5 ml / min, and the column temperature was room temperature. The elution profile was monitored at 280 nm. For multi-angle light scattering (MALS) detection, pertuzumab samples were separated using two sequential columns connected in line to a WYATT DAWN HELEO ™ MALS detector (using a 658 nm laser, 17 detectors) and a rex index detector of rex WYATT OPTILAB ™; ALS.

CE-SDS: each batch of pertuzumab was derived with 5-carboxytetramethylrodamine ester, succinimidyl, a fluorescent dye. After removing the free dye using NAP-5 columns, the non-reduced samples were prepared by adding 40 mM of iodoacetamide and heating at 70 C for 5 minutes. For the analysis of the reduced samples, the pertuzumab derivative was mixed with sodium dodecyl sulfate (SDS) and 1 M DTT to a final concentration of 1% SDS (volume / volume). The samples were then heated at 70 ° C for 20 minutes. Samples prepared in an EC system were analyzed using a fused silica capillary with an inside diameter of 50 pm x 31.2 cm maintained at 20 ° C throughout the analysis. Samples were introduced into the capillary by electrokinetic injection at 10 kV for 40 seconds. The separation was carried out at a constant voltage of 15 kV in the reverse polarity mode (from negative to positive) using buffer 5 in use of CE-SDS as a screening medium. An argon ion laser operating at 488 nm was used for fluorescence excitation, and the resulting emission signal was monitored at 560 nm.

RESULTS AND ANALYSIS

The SE-HPLC provides quantitative information about the molecular size distribution of a native protein. The SE-HPLC profiles for pertuzumab batches are shown in Figure 25, and an enlarged view of the profiles is shown in Figure 26. The relative peak area distribution of size exclusion peaks has been reported in Table 10

Table 10: Relative distribution of pertuzumab sizes by size exclusion chromatography

<td rowspan="2">Lot name</td><td colspan="3">Peak</td>

<td>HMWS (%)</td><td>Peak Principal (%)</td><td>LMWS (%)</td>

<td>ant2C4-</td><td></td><td></td><td></td>

<td></td><td> 0,1</td><td> 99,8</td><td> 0,1</td>

<td> 900-1</td><td></td><td></td><td></td>

<td>ant2C4907-</td><td></td><td></td><td></td>

<td></td><td> 0,2</td><td> 99,8</td><td> 0,0</td>

<td> 2^to</td><td></td><td></td><td></td>

<td>Cumshot 1</td><td> 0,2</td><td> 99,8</td><td> 0,0</td>

<td>Cumshot 3</td><td> 0,2</td><td> 99,8</td><td> 0,0</td>

<td>Cumshot 4</td><td> 0,1</td><td> 99,8</td><td> 0,0</td>

<td>Cumshot 5</td><td> 0,2</td><td> 99,8</td><td> 0,0</td>

<td>Cumshot 6</td><td> 0,2</td><td> 99,8</td><td> 0,0</td>

<td>Cumshot 7</td><td> 0,2</td><td> 99,8</td><td> 0,0</td>

^to Values ​​obtained from the reference standard ant¡2C4907-2.

Note: The total percentage may not add up to 100% due to rounding.

HMWS = high molecular weight species; LMW = low molecular weight species.

The proportion of pertuzumab eluting at the Main Peak was greater than 99% for all materials. The amount of high molecular weight species (HMWS) varied from 0.1% to 0.2%, and for low molecular weight species (LMWS) it was £ 0.1%. All batches show similar chromatographic profiles. A purified fraction of HMWS, including dimers and higher aggregates, was shown to have a potency of 46% with respect to the reference standard lot ant2C4907.

The SE-HPLC was carried out on both samples, pure and diluted, kept at 30 ° C to examine pertuzumab H; WS for both fast and slow dissociation aggregates that could result from dilution and / or prolonged exposure to room temperature high. There was no decrease in the HMWS content of diluted and / or hot batch reference standard anti2C4907, compared to the control.

The SE-HPLC separation combined with MALS carried out in the batch of reference standard ant2C4907 2 confirmed that the SEHPLC Main Peak is monomer, with a molecular weight of approximately 150 kDa.

