METHODS RELATED TO ADALIMUMAB

Abstract

The present invention relates to the characterization and production of adalimumab.

Claims

1.-34. (canceled)

35. A method, comprising: acquiring, from a test glycoprotein preparation, a value for each of a plurality of adalimumab parameters, wherein the plurality of adalimumab parameters comprises 6 to 22 parameters listed in Table 1, and wherein an adalimumab signature distinguishes adalimumab from a non-adalimumab glycoprotein, and the adalimumab signature consists of the 6 to 22 parameters and corresponding reference criteria in Table 1; and manufacturing an adalimumab drug product by formulating at least a portion of the test glycoprotein preparation as an adalimumab drug product if all of the values acquired for the parameters in the signature meet the corresponding reference criteria; and discarding the test glycoprotein preparation if any one of the values acquired for the parameters in the signature do not meet the corresponding reference criteria; wherein the test glycoprotein preparation comprises a recombinant antibody composition including a first polypeptide having the amino acid sequence of SEQ ID NO:1 and a second polypeptide having the amino acid sequence of SEQ ID NO:2, and wherein the first and second polypeptides together form a recombinant antibody.

36. The method of claim 35, wherein the value for a parameter in the signature is directly acquired by performing an analytical test on the test glycoprotein preparation.

37. The method of claim 36, wherein the value for a parameter in the signature is directly acquired using a method provided in Table 2.

38. The method of claim 35, wherein formulating comprises combining the test glycoprotein preparation with an excipient or buffer.

39. The method of claim 35, wherein the adalimumab drug product is approved under Section 351(k) of the Public Health Service (PHS) Act.

40. The method of claim 35, wherein the adalimumab drug product is not approved under biologics license application (BLA) under Section 351(a) of the Public Health Service (PHS) Act.

41. The method of claim 35, wherein the value for at least one parameter in the signature comprises an average of values or a range of values for the parameter acquired from multiple batches or samples of the test glycoprotein preparation.

42. The method of claim 35, wherein the reference criteria in Table 1 are a specification for commercial release of a drug product under Section 351(k) of the Public Health Service (PHS) Act.

43. The method of claim 35, wherein the values for each of the parameters in the signature is acquired from one or more samples or batches of the test glycoprotein preparation.

44. The method of claim 35, wherein providing the test glycoprotein preparation comprises: providing a host cell that is genetically engineered to express the first and second polypeptides, culturing the host cell under conditions that allow the cell to express the first and second polypeptides and form recombinant antibodies, and harvesting the recombinant antibodies from the host cell culture to produce the test glycoprotein preparation.

45. The method of claim 35, wherein the adalimumab drug product is an interchangeable version, under Section 351(k) of the Public Health Services (PHS) Act, of a adalimumab product approved by the FDA.

46. The method of claim 35, wherein the reference criteria in Table 1 are a product acceptance criterion.

Description

DESCRIPTION OF THE DRAWINGS

[0031] FIG. 1|Amino acid sequence of heavy chain of adalimumab (SEQ ID NO: 1).

[0032] FIG. 2|Amino acid sequence of light chain of adalimumab (SEQ ID NO: 2).

DETAILED DESCRIPTION

[0033] Detailed, high resolution, structural information about Humira (e.g., related to the presence of signature glycan species or quantitative analyses ascribing site-specificity for backbone modifications) is useful to be able to make and test products that qualify as adalimumab, e.g., that are interchangeable versions of Humira. Such information is also useful in monitoring product changes and controlling structural drift that may occur as a result of manufacturing changes. The art supports, however, that information necessary to be able to make and test products that qualify as adalimumab, e.g., that are interchangeable versions of Humira, or any other branded biologic, is unavailable (see, e.g., Nowicki, Basic Facts about Biosimilars, Kidney Blood Press. Res., 30:267-272 (2007); Hincal An Introduction To Safety Issues In Biosimilars/Follow-On Biopharmaceuticals, J. Med. CBR Def., 7:1-18, (2009); Roger, Biosimilars: current status and future directions, Expert Opin. Biol. Ther., 10(7):1011-1018 (2010); Schellekens et al., Nat. Biotechnol. 28:28-31 (2010); Sekhon et al., Biosimilars, 1:1-11 (2011)). One exemplary report states that [t]he size and complexity of . . . therapeutic proteins make the production of an exact replica almost impossible; therefore, there are no true generic forms of these proteins . . . Verification of the similarity of biosimilars to innovator medicines remains a key challenge (Hincal, supra). This disclosure provides, in part, methods and compositions sufficient to make and test products that qualify as adalimumab, e.g., that are interchangeable versions of Humira.

