METHODS FOR REDUCING HOST CELL PROTEIN CONTENT IN ANTIBODY PURIFICATION PROCESSES AND ANTIBODY COMPOSITIONS HAVING REDUCED HOST CELL PROTEIN CONTENT

Abstract

The present disclosure relates to methods for reducing host cell protein content in antibody preparation recombinantly produced in a host cell in the manufacturing process of antibodies intended for administration to a patient. The disclosed methods may be performed in order to prepare therapeutic antibody preparations having reduced host cell protein.

Claims

1-172. (canceled)

173. A pharmaceutical composition comprising an antibody that binds to human N3pGlu A (anti-N3pGlu A antibody), wherein the anti-N3pGlu A antibody was prepared by a process comprising purifying the anti-N3pGlu antibody from a mammalian host cell, and wherein the total content of host cell proteins (HCPs) in the composition is less than about 100 ppm (as measured by LCMS) and the composition comprises one of, combinations of, or all of the following host cell proteins: protein S100-A6, protein S100-A11, phospholipase B-like 2 protein, lysosomal protective protein, ubiquitin-40S ribosomal protein S27a, kallikrein-11, serine protease HTRA1 isoform X1, complement C1r subcomponent, actin, aortic smooth muscle isoform X1, heat shock cognate 71 kDa protein, and peroxiredoxin-1.

174. A pharmaceutical composition according to claim 173, wherein the mammalian cell is a CHO cell.

175. A pharmaceutical composition according to claim 173, wherein the anti-N3pGlu A antibody is a monoclonal antibody, a chimeric antibody, a humanized antibody, a human antibody, a bispecific antibody, or an antibody fragment.

176. A pharmaceutical composition according to claim 175, wherein the anti-N3pGlu A antibody is an IgG1 antibody.

177. A pharmaceutical composition according to claim 173, wherein the anti-N3pGlu A antibody comprises a heavy chain (HC) and a light chain (LC), wherein the light chain comprises a light chain variable region (LCVR) and the heavy chain comprises a heavy chain variable region (HCVR), wherein the LCVR comprises amino acid sequences LCDR1, LCDR2, and LCDR3, and the HCVR comprises amino acid sequences HCDR1, HCDR2, and HCDR3, wherein LCDR1 is KSSQSLLYSRGKTYLN (SEQ ID NO:17), LCDR2 is AVSKLDS (SEQ ID NO:18), LCDR3 is VQGTHYPFT (SEQ ID NO:19), HCDR1 is GYDFTRYYIN (SEQ ID NO:20), HCDR2 is WINPGSGNTKYNEKFKG (SEQ ID NO:21), and HCDR3 is EGITVY (SEQ ID NO:22).

178. A pharmaceutical composition according to claim 173, wherein the LC of the anti-N3pGlu A antibody comprises a LCVR and the HC of the anti-N3pGlu A antibody comprises a HCVR, wherein the LCVR is TABLE-US-00042 (SEQIDNO:13) DIVMTQTPLSLSVTPGQPASISCKSSQSLLYSRGKTYLNWLLQKPGQSP QLLIYAVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCVQGTH YPFTFGQGTKLEIK and the HCVR is TABLE-US-00043 (SEQIDNO:14) QVQLVQSGAEVKKPGSSVKVSCKASGYDFTRYYINWVRQAPGQGLEWMG WINPGSGNTKYNEKFKGRVTITADESTSTAYMELSSLRSEDTAVYYCAR EGITVYWGQGTTVTVSS

179. A pharmaceutical composition according claim 173, wherein the LC of the anti-N3pGlu A antibody is TABLE-US-00044 (SEQIDNO:15) DIVMTQTPLSLSVTPGQPASISCKSSQSLLYSRGKTYLNWLLQKPGQSP QLLIYAVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCVQGTH YPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPR EAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGEC and the HC of the anti-N3pGlu A antibody is TABLE-US-00045 (SEQIDNO:16) QVQLVQSGAEVKKPGSSVKVSCKASGYDFTRYYINWVRQAPGQGLEWMG WINPGSGNTKYNEKFKGRVTITADESTSTAYMELSSLRSEDTAVYYCAR EGITVYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRE PQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPG.

180. A pharmaceutical composition according to claim 173, wherein the anti-N3pGlu A antibody is donanemab.

181. (canceled)

182. A pharmaceutical composition according to claim 173, wherein the composition comprises less than about 5 ppm of protein S100-A6 (as measured by LCMS).

183. A pharmaceutical composition according to claim 173, wherein the composition comprises less than about 5 ppm of protein S100-A11 (as measured by LCMS).

184. A pharmaceutical composition according to claim 173, wherein the composition comprises less than about 10 ppm of phospholipase B-like 2 protein (as measured by LCMS).

185. A pharmaceutical composition according to claim 173, wherein the composition comprises less than about 5 ppm of lysosomal protective protein (as measured by LCMS).

186. A pharmaceutical composition according to claim 173, wherein the composition comprises less than about 5 ppm of ubiquitin-40S ribosomal protein S27a (as measured by LCMS).