The AUC was used in the sedimentation rate mode to characterize the HMWS present in pertuzumab samples. Sedimentation rate is an independent size exclusion chromatography technique that measures HMWS levels in a sample in the absence of a solid column matrix. AUC was performed on pertuzumab samples with increasing levels of HMWS to determine if the SE-HPLC is capable of detecting all major pertuzumab HMWS in a compatible manner by comparing levels and aggregate species determined by the speed of sedimentation with those determined by SE-HPLC. Five samples ranging from 0.2% to 7.2% of total HMWS (determined by SE-HPLC) were characterized by sedimentation rate and labeled as EA in Table 11 and in Figure 27.

These samples consisted of a representative batch of pertuzumab drug product (labeled A) and four samples with enriched HMWS. The samples with enriched HMWS were chosen as representative of a wide range of degradation mechanisms (exposure to light, exposure to acidic pH, and basic variants of purified IE-HPLC).

The SE-HPLC shows a main peak of HMWS for samples A, B, C and E and two peaks of HMWS for sample D (Figure 27). For samples A, B, C and E, the AUC showed only one HMWS peak with a sedimentation coefficient at approximately 9.1S. In sample D, the AUC showed two peaks of HMWS with sedimentation coefficients of approximately 9.1 S and 10.8 S. The HMWS detected by the AUC are compatible with the results of SE-HPLC; both methods show a major degradation product, with lower levels of a larger HMWS in sample D.

A comparison of the quantitative results of these samples obtained by the two methods is shown in Table 11.

Table 11: Comparison of the results of AUC and SE-HPLC

<td rowspan="2">Shows</td><td colspan="2">% HMWS (total)</td>

<td>AUC</td><td>SE-HPLC</td>

<td>TO</td><td>1.2 [31.4% RSD]^to</td><td> 0,2</td>

<td>B</td><td> 1,9</td><td> 1,3</td>

<td>C</td><td> 4,8</td><td> 5,5</td>

<td>D</td><td> 6,4</td><td> 6,6</td>

<td>AND</td><td>7.6 [7.9% RSD]</td><td> 7,2</td>

Note 1: Sample A consists of a representative pertuzumab drug product batch, sample B is composed of a batch of pertuzumab subjected to light exposure at a rate of 1.2 mlux hours, sample C is composed of a batch of pertuzumab subjected to light exposure at a rate of 3.6 mlux hours, sample D consists of a batch of pertuzumab subjected to acid treatment at a pH of 3.2, and sample E is composed of purified basic variants of IEHPLC.

Note 2: Refer to Figure 27 for the corresponding SEHPLC chromatograms.

AUC = analytical ultracentrifugation; HMWS = high molecular weight species; RSD = relative standard deviation; SE-HPLC = high performance liquid chromatography of size exclusion.

Samples A and B have HMWS levels that are below the quantification limit of the AUC technique.

For samples C, D, and E there is a good agreement on the percentage of HMWS measured by both techniques. The low level of HMWS present in samples A and B prevents an exact quantification of the species by the AUC, which has an estimated quantification limit of 3.7% (Gabrielson and Arthur, Methods 54:83 91 (2011)). This is reflected in an apparent discrepancy in the percentage of HMWS between SE-HPLC and AUC (Table 11). A correlation was evaluated across a range of HMWS levels. The correlation coefficient (Lin, L, Biometrics 45: 255-68 (1989)) calculated was 0.97 (n = 5) which indicates a good concordance between the AUC and the SE-HPLC for the quantification of HMWS ( Figure 28).

These results confirm that SE-HPLC is robust for measuring HMWS for pertuzumab. The SE-HPLC is capable of accurately detecting and quantifying all the HMWS species observed by the AUC.

Heterogeneity based on size, analyzed by SE-HPLC, SDSPAGE, and CE-SDS, was consistent across batches. The SE-HPLC assay showed similar levels of HMWS (0.1% -0.2%) and LMW (0.0% -0.1%) for all batches tested. The band patterns developed by the SDSPAGE analysis for reduced and reduced samples were consistent, as well as the electrophoretic profiles generated by CE-SDS.