[0034] Glycoprotein preparations can be obtained from any source. In some instances, providing or obtaining a glycoprotein preparation (e.g., such as a glycoprotein drug substance or a precursor thereof), e.g., that is or includes a glycoprotein, can include providing a host cell, e.g., a mammalian host cell (e.g., a CHO cell) that is genetically engineered to express a glycoprotein having an amino acid sequence at least 85% identical to SEQ ID NO:1 and an amino acid sequence at least 85% identical to SEQ ID NO:2 (e.g., a genetically engineered cell); culturing the host cell under conditions suitable to express the glycoprotein (e.g., mRNA and/or protein); and, optionally, purifying the expressed glycoproteins, e.g., in the form of a recombinant antibody) from the cultured cell, thereby producing a glycoprotein preparation. In some instances, the host cell is genetically engineered to express a glycoprotein having an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:1 and an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:2, wherein the expressed amino acid sequences form a recombinant antibody composition.

[0035] As used herein percent (%) sequence identity with respect to a sequence is defined as the percentage of amino acid residues or nucleotides in a candidate sequence that are identical with the amino acid residues or nucleotides in the reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. (E.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). Alignment for purposes of determining percent sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. In one embodiment, the length of a reference sequence aligned for comparison purposes is at least 30%, e.g., at least 40%, e.g., at least 50%, 60%, 70%, 80%, 90%, or 100% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. In some instances a product will include amino acid variants, e.g., species that differ at terminal residues, e.g., at one or two terminal residues. In instances of such cases the sequence identity which is compared is the identity between the primary amino acid sequences of the most abundant active species in each of the products being compared. In some instances sequence identity refers to the amino acid sequence encoded by a nucleic acid that can be used to make the product.

[0036] In some instances, an adalimumab signature disclosed herein can include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 1 or 22 of the adalimumab parameters (e.g., the reference criterion therefor) shown in Table 1 (e.g., including any combination of 2 or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22) of parameter numbers 1-22 shown in Table 1).

[0037] In some instances, an adalimumab signature disclosed herein can include, other structures or characteristics (whether intrinsic or extrinsic) of adalimumab, e.g., that distinguish adalimumab from non-adalimumab glycoprotein (see application entitled Methods of Evaluating and Making Biologics, filed on Jun. 1, 2012, as U.S. Ser. No. 61/654,467, for exemplary structures or characteristics). Examples of structures or characteristics include: the amount of GalNAc in the preparation (e.g., relative to total glycans of the preparation); the amount of truncated core glycans; the amount of aglycosylated glycans; the amount of each species of high mannose glycans; the amount of sialylated glycans or particular species of sialylated glycans; the ratio of monosialylated:diasylated glycans, the amount of diacetylated sialic acids (NeuXAc2), the amount of one or more of: NeuGc; NeuAc; Neu5,7,Ac2; Neu5Gc,9Ac; Neu5,8Ac2; Neu5,9Ac2; Neu4,5Ac2. Examples of parameters related to the glycan linkage composition of a glycoprotein preparation can be: the presence or amount of one or more of terminal fucose; terminal mannose; terminal galactose; 2 linked mannose; 3.6 linked mannose; terminal GlcNAc; terminal GalNAc; 4 linked GlcNAc; 4,6 linked GlcNAc. A parameter may also be the ratio of one of these to another or to another property. Examples of parameters related to the glycoform composition of a glycoprotein preparation include: the absence or presence of one or more specific glycoforms (e.g., one or more glycoforms described in Table 1); the amount or abundance of a specific glycoform in the preparation relative to total glycoforms (e.g., in a w/w basis); the ratio of one particular glycoform to another. Examples of parameters related to post-translational modification in the preparation include: the absence or presence of one or more specific post-translational modification; the abundance or distribution of one or more specific post-translational modification.