187. A pharmaceutical composition according to claim 173, wherein the composition comprises less than about 5 ppm of kallikrein-11 (as measured by LCMS).

188. A pharmaceutical composition according to claim 173, wherein the composition comprises less than about 5 ppm serine protease HTRA1 isoform X1 (as measured by LCMS).

189. A pharmaceutical composition according to claim 173, wherein the composition comprises less than about 5 ppm complement C1r subcomponent (as measured by LCMS).

190. A pharmaceutical composition according to claim 173, wherein the composition comprises less than about 5 ppm actin (as measured by LCMS).

191. A pharmaceutical composition according to claim 173, wherein the composition comprises less than about 5 ppm aortic smooth muscle isoform X1 (as measured by LCMS).

192. A pharmaceutical composition according to claim 173, wherein the composition comprises less than about 5 ppm heat shock cognate 71 kDa protein (as measured by LCMS).

193. A pharmaceutical composition according to claim 173, wherein the composition comprises less than about 5 ppm peroxiredoxin-1 (as measured by LCMS).

194-196. (canceled)

197. A pharmaceutical composition comprising an antibody that binds to human N3pGlu Ab (anti-N3pGlu Ab antibody), wherein the anti-N3pGlu Ab antibody was prepared by a process comprising purifying the anti-N3pGlu antibody from a mammalian host cell, and wherein the total content of host cell proteins (HCPs) in the composition is less than about 10 ppm (as measured by LCMS) and the composition comprises one of, combinations of, or all of the following host cell proteins: polyubiquitin, lysosomal protective protein, glutathione S-transferase Y1, 40S ribosomal protein S28, thioredoxin isoform X1, basement membrane-specific heparan sulfate proteoglycan core protein isoform X1, tubulointerstitial nephritis antigen-like protein, actin-partial cytoplasmic 2 isoform X2, galectin-1, peroxiredoxin-1, and cornifin alpha.

198. (canceled)

199. A pharmaceutical composition according to claim 197, wherein the composition comprises less than about 1 ppm of polyubiquitin (as measured by LCMS).

200. A pharmaceutical composition according to claim 197, wherein the composition comprises less than about 1 ppm of lysosomal protective protein (as measured by LCMS).

201. A pharmaceutical composition according to claim 197, wherein the composition comprises less than about 1 ppm of glutathione S-transferase Y1 (as measured by LCMS).

202. (canceled)

203. A pharmaceutical composition according to claim 197, wherein the composition comprises less than about 1 ppm of 40S ribosomal protein S28 (as measured by LCMS).

204. A pharmaceutical composition according to claim 197, wherein the composition comprises less than about 1 ppm of thioredoxin isoform X1 (as measured by LCMS).

205. A pharmaceutical composition according to claim 197, wherein the composition comprises less than about 1 ppm of basement membrane-specific heparan sulfate proteoglycan core protein isoform X1 (as measured by LCMS).

206. A pharmaceutical composition according to claim 197, wherein the composition comprises less than about 1 ppm of tubulointerstitial nephritis antigen-like protein (as measured by LCMS).

207. A pharmaceutical composition according to claim 197, wherein the composition comprises less than about 1 ppm of actin-partial cytoplasmic 2 isoform X2 (as measured by LCMS).

208. A pharmaceutical composition according to claim 197, wherein the composition comprises less than about 1 ppm of galectin-1 (as measured by LCMS).

209. A pharmaceutical composition according to claim 197, wherein the composition comprises less than about 1 ppm of peroxiredoxin-1 (as measured by LCMS).

210. A pharmaceutical composition according to claim 197, wherein the mammalian cell is a CHO cell.

211. A pharmaceutical composition according to claim 197, wherein the anti-N3pGlu A antibody is a monoclonal antibody, a chimeric antibody, a humanized antibody, a human antibody, a bispecific antibody, or an antibody fragment.

212. A pharmaceutical composition according to claim 197, wherein the anti-N3pGlu A antibody is an IgG1 antibody.

213. The pharmaceutical composition according to claim 197, wherein the anti-N3pGlu A antibody comprises a heavy chain (HC) and a light chain (LC), wherein the light chain comprises a light chain variable region (LCVR) and the heavy chain comprises a heavy chain variable region (HCVR), wherein the LCVR comprises amino acid sequences LCDR1, LCDR2, and LCDR3, and the HCVR comprises amino acid sequences HCDR1, HCDR2, and HCDR3, wherein LCDR1 is RASQSLGNWLA (SEQ ID NO: 27), LCDR2 is YQASTLES (SEQ ID NO: 28). LCDR3 is QHYKGSFWT (SEQ ID NO: 29), HCDR1 is AASGFTFSSYPMS (SEQ ID NO: 30), HCDR2 is AISGSGGSTYYADSVKG (SEQ ID NO: 31), and HCDR3 is AREGGSGSYYNGFDY (SEQ ID NO: 32).

