Methods Related to Alemtuzumab
20200354465 ยท 2020-11-12
Inventors
- John Robblee (Newton, MA)
- Brian Edward Collins (Arlington, MA)
- Ganesh Kaundinya (Bedford, MA)
- Carlos J. Bosques (Arlington, MA)
Cpc classification
C07K2317/41
CHEMISTRY; METALLURGY
International classification
Abstract
The present invention relates to the characterization and production of alemtuzumab.
Claims
1. (canceled)
2. A method of manufacturing an alemtuzumab drug product, comprising: providing or obtaining a test glycoprotein preparation; acquiring a value for a plurality of alemtuzumab parameters listed in the following table for the test glycoprotein preparation, wherein the plurality of alemtuzumab parameters comprises five or more of the alemtuzumab parameters listed in the table; TABLE-US-00006 Parameter Para- Reference meter Parameter
Sialic Criterion # Category Mannose Fucose GlcNAc Galactose Acid (rule) 1 HM6
3. The method of claim 2, wherein the plurality comprises: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19, of the alemtuzumab parameters listed in the table.
4. The method of claim 29, wherein the processing step comprises one or more of: formulating the test glycoprotein preparation; processing the test glycoprotein preparation into a drug product; combining the test glycoprotein preparation with a second component; changing the concentration of the test glycoprotein in the preparation; lyophilizing the test glycoprotein preparation; combining a first and second aliquot of the test glycoprotein to provide a third, larger, aliquot; dividing the test glycoprotein preparation into smaller aliquots; disposing the test glycoprotein preparation into a container; packaging the test glycoprotein preparation; associating a container comprising the test glycoprotein preparation with a label shipping or moving the test glycoprotein preparation to a different location.
5. The method of claim 2, comprising: providing a host cell that is genetically engineered to express a first amino acid sequence having a sequence with 100% identity to SEQ ID NO: 1 and a second amino acid sequence having a sequence with 100% identity to SEQ ID NO:2, wherein the expressed amino acid sequences form a recombinant antibody composition, culturing the host cell under conditions whereby the cell expresses the first and second amino acid sequences, wherein the expressed first and second amino acid sequences form recombinant antibodies, harvesting the recombinant antibodies from the host cell culture to produce an antibody preparation.
6. A method of manufacturing an alemtuzumab drug product, comprising: providing or obtaining a test glycoprotein preparation; acquiring a value for a plurality of alemtuzumab parameters listed in the following table for the test glycoprotein preparation, wherein the plurality of alemtuzumab parameters comprises all of the alemtuzumab parameters listed in the table; TABLE-US-00007 Parameter Reference Parameter Parameter
Sialic Criterion # Category Mannose Fucose GlcNAc Galactose Acid (rule) 1 HM6
Description
DESCRIPTION OF THE DRAWINGS
[0031]
[0032]
DETAILED DESCRIPTION
[0033] Detailed, high resolution, structural information about Campath (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 alemtuzumab, e.g., that are interchangeable versions of Campath. 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 alemtuzumab, e.g., that are interchangeable versions of Campath, 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 alemtuzumab, e.g., that are interchangeable versions of Campath.
[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 alemtuzumab signature disclosed herein can include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 of the alemtuzumab 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, or 19) of parameter numbers 1-19 shown in Table 1).
[0037] In some instances, an alemtuzumab signature disclosed herein can include, other structures or characteristics (whether intrinsic or extrinsic) of alemtuzumab, e.g., that distinguish alemtuzumab from non-alemtuzumab 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 alemtuzumab signature, e.g., by comparing the information with the alemtuzumab signature and/or confirming that the information has a defined (e.g., predefined) relationship with the alemtuzumab signature.
[0039] In some instances, methods disclosed herein can be used to confirm the identity and/or quality of alemtuzumab 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 alemtuzumab, and, optionally, qualifying the test protein as alemtuzumab if qualifying criteria (e.g. predefined qualifying criteria) are met; thereby evaluating, identifying, and/or producing (e.g., manufacturing) alemtuzumab.
[0040] Methods of the disclosure have a variety of applications and include, e.g., quality control at different stages of manufacture, analysis of alemtuzumab 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 alemtuzumab. In an embodiment the alemtuzumab 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 alemtuzumab. 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 alemtuzumab; 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).
[0043] Parameter Evaluation
[0044] The amino acid sequence of the heavy chain of alemtuzumab (Campath) is disclosed herein as SEQ ID NO:1. The amino acid sequence of the light chain of alemtuzumab (Campath) is disclosed herein as SEQ ID NO:2.
[0045] 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.
[0046] 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.
[0047] In some instances, methods for evaluating one or more alemtuzumab-specific parameters, e.g., in a glycoprotein preparation, e.g., one or more of alemtuzumab parameters disclosed in Table 1 in a glycoprotein preparation are known in the art and/or are disclosed in Table 2:
TABLE-US-00002 TABLE 2 Method(s) Relevant literature Parameter C18 UPLC Chen and Flynn, Anal. Glycan(s) Mass Spec.* Biochem., 370: 147-161 (2007) (e.g., N-linked glycan, exposed N-linked Chen and Flynn, J. Am. glycan, glycan detection, glycan Soc. Mass Spectrom., identification, and characterization; site 20: 1821-1833 (2009) specific glycation; glycoform detection (e.g., parameters 1-14); percent glycosylation; and/or aglycosyl) Peptide LC-MS Dick et al., Biotechnol. C-terminal lysine (e.g., parameter 15) (reducing/non- Bioeng., 100: 1132-1143 (2008) reducing) 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 Dick et al., Biotechnol. (reducing/non- Bioeng., 100: 1132-1143 (2008) reducing/ Goetze et al., Glycobiol., alkylated) 21: 949-959 (2011) Weak cation Dick et al., Biotechnol. exchange (WCX) Bioeng., 100: 1132-1143 (2008) chromatography LC-MS Dick et al., Biotechnol. N-terminal pyroglu (e.g., parameters 16-17) (reducing/non- Bioeng., 100: 1132-1143 (2008) reducing/ Goetze et al., Glycobiol., alkylated) 21: 949-959 (2011) PeptideLC-MS Yan et al., J. Chrom. A., (reducing/non- 1164: 153-161 (2007) reducing) Chelius et al., Anal. Chem., 78: 2370-2376 (2006) Miller et al., J. Pharm. Sci., 100: 2543-2550 (2011) Peptide LC-MS Wang et al., Anal. Chem., Free cysteine (e.g., parameters 18-19) (reducing/non- 83: 3133-3140 (2011); reducing) Chumsae et al., Anal. Chem., 81: 6449-6457 (2009)
[0048] 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 Alemtuzumab
[0049] Campath sample was analyzed to determine the amino acid sequences of the heavy and light chains of the alemtuzumab 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.