[0038] In some instances, the present disclosure includes determining whether information evaluated for a glycoprotein preparation meets an adalimumab signature, e.g., by comparing the information with the adalimumab signature and/or confirming that the information has a defined (e.g., predefined) relationship with the adalimumab signature.

[0039] In some instances, methods disclosed herein can be used to confirm the identity and/or quality of adalimumab preparations. For example, methods can include assessing preparations (e.g., samples, lots, and/or batches) of a test glycoprotein to confirm whether the test glycoprotein qualifies as adalimumab, and, optionally, qualifying the test protein as adalimumab if qualifying criteria (e.g. predefined qualifying criteria) are met; thereby evaluating, identifying, and/or producing (e.g., manufacturing) adalimumab.

[0040] Methods of the disclosure have a variety of applications and include, e.g., quality control at different stages of manufacture, analysis of adalimumab preparations prior to or after completion of manufacture (e.g., prior to or after distribution to a fill/finish environment or facility), prior to or after release into commerce (e.g., before distribution to a pharmacy, a caregiver, a patient, or other end-user). Thus, the preparation can be any preparation that potentially comprises adalimumab. In an embodiment the adalimumab preparation is a drug substance (an active pharmaceutical ingredient or API) or a drug product (an API formulated for use in a subject such as a human patient). In an embodiment the preparation is from a stage of manufacture or use that is prior to release to care givers or other end-users; prior to packaging into individual dosage forms, such as syringes, pens, vials, or multi-dose vials; prior to determination that the batch can be commercially released, prior to production of a Certificate of Testing, Material Safety Data Sheet (MSDS) or Certificate of Analysis (CofA) of the preparation. In an embodiment the glycoprotein preparation from an intermediate step in production, e.g., it is after secretion of the glycoprotein from a cell but prior to purification of drug substance.

[0041] Evaluations from methods of the invention are useful for guiding, controlling or implementing a number of activities or steps in the process of making, distributing, and monitoring and providing for the safe and efficacious use of adalimumab. Thus, in an embodiment, e.g., responsive to the evaluation, e.g., depending on whether a criterion is met, a decision or step is taken. The method can further comprise one or both of the decision to take the step and/or carrying out the step itself. E.g., the step can comprise one in which the preparation (or another preparation for which the preparation is representative) is: classified; selected; accepted or discarded; released or processed into a drug product; rendered unusable for commercial release, e.g., by labeling it, sequestering it, or destroying it; passed on to a subsequent step in manufacture; reprocessed (e.g., the preparation may undergo a repetition of a previous process step or subjected to a corrective process); formulated, e.g., into drug substance or drug product; combined with another component, e.g., an excipient, buffer or diluent; disposed into a container; divided into smaller aliquots, e.g., unit doses, or multi-dose containers; combined with another preparation of adalimumab; packaged; shipped; moved to a different location; combined with another element to form a kit; combined, e.g., placed into a package with a delivery device, diluent, or package insert; released into commerce; sold or offered for sale; delivered to a care giver or other end-user; or administered to a subject. E.g., based on the result of the determination or whether one or more subject entities is present, or upon comparison to a reference standard, the batch from which the preparation is taken can be processed, e.g., as just described.

[0042] Methods described herein may include making a decision: (a) as to whether a preparation may be formulated into drug substance or drug product; (b) as to whether a preparation may be reprocessed (e.g., the preparation may undergo a repetition of a previous process step); or (c) that the preparation is not suitable for formulation into drug substance or drug product. In instances the method comprises: formulating as referred to in step (a), reprocessing as referred to in step (b), or rendering the preparation unusable for commercial release, e.g., by labeling it or destroying it, as referred to in step (c).

Parameter Evaluation

[0043] The amino acid sequence of the heavy chain of adalimumab (Humira) is disclosed herein as SEQ ID NO:1. The amino acid sequence of the light chain of adalimumab (Humira) is disclosed herein as SEQ ID NO:2.