214. The pharmaceutical composition of claim 197, wherein the LC of the anti-N3pGlu A antibody comprises a LCVR and the HC of the anti-N3pGlu A antibody comprises a HCVR, wherein the LCVR is TABLE-US-00046 (SEQIDNO:23) DIQMTQSPSTLSASVGDRVTITCRASQSLGNWLAWYQQKPGKAPKLLIY QASTLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQHYKGSFWTF GQGTKVEIK and the HCVR is TABLE-US-00047 (SEQIDNO:24) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYPMSWVRQAPGKGLEWVS AISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR EGGSGSYYNGFDYWGQGTLVTVSS.

215. The pharmaceutical composition of claim 197, wherein the LC of the anti-N3pGlu A antibody is TABLE-US-00048 (SEQIDNO:25) DIQMTQSPSTLSASVGDRVTITCRASQSLGNWLAWYQQKPGKAPKLLIY QASTLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQHYKGSFWTF GQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ WKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC and the HC of the anti-N3pGlu A antibody is TABLE-US-00049 (SEQIDNO:26) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYPMSWVRQAPGKGLEWVS AISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR EGGSGSYYNGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAAL GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSS SLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPG.

Description

EXAMPLES

[0132] Host cell protein (HCP) measurements by LCMS: to assess purification impact on host cell protein (HCP) levels in the examples which follow, samples are analyzed by peptide mapping/LC-MS/MS HCP profiling via, e.g., a Ultra Performance Liquid Chromatography (UPLC) coupled to a Thermo Scientific mass spectrometer. Methods for detecting HCPs have been disclosed in the art. (See, e.g., Huang et al., A Novel Sample Preparation for Shotgun Proteomics Characterization of HCPs in Antibodies, Anal. Chem. 2017, 89, 5436-5444.) In this analysis, the samples are subjected to digestion by trypsin, reduced/precipitated with dithiothreitol (DTT), followed by transfer and acidification of the supernatant in a HPLC vial for LC-MS/MS analysis. The LC-MS/MS data is analyzed by Proteome Discoverer against CHO-K1 protein database with added antibody, spike, and control protein sequences. The HCP concentration is reported as total parts per million (ppm) of HCP per sample for total HCP content (e.g., ng of HCP per mg of product). Additionally, the concentrations of certain HCPs, (e.g., phospholipase B-like 2 protein (PLBL2) and lysosomal protective protein) are also provided.
HCP measurements by ELISA: HCP levels concentration in the samples are also assessed in the examples which follow by an ELISA assay using a Gyrolab CHO-HCP Kit 1 (Cygnus Technologies, performed per manufacturer instructions). The HCP results concentration are reported as total parts per million (ppm) of HCP per sample for total HCP content.

Example 1HCP Reduction in mAb1 (Etesevimab) Purification Process

[0133] Protein Capture step: A sanitized Protein A column (MabSelect SuRe Protein A media) is equilibrated and mAb1 (etesevimab) cell-free bioreactor harvest is loaded onto the Protein A column and three washes of the Protein A column are performed using 20 mM Tris (pH 7.0) as the last wash. mAb1 is eluted from the column using 5 column volumes (CVs) of 20 mM acetic acid+5 mM phosphoric acid. The main product fraction is collected into a single bulk fraction by using absorbance-based peak cutting on the frontside and backside.
Low pH Viral Inactivation Step and Neutralization Step: The pH of the main product fraction (protein capture eluate bulk fraction) containing mAb1 is adjusted to a pH between 3.30 and 3.60 by the addition of 20 mM HCl for low pH viral inactivation. The mixture is incubated at 18 C. to 25 C. for 180 min. The mixture is then neutralized to a pH of 7.0 using 250 mM Tris base pH unadjusted buffer.
Depth Filtration Step: A depth filter (X0SP, Millipore) is flushed with water for injection (WFI). The mAb1 mixture, obtained from the low pH viral inactivation step and neutralization step, is applied to the depth filter with a loading of 1200 g/m.sup.2 (grams of mAb per m.sup.2 of depth filter membrane area). The loaded depth filter is flushed with WFI. The filtrate from the depth filter, optionally inclusive of the post-loading WFI flush, is neutralized to pH 8.0 using 250 mM Tris base pH unadjusted buffer.
Anion Exchange (AEX) Chromatography Step: A sanitized column (Q Sepharose Fast Flow Anion Exchange Chromatography Media, or QFF) is equilibrated with 2 CVs of 20 mM Tris (pH 8.0). The mAb1 solution, obtained from the depth filtration step, is loaded onto the column at a loading of 25 to 100 g per liter of resin, and an additional wash is performed with the equilibration buffer. mAb1 is collected by absorbance-based peak cutting on the frontside and backside of the peak area formed by the unbound fraction plus the additional wash.
Results: Using the purification process described, the total HCP level as measured by LC-MS is: [0134] 23299 ppm after Protein A elution; [0135] 13 ppm after X0SP depth filtration; [0136] 2 ppm after AEX chromatography.
Depth filter Set 1 assessment for mAb1: mAb1 is processed through Protein A, low pH viral inactivation, neutralization, and depth filtration steps essentially as described above. Four different depth filters: Emphaze AEX Hybrid Purifier, Zeta Plus BC25-60ZB05A, Zeta Plus BC25-90ZB05A, and Zeta Plus BC25-90ZB08A (3M) are tested at a loading of 2000 g/m.sup.2 as shown in Table 1. The results in Table 1 show a significant reduction in total HCP content after depth filtration by LCMS and/or ELISA for the 4 depth filters tested when compared to the total HCP content observed after Protein A elution.