In one embodiment, the quantities of pertuzumab and HMWS variant and LMWS variant species evaluated by SEHPLC are as follows:

£ 96% Main Peak for example, 2 96.7% Main Peak, for example, 2: 97.3% Main Peak for example, 2 97.4% Main Peak £ 2% HMWS, for example, &lt;1.7% HMWS, for example, &lt;1.5% HMWS, for example, £ 1.4% HMWS, for example £ 0.8% HMWS.

&lt;2% LMWS, for example, 1.6% LMWS, for example, £ 1.2% LMWS, for example at 0.6% LMWS.

Both the HMWS and LMWS fractions of pertuzumab purified by SE-HPLC showed decreased antiproliferation activity compared to the Main Peak and the control, which was completely potent. All size variants showed an HER2 binding activity and an FcRn binding activity comparable to the control, except for the LMWS, which showed a lower FcRn binding. Since the LMWS sample contains 2/3 of Fab fragments and 1/3 of Fe fragments, the lowest antiproliferation activity and the lowest FcRn binding activity were as expected. The HMWS showed a greater activity of binding to FcRIIIa (CD16) V158, but a lower activity of ADCC. The LMWS showed a lower FcRIIIa (CD16) V158 binding activity, and no ADCC activity was observed for this variant (Table 12).

Table 12: Biological activities of pertuzumab Main Peak,

HMWS, and LMWS

Average Activity Samples% (n = 3) pertuzumab and Union-to-Union Antiproliferation under conditions with Union to HER2 FcyRIIIa ADCC FcRn

<td>Control</td><td> 103</td><td> 108</td><td> 91^b</td><td> 80^b</td><td> 80</td>

<td>Main Peak</td><td> 104</td><td> 96</td><td> 96</td><td> 79</td><td> 87</td>

<td>HMWS</td><td> 46</td><td> 82</td><td> 522</td><td> 38</td><td> 73</td>

<td>LMWS</td><td> 12^to</td><td> 73^to</td><td> 23^to</td><td>Without activity</td><td> 7^to</td>

Note: percentage of activity reported in relation to pertuzumab Standard Reference (lot ant2C4907 2).

ADCC = cell-mediated antibody dependent cytotoxicity; HMWS = high molecular weight species; LMW = low molecular weight species.

^to The LMWS sample consists of 2/3 Fab and 1/3 Fe fragments. The value shown reflects the nM / nM adjustment based on molecular weight (Fab = 47,644 Da, Fe = 52,800 Da, and the full length antibody = 148,088 Da.

b The pertuzumab reference standard (lot anti2C4907-2) has a G0-F level of 2.2%, while the control sample had a G0-F of 1.7%. The results have not been corrected by the difference in the level of afucosylated material.

Capillary electrophoresis with sodium dodecyl sulfate (CE-SDS): CE-SDS detection with laser-induced fluorescence (LIF) is a high sensitivity assay that provides a means to quantitatively assess the molecular size distribution of proteins under denaturing conditions. In the EC SDS analysis of non-reduced samples (Figure 29), pertuzumab migrates as a prominent peak consisting of 96% -98% of the total peak surface area with smaller peaks representing LMWS and HMWS. The amount of HMWS determined by this technique was 0.6% for all materials tested. The remaining species migrated as LMWS as shown in Figure 30 (enlarged view). Fragmentation induced by heating the samples is minimized, with alkylation (SalasSolano et al. Anal Chem. 78: 6583-6594 ​​(2006)). The relative distribution of species separated by CE-SDS is listed in Table 13.