[0044] Parameters disclosed herein can be analyzed by any available suitable method. In some instances, glycan structure and composition as described herein are analyzed, for example, by one or more, enzymatic, chromatographic, mass spectrometry (MS), chromatographic followed by MS, electrophoretic methods, electrophoretic methods followed by MS, nuclear magnetic resonance (NMR) methods, and combinations thereof. Exemplary enzymatic methods include contacting a glycoprotein preparation with one or more enzymes under conditions and for a time sufficient to release one or more glycans (e.g., one or more exposed glycans). In some instances, the one or more enzymes includes PNGase F. Exemplary chromatographic methods include, but are not limited to, Strong Anion Exchange chromatography using Pulsed Amperometric Detection (SAX-PAD), liquid chromatography (LC), high performance liquid chromatography (HPLC), ultra performance liquid chromatography (UPLC), thin layer chromatography (TLC), amide column chromatography, and combinations thereof. Exemplary mass spectrometry (MS) include, but are not limited to, tandem MS, LC-MS, LC-MS/MS, matrix assisted laser desorption ionisation mass spectrometry (MALDI-MS), Fourier transform mass spectrometry (FTMS), ion mobility separation with mass spectrometry (IMS-MS), electron transfer dissociation (ETD-MS), and combinations thereof. Exemplary electrophoretic methods include, but are not limited to, capillary electrophoresis (CE), CE-MS, gel electrophoresis, agarose gel electrophoresis, acrylamide gel electrophoresis, SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western blotting using antibodies that recognize specific glycan structures, and combinations thereof. Exemplary nuclear magnetic resonance (NMR) include, but are not limited to, one-dimensional NMR (1D-NMR), two-dimensional NMR (2D-NMR), correlation spectroscopy magnetic-angle spinning NMR (COSY-NMR), total correlated spectroscopy NMR (TOCSY-NMR), heteronuclear single-quantum coherence NMR (HSQC-NMR), heteronuclear multiple quantum coherence (HMQC-NMR), rotational nuclear overhauser effect spectroscopy NMR (ROESY-NMR), nuclear overhauser effect spectroscopy (NOESY-NMR), and combinations thereof.

[0045] In some instances, techniques described herein may be combined with one or more other technologies for the detection, analysis, and or isolation of glycans or glycoproteins. For example, in certain instances, glycans are analyzed in accordance with the present disclosure using one or more available methods (to give but a few examples, see Anumula, Anal. Biochem. 350(1):1, 2006; Klein et al., Anal. Biochem., 179:162, 1989; and/or Townsend, R.R. Carbohydrate Analysis High Performance Liquid Chromatography and Capillary Electrophoresis., Ed. Z. El Rassi, pp 181-209, 1995, each of which is incorporated herein by reference in its entirety). For example, in some instances, glycans are characterized using one or more of chromatographic methods, electrophoretic methods, nuclear magnetic resonance methods, and combinations thereof.

[0046] In some instances, methods for evaluating one or more adalimumab-specific parameters, e.g., in a glycoprotein preparation, e.g., one or more of adalimumab parameters disclosed in Table 1 in a glycoprotein preparation are known in the art and/or are disclosed in Table 2:

TABLE-US-00001 TABLE 2 Method(s) Relevant literature Parameter C18 UPLC Mass Spec.* Chen and Flynn, Anal. Glycan(s) Biochem., 370: 147-161 (2007) (e.g., N-linked glycan, exposed N- Chen and Flynn, J. Am. Soc. linked glycan, glycan detection, Mass Spectrom., 20: 1821-1833 glycan identification, and (2009) characterization; site specific glycation; glycoform detection (e.g., parameters 1-16); percent glycosylation; and/or aglycoosyl) Peptide LC-MS Dick et al., Biotechnol. C-terminal lysine (e.g., parameter (reducing/non-reducing) Bioeng., 100: 1132-1143 (2008) 18) Yan et al., J. Chrom. A., 1164: 153-161 (2007) Chelius et al., Anal. Chem., 78: 2370-2376 (2006) Miller et al., J. Pharm. Sci., 100: 2543-2550 (2011) LC-MS (reducing/non- Dick et al., Biotechnol. reducing/alkylated) Bioeng., 100: 1132-1143 (2008) Goetze et al., Glycobiol., 21: 949-959 (2011) Weak cation exchange Dick et al., Biotechnol. (WCX) chromatography Bioeng., 100: 1132-1143 (2008) LC-MS (reducing/non- Dick et al., Biotechnol. N-terminal pyroglu (e.g., reducing/alkylated) Bioeng., 100: 1132-1143 (2008) parameters 19-20) Goetze et al., Glycobiol., Succinimide formation at aspartic 21: 949-959 (2011) acid 17 on the light chain (e.g., PeptideLC-MS Yan et al., J. Chrom. A., parameter 21) (reducing/non-reducing) 1164: 153-161 (2007) Chelius et al., Anal. Chem., 78: 2370-2376 (2006) Miller et al., J. Pharm. Sci., 100: 2543-2550 (2011) Peptide LC-MS Miller et al., J. Pharm. Sci., Site specific glycation (e.g., (reducing/non-reducing) 100: 2543-2550 (2011) parameter 22)