TABLE-US-00029 TABLE 1 mAb1 total HCP content before and after depth filtration Total HCP content Total HCP content after Protein A after depth elution (ppm) filtration (ppm) LCMS ELISA Depth filter LCMS ELISA 28901 527 Emphaze AEX not available 16 Hybrid Purifier Zeta Plus BC25 - 31 8 (60ZB05A) Zeta Plus BC25 - 29 7 (90ZB05A) Zeta Plus BC25 - 24 6 (90ZB08A)

Example 2HCP Reduction in mAb2 (Bamlanivimab) Purification Process

[0137] Protein A elution buffer comparison: mAb2 is prepared essentially as described for mAb1 in Example 1 with the following exceptions: 1) after low pH viral inactivation and before depth filtration, the solution is neutralized to a pH of 7.25 instead of 7.0 using 250 mM Tris base pH unadjusted buffer, 2) mAb 2 is eluted from the Protein A capture column using the buffer combinations listed in Table 2, and 3) the AEX chromatography is performed using Poros XQ resin. HCP content (both total HCP levels and PLBL2 levels) is assessed via LCMS, after purification unit operations as listed in Tables 2 and 3. The results in Tables 2 and 3, show that total HCP and PLBL2 content is reduced for all 3 buffer combinations tested, after the depth filtration step. Specifically, the 20 mM acetic acid+5 mM phosphoric acid and 20 mM acetic acid+5 mM L-lactic acid showed a greater reduction of total HCP and PLBL2 of less than 20 ppm when compared to the 20 mM acetic acid+5 mM citric acid combination after depth filtration.

TABLE-US-00030 TABLE 2 mAb2 total HCP content using different Protein A elution buffers Total HCP by Total HCP by Total HCP by LCMS detection Protein A LCMS detection LCMS detection after AEX elution after Protein A after X0SP depth chromatography buffer elution (ppm) filtration (ppm) (ppm) 20 mM acetic 71022 469 55 acid + 5 mM citric acid 20 mM acetic 77892 7 11 acid + 5 mM phosphoric acid 20 mM acetic 78669 16 Below limit of acid + 5 mM quantitation L-lactic acid

TABLE-US-00031 TABLE 3 mAb2 PLBL2 content using different Protein A elution buffers PLBL2 by LCMS PLBL2 by LCMS PLBL2 by LCMS detection after Protein A detection after detection after AEX elution Protein A elution X0SP depth chromatography buffer (ppm) filtration (ppm) (ppm) 20 mM acetic 356 454 8 acid + 5 mM citric acid 20 mM acetic 351 Below limit of Below limit of acid + 5 mM quantitation quantitation phosphoric acid 20 mM acetic 404 Below limit of Below limit of acid + 5 mM quantitation quantitation L-lactic acid
Depth filter set 2 assessment: mAb 2 is prepared essentially as described for mAb1 with the following exceptions: 1) after low pH viral inactivation and before depth filtration, neutralize the pH of the solution to a pH of 7.25 instead of 7.0 using 250 mM Tris base pH unadjusted buffer, and 2) depth filtration is performed with the depth filters shown in Table 4. Table 4 shows total HCP and PLBL2 content after depth filtration using various depth filters at a loading of 1500 g/m.sup.2. All 3 set 2 depth filters tested (X0SP, C0SP, X0HC, (Millipore)) show significant reduction in total HCP and PLBL2 content of less than 20 ppm after depth filtration.

TABLE-US-00032 TABLE 4 mAb2 HCP total and PLBL2 content before and after depth filtration Total HCP PLBL2 Total HCP PLBL2 content by content by content by content by LCMS LCMS LCMS after LCMS after after depth after depth Protein A Protein A Depth filtration filtration elution (ppm) elution (ppm) filter (ppm) (ppm) 74528 543 X0SP 3 Below limit of quantitation C0SP 18 5 X0HC 2 Below limit of quantitation

Example 3. HCP Reduction in mAb3 (Bebtelovimab) Purification Process

[0138] mAb3 is prepared using the protein capture, low pH viral inactivation, neutralization, and depth filtration steps essentially as described for mAb1 in Example 1, except using a X0SP depth filter with a loading of 900 g/m.sup.2. Using the described purification process the total HCP level as measured by LCMS is: [0139] 179964 ppm after the Protein A elution, [0140] 77 ppm after X0SP (Millipore) depth filtration.

Example 4. HCP Reduction in Bispecific Antibody (mAb4) Purification Process

[0141] A bispecific antibody mAb4 is prepared using the protein capture step essentially as described for mAb1 in Example 1, except using a Protein L affinity capture column (Cytiva) and eluting with the buffer systems shown in Table 5. The total HCP content is measured by ELISA giving a range of about 1300 to about 2500 ppm. Following protein capture, low pH viral inactivation is performed essentially as described for mAb1 in Example 1, except using the titrants listed in Table 5, followed by neutralization up to pH 7.0 using 500 mM Tris base pH unadjusted buffer. Then, the depth filtration step is performed as described for mAb1 in Example 1 using a X0SP depth filter at a loading of 1200 g/m.sup.2. The HCP content is measured after depth filtration by ELISA.