Table 13: Relative distribution of pertuzumab not reduced by CESDS (Percent peak area)

Peak

<td>Lot name</td><td> 1</td><td> 2</td><td> 3</td><td> 4</td><td> 5</td><td> 6</td><td>Princ Pal</td><td>HMW S</td>

<td>Ant2C4-900- one</td><td> 0,1</td><td> 0,2</td><td> 0,1</td><td> 0,2</td><td> 0,2</td><td> 0,9</td><td> 97,8</td><td> 0,6</td>

<td>ant2C4907-2</td><td> 0,1</td><td> 0,3</td><td> 0,1</td><td> 0,3</td><td> 0,2</td><td> 1,9</td><td> 96,5</td><td> 0,6</td>

<td>Cumshot 1</td><td> 0,1</td><td> 0,3</td><td> 0,1</td><td> 0,4</td><td> 0,2</td><td> 1,7</td><td> 96,7</td><td> 0,6</td>

<td>Cumshot 3</td><td> 0,1</td><td> 0,4</td><td> 0,1</td><td> 0,3</td><td> 0,2</td><td> 2,3</td><td> 96,0</td><td> 0,6</td>

<td>Cumshot 4</td><td> 0,1</td><td> 0,4</td><td> 0,1</td><td> 0,3</td><td> 0,2</td><td> 2,3</td><td> 96,1</td><td> 0,6</td>

<td>Cumshot 5</td><td> 0,1</td><td> 0,3</td><td> 0,1</td><td> 0,2</td><td> 0,2</td><td> 2,0</td><td> 96,4</td><td> 0,6</td>

<td>Cumshot 6</td><td> 0,1</td><td> 0,4</td><td> 0,1</td><td> 0,3</td><td> 0,2</td><td> 2,2</td><td> 96,2</td><td> 0,6</td>

<td>Cumshot 7</td><td> 0,1</td><td> 0,4</td><td> 0,0</td><td> 0,2</td><td> 0,2</td><td> 2,1</td><td> 96,3</td><td> 0,6</td>

Note: that the total% may not add exactly the

100% due to rounding.

CE-SDS = capillary electrophoresis of sodium dodecyl sulfate;

HMWS = high molecular weight species.

A minor difference was observed in which Peak 6 increased from 0.9% in the Standard Reference lot anti2C4 900 1 (Process phase l / ll) to 1.7% 2.3% for the Standard Reference lot ant2C4907 2, Run 1, and Run 3-7.

In one embodiment, the pertuzumab Main Peak (excluding LMWS and HMWS), separated or isolated by NR-CE-SDS is from about 95% to about 99%, for example, from about 96.0% to about 97, 8% Optionally, the amount of HMWS is £ 1%, for example, £ 0.6% and the amount of LMWS is £ 4%, for example, &lt;3.4% separated or isolated by CE-SDS.

EXAMPLE 6

Detection and quantification of pertuzumab fragmentation

The purpose of this example was to evaluate the following methods: size exclusion chromatography (SE-HPLC), reduced capillary electrophoresis with sodium dodecyl sulfate (R-CE-SDS), and non-reduced CE-SDS (NR-CESDS ) for the detection of pertuzumab fragments.

MATERIALS AND METHODS

The samples evaluated in this study are summarized below. These include samples of pertuzumab that have undergone various stress conditions that could result in increased fragmentation;

• Reference standard (2C4907-2) • Thermal stress (42 days, 40 ° C) • Acid treatment (pH 3.2, 1 day, 40 ° C) • Accelerated stability (30 days at 40 ° C and then stored at approximately 5 ° C) • Stability of the Drug Product in real time (T = 0 and T = 548 days and

100 stored at approximately 5 ° C) and the corresponding drug substance (DS, Drug Substance)

The SE-HPLC was carried out as described in Example 5 above, with the following reportable values: LMWS, Main Peak, HMWS, and all other significant peaks above the quantification limit (LOQ).

The reduced CE-SDS (R-CE-SDS) was carried out according to the above-mentioned Example 5, with reportable values: Peak 1, LC, Peak 2, Peak 3, NGHC, HC, Peak 5, Inc. Network. , and other significant peaks above LOQ.

Non-reduced CE-SDS (NR-CE-CDS) was carried out as in Example 5 above, with sample preparation excluding the step of antibody reduction to allow non-reduced analysis by removed dithiothreitol ( DTT, dithiothreitol) of the SDS complex formation stage.