[0047] Literature shown in Table 2 are hereby incorporated by reference in their entirety or, in the alternative, to the extent that they pertain to one or more of the methods disclosed in Table 2.

EXAMPLES

Example 1: Characterization of Adalimumab

[0048] Humira samples were analyzed to determine the amino acid sequences of the heavy and light chains of the adalimumab antibody. The sequence of the heavy chain is shown as SEQ ID NO:1 and the sequence of the light chain is shown as SEQ ID NO:2.

[0049] Characterization of Humira/adalimumab was performed by orthogonal methods. 16 distinct lots of adalimumab were analyzed and measurements made included use of glycan profiling, glycoform analysis, post-translational modification analysis, and analysis of other intrinsic and extrinsic structures or features. Of 113 adalimumab structures or features that were measured or determined, 22 were determined to be adalimumab parameters, i.e., parameters of adalimumab that distinguish adalimumab from non-adalimumab antibody products. Parameters and values are listed in Table 3 below for a sample of adalimumab.

TABLE-US-00002 TABLE 3 Parameter Parameter # Category.sup.1 Value.sup.2 1 HM5 4.19 2 HM6 1.47 3 HM3 0.53 4 HM4 0.44 5 HM9 0.10 6 Sialylated 0.10 7 Sialylated 0.04 8 Complex G0F 67.85 9 Complex G1F 12.31 10 Complex G1F 3.76 11 Complex 2.00 12 Complex G2F 1.11 13 Complex G0 0.72 14 Complex G1 0.07 15 Complex G1 0.01 16 Hybrid 0.33 17 Hybrid 0.06 18 C-terminal-lysine 16.70 19 HC-pyroglu 1.37 20 LC-pyroglu 0.00 21 LC-D17-Suc 0.13 22 LC-K149-Glyc 0.20 .sup.1Detailed descriptions of the structures/features of each parameter are provided in Table 1. .sup.2See Table 1 for unit information.

[0050] Information (values) obtained for the 16 lots of adalimumab 3 were used to formulate a reference criterion or rule for each adalimumab parameter (shown in Table 1).

Example 2: Qualification of Glycoprotein Preparations

[0051] The reference criterion or rules described in Table 1 were used to determine whether glycoprotein preparations (samples A and B) qualify as adalimumab.

[0052] Sample A was analyzed and values were obtained for each of the adalimumab parameters in Table 1. The values of these parameters in sample A are presented in Table 4 below. In addition, values obtained for sample A were compared to the reference criteria for adalimumab as shown in Table 4:

TABLE-US-00003 TABLE 4 Comparison of A Values Parameter Sample A Reference and reference Parameter # Category.sup.1 Value Criterion.sup.2 criterion 1 HM5 3.1 >3.00% 2 HM6 2.59 >1.20% 3 HM3 0.01 >0.20% 4 HM4 0.01 >0.20% 5 HM9 0.01 >0.05% 6 Sialylated 0.35 <0.10% 7 Sialylated 0.04 <0.20% 8 Complex G0F 45.64 >60.00% 9 Complex G1F 22.83 <13.00% 10 Complex G1F 5.9 <4.50% 11 Complex 1.07 >1.50% 12 Complex G2F 3.47 <1.80% 13 Complex G0 3.72 <1.00% 14 Complex G1 0.84 <0.10% 15 Complex G1 0.38 <0.10% 16 Hybrid 0.25 >0.10% 17 Hybrid 0.21 >0.05% 18 C-terminal-lysine 45.20 >12.00% 19 HC-pyroglu 100.00 <10.00% 20 LC-pyroglu 70.00 <3.00% 21 LC-D17-Suc 0.00 >0.10% 22 LC-K149-Glyc 0.90 <0.30% .sup.1Detailed descriptions of the structures/features of each parameter are provided in Table 1. .sup.2See Table 1 for unit information. Illustrates that a value meets the reference criterion/rule.