[0142] The results in Table 5, show significant reduction in total HCP content to less than 50 ppm for Entries 1 to 7 following depth filtration, where the ionic strength of the mixtures applied to the depth filter was less than 45 mM. In addition, a correlation between the ionic strength of the mixtures applied to the depth filter and the total HCP content after the depth filtration. Furthermore, Entry 2 shows that ionic strength can be decreased by diluting the buffer, providing low HCP content after depth filtration, however the volume increase from dilution can be disadvantageous to manufacturing processes.

TABLE-US-00033 TABLE 5 HCP levels in mAb4 preparations following Protein L elution and depth filtration Ionic strength Total HCP of mixture content by Low pH viral applied to ELISA after Protein L inactivation depth filter X0SP depth Entry elution buffer titrant (mM) filtration (ppm) 1 20 mM acetic 20 mM acetic 38 38 acid + 10 mM acid + 10 mM phosphoric acid phosphoric acid 2 20 mM acetic 20 mM acetic 13 (after 1:2 18 acid + 10 mM acid + 10 mM H.sub.2O dilution)* phosphoric acid phosphoric acid 3 20 mM acetic 20 mM HCl 36 35 acid + 10 mM phosphoric acid 4 20 mM acetic 20 mM HCl 27 30 acid + 5 mM phosphoric acid 5 20 mM acetic 20 mM HCl 23 26 acid + 5 mM formic acid 6 20 mM acetic 200 mM 43 50 acid + 10 mM phosphoric acid phosphoric acid 7 20 mM acetic 15 mM 37 36 acid + 10 mM phosphoric acid phosphoric acid 8 20 mM acetic 1000 mM 64 209 acid + 10 mM citric acid phosphoric acid *following low pH viral inactivation and neutralization to pH 7.0 with 500 mM Tris, the mAb4 solution is diluted with 2 parts water (1:2 ratio of mAb4 solution:H.sub.2O)

Example 5. HCP Reduction in mAb5 (Donanemab) Purification Processes

[0143] A mAb5 preparation is prepared using the steps as essentially described below: protein capture, low pH viral inactivation and neutralization, depth filtration, anion exchange (AEX) chromatography, cation exchange (CEX) chromatography, viral filtration and tangential flow filtration (TFF).

Protein Capture Step:

[0144] Capture and purify the antibody by reducing process-related impurities such as residual HCPs and residual DNA. A sanitized Protein A column (MabSelect Protein A media) is equilibrated and a monoclonal antibody (mAb5 (donanemab) expressed from CHO cell) cell-free bioreactor harvest is loaded onto the Protein A column and three washes of the Protein A column are performed using 20 mM Tris (pH 7.0) as the last wash. The antibody is eluted from the column using 5 column volumes (CVs) of 20 mM acetic acid+5 mM citric acid. The main product fraction is collected into a single bulk fraction by using absorbance-based peak cutting on the frontside and backside.

Low pH Viral Inactivation Step and Neutralization Step:

[0145] Inactivate low pH susceptible viruses, reduce residual HCP, residual protein A, residual DNA and total aggregates. Viral inactivation is conducted by adjusting the pH of the collected main product fraction (protein capture eluate bulk fraction) containing the mAb to a pH between 3.30 and 3.60 by the addition of 20 mM acetic acid, 5 mM citric acid. The mixture is incubated at 18 C. to 25 C. for about 180 min. The mixture is then neutralized to a pH of 5 to 7.0, preferably pH 5.0, using 250 mM Tris base pH unadjusted buffer.

Depth Filtration Step:

[0146] A separate depth filter (B1HC, Millipore) is flushed with water for injection (WFI) for each test condition (pH 5 with B1HC). The mAb mixture, obtained from the low pH viral inactivation step and neutralization step, is applied to the depth filter with a target loading of approximately 500-1500 g/m.sup.2 (grams of mAb per m.sup.2 of depth filter membrane area). The loaded depth filter is flushed with WFI. The filtrate from the depth filter, optionally inclusive of the post-loading WFI flush, is neutralized to pH 7.25 using 250 mM Tris base pH unadjusted buffer. A calculated volume of 20 mM Tris, 1 M NaCl, pH 7.0 buffer to added to a final NaCl concentration of 50 mM.

Anion Exchange (AEX) Chromatography Step:

[0147] Reduce potential viral contaminants. A sanitized Poros XQ (or Sartobind Q or Poros HQ) anion exchange (AEX) column (is pre-equilibrated with 2 CV of 20 mM Tris, 1 M NaCl, pH 7.0 buffer followed by 3 CVs of equilibration buffer 20 mM Tris 50 mM NaCl, (pH 7.25). The mAb solutions from each of the of depth filter conditions were flowed through the AEX column in discrete runs based upon depth filter condition, obtained from the depth filtration step, is loaded onto the column at a loading of approximately 100 g-200 g per liter of resin (e.g., approximately 150 g per liter of resin), and an additional wash is performed with the equilibration buffer. mAb is collected from the start of loading until the end of wash.