The qualitative results obtained by SE-HPLC, NR-CE-SDS, and RCE-SDS are presented in Figures 31A-31B, Figures 32A-32B, and Figures 33A-33B, respectively, as well as in Tables 14, 15, and 16, respectively

Peak identifications are based on Hunt &amp; Nashabeh Analytical Chemistry 71: 2390-2397 (1999), and Ma &amp; Nashabeh Chromatographia Supplement 53: S75-S89 (2001). For NR-CE-SDS analysis, typically a small peak after Peak 2 is included as part of Peak 2 during the

101 data presentation. For this study, this small peak is presented separately as Pico 2a to differentiate the light chain fragment (LC).

Table 14: Quantitative data of SE-HPLC (% of peak area)

<td></td><td>HMWS (%)</td><td>Peak Principal (%)</td><td>LMWS (%)</td>

<td>2C4907-2</td><td> 0,18</td><td> 99,77</td><td> 0,04</td>

<td>Thermal</td><td> 0,40</td><td> 98,96</td><td> 0,64</td>

<td>Acid treated</td><td> 7,09</td><td> 92,65</td><td> 0,26</td>

<td>Accelerated stability</td><td> 0,26</td><td> 99,23</td><td> 0,51</td>

<td>DP stability</td><td> 0,19</td><td> 99,68</td><td> 0,13</td>

102

Table 15: quantitative data of NR-CE-SDS (% of CPA)

<td></td><td>Foot or 1</td><td>Foot or 2 (LC)</td><td>Foot 0 2nd</td><td>Peak 3 (Fab )</td><td>Foot or 4 (HL )</td><td>Peak 5 (HH / desFa b)</td><td>Peak 6 (HHL )</td><td>Peak Principal</td><td>HMW S</td>

<td>2C4907-2</td><td> 0,1 2</td><td> 0,2 9</td><td> 0,0 5</td><td> 0,07</td><td> 0,3 5</td><td> 0,18</td><td> 1,90</td><td> 96,32</td><td> 0,73</td>

<td>Thermal</td><td> 0,4 2</td><td> 0,7 0</td><td> 0,0 9</td><td> 0,61</td><td> 0,5 2</td><td> 1,36</td><td> 3,62</td><td> 91,99</td><td> 0,71</td>

<td>Treatment with acid</td><td> 0,1 5</td><td> 0,3 7</td><td> 0,1 7</td><td> 0,35</td><td> 0,4 5</td><td> 0,66</td><td> 3,19</td><td> 93,42</td><td> 1,25</td>

<td>Accelerated stability</td><td> 0,3 5</td><td> 0,5 9</td><td> 0,0 9</td><td> 0,48</td><td> 0,4 4</td><td> 1,07</td><td> 3,25</td><td> 92,90</td><td> 0,84</td>

<td>Stability DP</td><td> 0,2 2</td><td> 0,3 7</td><td> 0,0 6</td><td> 0,20</td><td> 0,3 3</td><td> 0,43</td><td> 2,53</td><td> 95,27</td><td> 0,60</td>

LC = light chain (Light Chain), HC = Heavy chain (Heavy Chain), L = light (Light), H = heavy (Heavy)

103

Table 16: CE-SDS quantitative data (% of CPA)

<td></td><td>Peak one</td><td>LC</td><td>Peak two</td><td>Peak 3</td><td>NGHC</td><td>HC</td><td>Peak 5</td><td>Inc. Net.</td>

<td>2C4907-2</td><td> 0,31</td><td> 25,30</td><td> 0,92</td><td> 2,44</td><td> 2,81</td><td> 66,82</td><td> 0,48</td><td> 0,91</td>

<td>Thermal</td><td> 0,54</td><td> 26,16</td><td> 1,12</td><td> 3,13</td><td> 2,81</td><td> 64,54</td><td> 0,27</td><td> 1,43</td>

<td>Acid treated</td><td> 0,38</td><td> 25,04</td><td> 5,58^c</td><td> 2,77</td><td> 2,41</td><td> 62,91</td><td> 0,19</td><td> 0,71</td>

<td>Accelerated stability</td><td> 0,51</td><td> 25,01</td><td> 0,98</td><td> 2,99</td><td> 3,16</td><td> 66,10</td><td> 0,25</td><td> 1,03</td>