[0053] Data plotted in Table 4 confirms that sample A is not adalimumab, according to the methods herein. Based on these data, sample A does not meet an adalimumab signature that comprises all 22 parameters and, thus, does not qualify as adalimumab.

[0054] Sample B was analyzed and values were obtained for each of the adalimumab parameters in Table 1. The values of these parameters in sample B are presented in Table 5 below. In addition, values obtained for sample B were compared to the reference criteria for adalimumab as shown in Table 5:

TABLE-US-00004 TABLE 5 Comparison of B Values Parameter Sample B Reference and reference Parameter # Category.sup.1 Value Criterion.sup.2 criterion 1 HM5 0.72 >3.00% 2 HM6 0.01 >1.20% 3 HM3 0.01 >0.20% 4 HM4 0.01 >0.20% 5 HM9 0.01 >0.05% 6 Sialylated 0.13 <0.10% 7 Sialylated 0.02 <0.20% 8 Complex G0F 68.46 >60.00% 9 Complex G1F 16.84 <13.00% 10 Complex G1F 4.8 <4.50% 11 Complex 1.28 >1.50% 12 Complex G2F 2.26 <1.80% 13 Complex G0 2.17 <1.00% 14 Complex G1 0.26 <0.10% 15 Complex G1 0.13 <0.10% 16 Hybrid 0.06 >0.10% 17 Hybrid 0.01 >0.05% 18 C-terminal-lysine 1.60 >12.00% 19 HC-pyroglu 2.30 <10.00% 20 LC-pyroglu 0.00 <3.00% 21 LC-D17-Suc 0.10 >0.10% 22 LC-K149-Glyc 0.40 <0.30% .sup.1Detailed descriptions of the structures/features of each parameter are provided in Table 1. .sup.2See Table 1 for unit information. Illustrates that a value meets the reference criterion/rule.

[0055] Data plotted in Table 5 confirms that sample B is not adalimumab. Based on these data, sample B does not meet an adalimumab signature that comprises all 22 parameters and, thus, does not qualify as adalimumab.

[0056] A control Humira sample was also analyzed and values were obtained for each of the adalimumab parameters in Table 1. The values of these parameters in the control are presented in Table 6 below. In addition, values obtained for the control were compared to the reference criteria for adalimumab, as shown in Table 6:

TABLE-US-00005 TABLE 6 Comparison of A Values Parameter Reference and reference Parameter # Category.sup.1 Value Criterion.sup.2 criterion 1 HM5 4.19 >4.00 2 HM6 1.47 >1.30 3 HM3 0.53 >0.20 4 HM4 0.44 >0.20 5 HM9 0.1 >0.05 6 Sialylated 0.1 <0.15 7 Sialylated 0.04 <0.10 8 Complex G0F 67.85 >65.00 9 Complex G1F 12.31 <13.00 10 Complex G1F 3.76 <4.00 11 Complex 2.0 >1.50 12 Complex G2F 1.11 <1.50 13 Complex G0 0.72 <1.00 14 Complex G1 0.07 <0.10 15 Complex G1 0.01 <0.10 16 Hybrid 0.33 >0.10 17 Hybrid 0.06 >0.05 18 C-terminal-lysine 16.70 >15.00 19 HC-pyroglu 1.37 <3.00 20 LC-pyroglu 0.00 <3.00 21 LC-D17-Suc 0.13 >0.10 22 LC-K149-Glyc 0.20 <0.30 .sup.1Detailed descriptions of the structures/features of each parameter are provided in Table 1. .sup.2See Table 1 for unit information. Illustrates that a value meets the reference criterion/rule.

[0057] As shown in Table 6, the control sample meets all listed reference criteria signatures for adalimumab. Accordingly, the control sample does meet an adalimumab signature that includes all 22 parameters and, thus, qualifies as adalimumab.

[0058] While the methods have been described in conjunction with various instances and examples, it is not intended that the methods be limited to such instances or examples. On the contrary, the methods encompass various alternatives, modifications, and equivalents, as will be appreciated by those of skill in the art.