Cation Exchange (CEX) Chromatography Step:

[0148] Reduce total aggregates, reduce residual HCP and reduce residual protein A. The different AEX intermediates were pH adjusted from approximately 7.25 to 5.0 with the addition of 0.1 N acetic acid before loading onto the equilibrated (20% Mobile Phase B or equivalent to 20 mM sodium acetate, 200 mM sodium chloride, pH 5.0) CEX chromatography resin (POROS HS or UNOsphere S). The AEX process intermediate at pH 5.0 is blended with 15% Mobile Phase B (corresponding to 193 mM sodium chloride) at the point of loading onto the CEX column. Column load was approximately 25 grams of mAb per liter of resin. After loading, the column is washed with 20% Mobile Phase B (equivalent to 20 mM sodium acetate, 200 mM sodium chloride, pH 5.0) to facilitate removal of unbound impurities. mAb is then eluted from the column with a linear gradient from 20%-55% Mobile Phase B over 10 column volumes (200 to 550 mM sodium chloride gradient in a 20 mM sodium acetate, pH 5.0 buffer). To ensure complete elution of product, the linear gradient may be followed by an isocratic hold at 55% Mobile Phase B (equivalent to 20 mM sodium acetate, 550 mM sodium chloride, pH 5.0). During elution, a UV-based cut on the front-side at NLT 4.8 AU/cm initiates CEX eluate collection and continues through the peak apex until the back-side cut is made at NLT 2.4 AU/cm. The column is regenerated and sanitized with a 1 N sodium hydroxide solution. The column may be stored in 0.01 N sodium hydroxide. The preparations then are analyzed for HCP content using LCMS.

Viral Filtration:

[0149] Remove potential viral contaminants. Viral filtration is performed through a Viresolve Pro, Planova 20N or Planova BioEX membrane.

Tangential Flow Filtration (TFF):

[0150] Exchange the viral filtrate process intermediate into the appropriate matrix for final drug substance (DS) preparation and concentrate the antibody to the appropriate range for final DS preparation. TFF is performed on a 30 kDa PES or 30 kDa Regenerated Cellulose membrane.

Drug Substance Dispensing:

[0151] After TFF, a surfactant is added to complete the drug substance formulation and dispensed into an approved container closure system for storage and transport at the appropriate temperature prior to drug product manufacture.

Measurement of HCP Content by LC-MS

[0152] HCP content was measured by LC-MS as described below. For mAb5 Batch 1 and mAb5 Batch 2, HCP content was measured after the Protein Capture Step, after low pH viral inactivation, after AEX and after CEX. For mAb5 Batches 3-5, HCP content was measured prior to drug substance dispensing. The results are shown in Tables 6a and 6b and Table 7 below.

Sample Preparation

[0153] The aliquot containing 1 mg protein of each sample or control was added to pure water to 193 mL. The solution was mixed with 5.0 mL of 1 M tris-HCl buffer, pH 8, 1.0 mL aliquot of four protein mixture and then treated with 1 mL of 2.5 mg/mL r-trypsin at 37 C. for overnight. Each digest was mixed with 2.0 mL of 50 mg/mL DTT solution and the heat at 90 C. for 15 minutes. The precipitate was observed. Vortexed the samples vigorously for 230s. Each sample was centrifuged at 13200 rpm for 3 minutes; 120 mL of the supernatant was transferred into HPLC vial. The samples in the HPLC vials were then mixed with 5.0 L of 20% TFA in H.sub.2O for LC/MS analysis.

LC/MS/MS Method

[0154] The prepared tryptic peptides were analyzed using UPLC-MS/MS. Samples were directly injected onto a Waters Acquity UPLC CSH C18 (Milford, MA, U.S.A.) (2.150 mm, 1.7 m particle size) at a volume of 50 L. The column was heated to 60 C. during analysis. Separation was performed on a Waters Acquity UPLC system with mobile phase A consisting of 0.1% formic acid in water and mobile phase B consisting of 0.1% formic acid in acetonitrile with equilibrating at 0% mobile phase B for 2 min at 200 L/min, linearly increasing from 0% to 10% over 23 min, to 20% B over 57 min, to 30% over 30 min at a flow rate of 50 L/min, followed with multiple zigzag wash cycles at a flow rate of 400 L/min. Mass spectrometric analysis was performed on a Thermo Scientific Q Exactive Plus mass spectrometer (Bremen, Germany). Data-dependent MS/MS was performed as follows: the first event was the survey positive mass scan (m/z range of 230-1500) followed by 10 HCD events (28% NCE) on the 10 most abundant ions from the first event. Ions were generated using a sheath gas flow rate of 15, an auxiliary gas flow rate of 5, a spray voltage of 4 kV, a capillary temperature of 320 C., and an S-Lens RF level of 50. Resolution was set at 35 000 (AGC target of 5E6) and 17 500 (AGC target of 5E4) for survey scans and MS/MS events, respectively. The maximum ion injection time was 250 ms for survey scan, 300 ms for the other scans. The dynamic exclusion duration of 60s was used with a single repeat count.