<td>Stability DP</td><td> 0,19</td><td> 26,34</td><td> 0,69</td><td> 3,21</td><td> 2,83</td><td> 65,93</td><td> 0,24</td><td> 0,57</td>

NGHC = Non-Glycosylated Heavy Chain, HC = Heavy Chain, Inc. Red. = Incompletely reduced

Data evaluation: The percentage of peak area (or corrected percentage peak area,% CPA, for CE-SDS) of the relevant fragments was compared to determine if any of the CE-SDS methods provide non-redundant information in comparison with SE-HPLC. Relevant fragments include peaks of unknown structure, or those known to contain products derived from cleavage of the polypeptide chain (s). These fragments are distinct from the dissociable disulfide-linked heavy and / or light chain fragments that are present in

104 the antibody products and that are commonly observed by CE-SDS. Fragment peaks must be resolved with respect to other peaks to allow sensitive detection and exact quantification.

Ability to detect small fragments, small fragments can be observed in both analyzes, R-CE-SDS and NR-CE-SDS, and are named as Peak 1 in both assays. These peaks retain the same general form and migration time, and increase similarly under stress conditions in both trials. Therefore, it is presumed that Peak 1 contains the same species in both trials. Both CE-SDS assays are capable of detecting small fragments, as indicated in Table 17.

Ability to detect fragments generated by acid hydrolysis (acid Clips): prolonged exposure to acidic conditions can generate fragments, especially in the Asp-Pro sequence (heavy chain residues of pertuzumab 272-273), which is supported by the Spectrometry analysis of the acid-treated sample showing masses 29,039 Da (HC 1-272) and 21,513 Da (HC 273-448 with glucan G0). The theoretical masses for these forms are 29,031 Da and 21,510 Da, respectively. Based on the expected migration time of these forms, a corresponding peak can be clearly seen in the reduced CE-SDS analysis of the acid-treated sample (Peak 2, Figure 34), but is detected at a much lower level ( lower signal) in the non-reduced test. It can be postulated that in the NR-CE-SDS test the Pico 2 fragment is presumably bound to disulfide, and therefore is not detected. Since the level of Peak 2 detected by R-CE-SDS (5.58%) in the acid treated sample

105 exceeds the LMWS detected by SE-HPLC (0.26%) for this sample, it can be concluded that SE-HPLC is also insufficient for the detection of these forms. Therefore, the reduced CE-SDS test is the only test presented herein capable of detecting the fragmentation generated as a result of acid hydrolysis, as indicated in Table 17.

Ability to detect Fab / DesFabs fragments: There is a good linear correlation (r² = 0.97) between the LMWS detected by SE-HPLC, and Peak 3 a of the non-reduced CE-SDS assay (Figure 35). The LMWS were identified to contain the Fab fragment through co-elution studies with enzymatically generated Fab. Similarly, Peak 3 and Peak 5 in the NR-CE-SDS assay were identified through co-migration studies with enzymatically generated Fab and DesFab, respectively (Ma and Nashabeh, supra). The desFab peak arises from the excision of the heavy chain that produces the Fab form, so it is presumed that it is present in a molar equivalent amount (corresponds to a 2: 1 mass ratio) in relation to the Fab fragment, and therefore, information about this form can also be obtained indirectly by SE-HPLC as indicated in Table 17.

106

Table 17: fragment detection capability by SE-HPLC, NR-CESDS, and R-CE-SDS

<td>Fragment</td><td>SE-HPLC</td><td>NR-CE-SDS</td><td>R-CE-SDS</td>

<td>Small figures CE-SDS Peak 1</td><td>Unknown</td><td>yes</td><td>Yes</td>

<td>Acidic clips (R-CE-SDS Peak two)</td><td>Do not</td><td>Do not</td><td>Yes</td>

<td>Fab / DesFab</td><td>Yes (indirectly)</td><td>Yes</td><td>Do not</td>

Peak 3 of CE-SDS Reduced: Peak 3 of R-CE-SDS peak 3 is unique for pertuzumab and has not been observed in the CE-SDS analysis of other antibodies. The extended characterization results support the conclusion that Peak 3 is not a product or impurity variant, but rather a method-induced artifact, specific for pertuzumab, consisting of a dissociable form of LC-LC dimer. Multiple techniques were used to characterize Peak 3.