[0050] Characterization of Campath was performed by orthogonal methods. 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 Campath/alemtuzumab structures or features that were measured or determined, 19 were determined to be alemtuzumab parameters, i.e., parameters of alemtuzumab that distinguish alemtuzumab from non-alemtuzumab antibody products. These 19 alemtuzumab parameters and values are listed in Table 3 for an illustrative sample of alemtuzumab.
TABLE-US-00003 TABLE 3 Acquired Values for Each Parameter Parameter # Parameter Category.sup.1 Value.sup.2 1 HM6 0.64 2 HM8 0.27 3 Sialyated 0.66 4 Sialyated 0.24 5 Sialyated 0.21 6 Sialyated 0.18 7 Complex G0F 43.19 8 Complex 0.34 9 Complex G2F 5.43 10 Complex 0.16 11 Complex G0 7.29 12 Complex G1 2.14 13 Complex G1 1.11 14 Complex G2 0.25 15 C-Terminal-lysine 1.00 16 HC-pyroglu 99.50 17 LC-pyroglu 0.00 18 HC148 5.50 19 HC204 3.60 .sup.1Detailed descriptions of the structures/features of each parameter are provided in Table 1. .sup.2See Table 1 for unit information.
[0051] The information (values) shown for each alemtuzumab parameter in Table 3 were used to formulate a reference criterion or rule for each alemtuzumab parameter (shown in Table 1).
Example 2: Qualification of Glycoprotein Preparations
[0052] The reference criterion or rules described in Table 1 were used to determine whether samples qualify as alemtuzumab. Multiple glycoprotein products were prepared and samples thereof were used for identity analysis (samples A and B).
[0053] Sample A was analyzed and values were obtained for each of the alemtuzumab 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 alemtuzumab as shown in Table 4:
TABLE-US-00004 TABLE 4 Acquired Values of Sample A Compared with Reference Values Comparison of A Values Parameter Sample Reference and reference Parameter # Category.sup.1 A Value Criterion.sup.2 criterion 1 HM6 2.59 >0.50% 2 HM8 1.4 >0.20%
3 Sialyated 0.35 >0.50% 4 Sialyated 0.04 >0.20% 5 Sialyated 0.05 >0.10% 6 Sialyated 0.01 >0.10% 7 Complex G0F 45.64 <50.00%
8 Complex 1.07 <0.50% 9 Complex G2F 3.47 >4.50% 10 Complex 0.93 <0.25% 11 Complex G0 3.72 >6.00% 12 Complex G1 0.84 >1.80% 13 Complex G1 0.38 >0.70% 14 Complex G2 0.07 >0.10% 15 C-Terminal-lysine 45.20 <5.00% 16 HC-pyroglu 100.00 >80.00%
17 LC-pyroglu 70.00 <3.00% 18 HC148 13.40 <10.00% 19 HC204 4.10 <5.00% .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.
[0054] Data plotted in Table 4 confirms that sample A is not alemtuzumab, according to the methods herein. Based on these data, sample A does not meet an alemtuzumab signature that comprises all 19 parameters and, thus, does not qualify as alemtuzumab
[0055] A control Campath sample was also analyzed and values were obtained for each of the alemtuzumab parameters in Table 1. The values of these parameters in the control are presented in Table 5 below. In addition, values obtained for the control were compared to the reference criteria for alemtuzumab as shown in Table 5:
TABLE-US-00005 TABLE 5 Comparison Control of A Values Param- Parameter Campath Reference and reference eter # Category.sup.1 sample Criterion.sup.2 criterion 1 HM6 0.64 >0.50% 2 HM8 0.27 >0.20%
3 Sialyated 0.66 >0.50%
4 Sialyated 0.24 >0.20%
5 Sialyated 0.21 >0.10%
6 Sialyated 0.18 >0.10%
7 Complex G0F 43.19 <50.00%
8 Complex 0.34 <0.50%
9 Complex G2F 5.43 >4.50%
10 Complex 0.16 <0.25%
11 Complex G0 7.29 >6.00%
12 Complex G1 2.14 >1.80%
13 Complex G1 1.11 >0.70%
14 Complex G2 0.25 >0.10%
15 C-Terminal-lysine 1.00 <5.00%
16 HC-pyroglu 99.50 >80.00%
17 LC-pyroglu 0.00 <3.00%
18 HC148 5.50 <10.00%
19 HC204 3.60 <5.00%
.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.
[0056] As shown in Table 5, the control Campath sample meets all listed reference criteria signatures for alemtuzumab. Accordingly, the control Campath sample does meet an alemtuzumab signature that includes all 19 parameters and, thus, qualifies as alemtuzumab.
[0057] 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.