HCP Identification and Quantification

[0155] A customized protein database composed of sequences obtained from the CHO-K1_refseq_2014 Protein.fasta database (downloaded 08/23/2014 from http://www.chogenome.org) was developed to predict the identities of HCPs from the MS/MS data. The MS/MS data was searched with a mass tolerance of 10 ppm and 0.02 Da, and a strict false discovery rate (FDR)<1% against this database using the Proteome Discoverer software package, version 1.4 or 2.3 (Thermo Scientific, Bremen, Germany) with Sequest HT searching. Further peptide/protein filtering was performed by eliminating proteins that had scored 0 and single spectrum hit, or single spectrum hit and 10 ppm and contaminated human proteins. Protein area from the top 3 peptides (if possible) for each HCP and the areas for the three spiked proteins, r-trypsin, PCSK9, and ADH1 were used to calculate individual HCP concentration (ppm or ng HCP/mg mAb).

TABLE-US-00034 TABLE 6a In process LC-MS HCP content for Batch 1 of mAb5 HCP content HCP content after after Low Protein pH viral HCP content HCP content EpiMatrix Capture inactivation after AEX after CEX HCP ID Score (ppm) (ppm) (ppm) (ppm) Total N/A 101023 1685 943 42.2 protein S100- 52.84 6.6 5.9 3.9 Below limit A6 of quantitation protein S100- 48.79 8.8 1.8 Below limit Below limit A11 of of quantitation quantitation phospholipase 32.89 547 24.4 14.3 12.2 B-like 2 protein lysosomal 29.45 227.7 102.7 26 Below limit protective of protein quantitation ubiquitin-40S 1.9 9.9 9.2 13.4 Below limit ribosomal of protein S27a quantitation Kallikrein-11 12.83 Below limit Below limit Below limit Below limit of of of of quantitation quantitation quantitation quantitation serine 13 1950.6 260.3 145.6 Below limit protease of HTRA1 quantitation isoform X1 thioredoxin 15.94 14.5 4.8 2.1 1.5 isoform X1 complement 23.01 644.9 28.5 23.6 16.8 C1r subcomponent actin, aortic 34.63 Below limit Below limit Below limit Below limit smooth of of of of muscle quantitation quantitation quantitation quantitation isoform X1 galectin-1 45.49 36.4 2.7 Below limit Below limit of of quantitation quantitation heat shock 47.2 579.3 14.8 32.4 Below limit cognate 71 of kDa protein quantitation peroxiredoxin-1 50.43 465.4 127.6 108.3 22.6 cornifin alpha 109.26 68.7 11.1 12.6 Below limit of quantitation

TABLE-US-00035 TABLE 6b In process LC-MS HCP content for Batch 2 of mAb5 HCP content HCP content after after Low Protein pH viral HCP content HCP content EpiMatrix Capture inactivation after AEX after CEX HCP ID Score (ppm) (ppm) (ppm) (ppm) Total N/A 104333 1384 933 70 protein S100- 52.84 63.4 5.7 5.8 Below limit A6 of quantitation protein S100- 48.79 14.3 1.8 Below limit Below limit A11 of of quantitation quantitation phospholipase 32.89 507.6 19.8 12.2 Below limit B-like 2 of protein quantitation lysosomal 29.45 229.6 75.9 19.9 12 protective protein ubiquitin-40S 1.9 8.3 Below limit Below limit Below limit ribosomal of of of protein S27a quantitation quantitation quantitation Kallikrein-11 12.83 Below limit Below limit Below limit Below limit of of of of quantitation quantitation quantitation quantitation serine 13 1850.2 150.9 88.2 6.9 protease HTRA1 isoform X1 thioredoxin 15.94 14.6 4.2 4.2 6.5 isoform X1 complement 23.01 542.8 24.1 23.1 15.4 C1r subcomponent actin, aortic 34.63 Below limit Below limit Below limit Below limit smooth muscle of of of of isoform X1 quantitation quantitation quantitation quantitation galectin-1 45.49 42.7 1.7 Below limit Below limit of of quantitation quantitation heat shock 47.2 590.3 17 49.4 Below limit cognate 71 of kDa protein quantitation peroxiredoxin-1 50.43 499.6 101.2 108.3 27 cornifin alpha 109.26 75 12.1 11.2 1.1

TABLE-US-00036 TABLE 7 Drug Substance LC-MS HCP content for Batches 3, 4 and 5 of mAb5 Batch 3 Batch 4 Batch 5 Drug Drug Drug Substance Substance Substance EpiMatrix HCP content HCP content HCP content HCP ID Score (ppm) (ppm) (ppm) Total N/A 39.7 52.2 51.7 protein S100- 52.84 0.4 0.4 0.4 A6 protein S100- 48.79 0.3 0.4 0.6 A11 phospholipase 32.89 4.3 6.2 3.5 B-like 2 protein lysosomal 29.45 7.1 6.8 6.1 protective protein ubiquitin-40S 1.9 1.1 0.6 1.3 ribosomal protein S27a Kallikrein-11 12.83 1.0 0.0 0.0 serine 13 1.8 1.6 1.7 protease HTRA1 isoform X1 thioredoxin 15.94 0.5 0.6 0.7 isoform X1 complement 23.01 5.2 4.3 5.6 C1r subcomponent actin, aortic 34.63 3.2 5.1 5.0 smooth muscle isoform X1 galectin-1 45.49 0.4 5.5 3.2 heat shock 47.2 3.2 2.7 3.8 cognate 71 kDa protein peroxiredoxin-1 50.43 7.9 9.9 9.1 cornifin alpha 109.26 0.0 0.0 0.2