Peak 3 is observed by R-CE-SDS with UV and LIF detection, suggesting that it is not an artifact of dye labeling or sample preparation.

107

After analysis by R-CE-SDS, purified pertuzumab light chain fractions produce Peak 3, which has an apparent molecular weight of approximately 2 times the theoretical size of the LC.

Peak 3 is not observed when electrophoretic conditions include a higher capillary temperature, and no other comigrant fragments are observed under these conditions.

Studies involving simple amino acid mutations have identified three amino acid residues in LC CDR1 and CDR2 correlated with the formation of LC-LC dimer. When any of these three residues is replaced by another amino acid, Peak 3 completely dissociates and is no longer observed by reduced CE-SDS.

Analysis by SDS-PAGE coupled with MALDI-TOF of PMF (Protein Mass Fingerprinting, Digital Protein Mass Printing) confirmed that there were no host cell proteins present in pertuzumab and that there was also no analog band detected at levels observed by CE -SDS.

Taken together, these results support the identification of Peak 3 as a specific LC-LC number for pertuzumab induced by the method.

ANALYSIS

The evaluation of the data obtained from this study indicates that:

(1) NR-CE-SDS provides non-redundant information compared to SE-HPLC for fragment detection;

(2) Non-redundant fragmentation information obtained through

108 The NR-CE-SDS method (as compared to SE-HPLC) can also be obtained using the R-CE-SDS method.

As shown in Table 16, the reduced assay detects the cleavage products resulting from exposure to low pH, which may occur during the manufacture of the drug substance. Table 18 contains the values ​​obtained for reduced CE-SDS of Peak 1 and Peak 2 for the pertuzumab reference standard, phase III material (n = 3), and batches produced using the commercial manufacturing process (n = 39). The 95/99 tolerance intervals (Tls) have been calculated for Peak 1 and Peak 2 using a k value of 3.2, and are presented in Table 18. The 95/99 tolerance range for Peak 1 is 0 , 0 to 0.4% of the takeover. The 95/99 tolerance range for Peak 2 is 0.3 to 0.9% OPA.

Table 18: R-CE-SDS quantitative data (% of OPA) on 43 batches tested

<td>n</td><td>Lot</td><td>Peak 1 (% of CPA)</td><td>Peak 2 (% of CPA)</td>

<td> 1</td><td>ant2C4907-2</td><td> 0,31</td><td> 0,92</td>

<td> 2</td><td>SSF0001</td><td> 0,25</td><td> 0,63</td>

<td> 3</td><td>SSF0002</td><td> 0,20</td><td> 0,62</td>

<td> 4</td><td>SSF0003</td><td> 0,27</td><td> 0,60</td>

<td> 5</td><td>W0002</td><td> 0,27</td><td> 0,47</td>

<td> 6</td><td>W0003</td><td> 0,28</td><td> 0,49</td>

109

<td> 7</td><td>W0004</td><td> 0,25</td><td> 0,51</td>

<td> 8</td><td>W0005</td><td> 0,41</td><td> 0,55</td>

<td> 9</td><td>W0006</td><td> 0,27</td><td> 0,50</td>

<td> 10</td><td>W0007</td><td> 0,29</td><td> 0,50</td>

<td> 11</td><td>W0008</td><td> 0,30</td><td> 0,53</td>

<td> 12</td><td>W0009</td><td> 0,26</td><td> 0,54</td>

<td> 13</td><td>W0013</td><td> 0,19</td><td> 0,54</td>

<td> 14</td><td>W0018</td><td> 0,17</td><td> 0,56</td>

<td> 15</td><td>W0020</td><td> 0,17</td><td> 0,58</td>

<td> 16</td><td>W0021</td><td> 0,18</td><td> 0,62</td>

<td> 17</td><td>W0023</td><td> 0,14</td><td> 0,62</td>

<td> 18</td><td>W0024</td><td> 0,13</td><td> 0,64</td>

<td> 19</td><td>W0025</td><td> 0,18</td><td> 0,69</td>

<td> 20</td><td>W0026</td><td> 0,13</td><td> 0,58</td>

<td> 21</td><td>W0028</td><td> 0,17</td><td> 0,60</td>

<td> 22</td><td>W0029</td><td> 0,18</td><td> 0,61</td>

<td> 23</td><td>W0031</td><td> 0,18</td><td> 0,65</td>

<td> 24</td><td>W0032</td><td> 0,16</td><td> 0,69</td>

<td> 25</td><td>W0033</td><td> 0,18</td><td> 0,69</td>

<td> 26</td><td>W0034</td><td> 0,18</td><td> 0,67</td>

<td> 27</td><td>W0035</td><td> 0,17</td><td> 0,59</td>

<td> 28</td><td>W0036</td><td> 0,17</td><td> 0,67</td>

110

<td> 29</td><td>W0037</td><td> 0,19</td><td> 0,74</td>

<td> 30</td><td>W0038</td><td> 0,19</td><td> 0,71</td>

<td> 31</td><td>W0039</td><td> 0,19</td><td> 0,73</td>

<td> 32</td><td>W0040</td><td> 0,19</td><td> 0,70</td>

<td> 33</td><td>W0041</td><td> 0,18</td><td> 0,72</td>

<td> 34</td><td>W0042</td><td> 0,19</td><td> 0,73</td>

<td> 35</td><td>W0043</td><td> 0,19</td><td> 0,61</td>

<td> 36</td><td>W0044</td><td> 0,20</td><td> 0,67</td>

<td> 37</td><td>W0046</td><td> 0,20</td><td> 0,62</td>

<td> 38</td><td>W0047</td><td> 0,22</td><td> 0,66</td>

<td> 39</td><td>W0048</td><td> 0,22</td><td> 0,63</td>

<td> 40</td><td>W0049</td><td> 0,20</td><td> 0,69</td>

<td> 41</td><td>W0050</td><td> 0,18</td><td> 0,49</td>

<td> 42</td><td>W0051</td><td> 0,16</td><td> 0,63</td>

<td> 43</td><td>W0052</td><td> 0,18</td><td> 0,60</td>

<td>Medium</td><td> 0,21</td><td> 0,62</td>

<td>Deviation</td><td> 0,06</td><td> 0,09</td>

<td>standard</td><td></td><td></td>

<td>Minimum</td><td> 0,13</td><td> 0,47</td>

<td>Maximum</td><td> 0,41</td><td> 0,92</td>

<td>N</td><td> 43</td><td> 43</td>

<td>K</td><td> 3,2</td><td> 3,2</td>

111

Lower IT 0.02

Higher IT 0.40

0,33

0,91

Here, a final acceptance criterion of Pico 1 &lt;0.5% and Pico 2 at 1.0% on the release of the drug substance is selected.

As the drug substance pertuzumab is stored frozen, there would be no expected changes in DS stability. Furthermore, based on the data in the R-CE-SDS data obtained for the drug product in both T = 0 and T548d (Table 19), no significant change is observed for any of the mentioned species.

Table 19: Quantitative data of R-CE-SDS (% CPA) for a DP stability sample

<td></td><td>Peak one</td><td>LC</td><td>Peak two</td><td>Peak 3</td><td>NGHC</td><td>HC</td><td>Peak 5</td><td>Inc. Net.</td>

<td>Liberation of DS</td><td> 0,25</td><td> 26,72</td><td> 0,51</td><td> 2,27</td><td> 2,76</td><td> 66,66</td><td> 0,27</td><td> 0,56</td>

<td>Stability DP T = 0</td><td> 0,13</td><td> 26,45</td><td> 0,54</td><td> 2,78</td><td> 2,78</td><td> 66,53</td><td> 0,30</td><td> 0,49</td>

<td>Stability DP T = 548d</td><td> 0,19</td><td> 26,34</td><td> 0,69</td><td> 3,21</td><td> 2,83</td><td> 65,93</td><td> 0,24</td><td> 0,57</td>

NGHC = non-glycosylated heavy chain; HC = heavy chain, Inc. Red. =

Incompletely reduced