Example 6. HCP Reduction in mAb7 (Antibody 201c in U.S. Pat. No. 10,647,759) Purification Processes

[0156] A mAb7 (Antibody 201c in U.S. Pat. No. 10,647,759)(LC is SEQ ID NO: 25; HC is SEQ ID NO: 26) preparation is prepared using the steps as essentially described above in respect of mAb5 with the following minor differences:

Protein Capture:

[0157] Protein A column: MabSelect SuRe [0158] Load: 20-40 g/L [0159] Elution: 20 mM Acetic Acid/5 mM Citric Acid

Low pH Viral Inactivation and Neutralization:

[0160] Titrant: 20 mM Acetic Acid/5 mM Citric Acid, pH 3.45 [0161] Time: 180 min [0162] Neutralization: pH 5.0, 500 mM Tris Base

Aex Chromatography:

[0163] Resin: POROS 50 XQ; [0164] Load: 100-200 g/L load [0165] pH: 7.0

Cex Chromatography:

[0166] Resin: POROS 50 HS [0167] Load: 20-40 g/L

[0168] HCP content was measured by LC-MS as described in Example 5. For mAb7 Batch 1 and mAb7 Batch 2, HCP content was measured after the Protein Capture Step, after low pH viral inactivation, after AEX, after CEX and after TFF. The results are shown in Tables 8a and 8b

TABLE-US-00037 TABLE 8a In process LC-MS HCP content for Batch 1 of mAb7 HCP HCP content content HCP HCP HCP after after Low content content content Protein pH viral after after after EpiMatrix Capture inactivation AEX CEX TFF HCP ID Score (ppm) (ppm) (ppm) (ppm) (ppm) Total N/A 14581 66.4 63.7 4 1.2 polyubiquitin 40.81 29.2 39 49 Below Below limit of limit of quantitation quantitation lysosomal 29.45 12.5 Below Below Below Below protective limit of limit of limit of limit of protein quantitation quantitation quantitation quantitation glutathione 24.04 Below Below Below Below Below S- limit of limit of limit of limit of limit of transferase quantitation quantitation quantitation quantitation quantitation Y1 40S 9.16 1 Below Below Below Below ribosomal limit of limit of limit of limit of protein S28 quantitation quantitation quantitation quantitation thioredoxin 15.94 4 Below Below 1 1 isoform X1 limit of limit of quantitation quantitation basement 29.68 2241 11 Below Below Below membrane- limit of limit of limit of specific quantitation quantitation quantitation heparan sulfate proteoglycan core protein isoform X1 tubulointerstitial 35.46 226 5 Below Below Below nephritis limit of limit of limit of antigen-like quantitation quantitation quantitation protein actin - 38.94 Below Below 2 Below Below partial limit of limit of limit of limit of cytoplasmic quantitation quantitation quantitation quantitation 2 isoform X2 galectin-1 45.49 32.6 1 Below Below Below limit of limit of limit of quantitation quantitation quantitation peroxiredoxin- 50.43 183.2 7 10 3 Below 1 limit of quantitation cornifin 109.26 46.8 4 2 Below Below alpha limit of limit of quantitation quantitation

TABLE-US-00038 TABLE 8b In process LC-MS HCP content for Batch 2 of mAb7 HCP HCP content content HCP HCP HCP after after Low content content content Protein pH viral after after after EpiMatrix Capture inactivation AEX CEX TFF HCP ID Score (ppm) (ppm) (ppm) (ppm) (ppm) Total N/A 8761 70.8 106.5 7.7 0 polyubiquitin 40.81 17 48 76 7 1 lysosomal 29.45 23 10 7 Below Below protective limit of limit of protein quantitation quantitation glutathione 24.04 Below Below 1 Below Below S-transferase limit of limit of limit of limit of Y1 quantitation quantitation quantitation quantitation 40S 9.16 1 1 Below Below Below ribosomal limit of limit of limit of protein S28 quantitation quantitation quantitation thioredoxin 15.94 3 Below 3 1 Below isoform X1 limit of limit of quantitation quantitation basement 29.68 951 Below Below Below Below membrane- limit of limit of limit of limit of specific quantitation quantitation quantitation quantitation heparan sulfate proteoglycan core protein isoform X1 tubulointerstitial 35.46 148 Below Below Below Below nephritis limit of limit of limit of limit of antigen-like quantitation quantitation quantitation quantitation protein actin - partial 38.94 398 Below Below Below Below cytoplasmic 2 limit of limit of limit of limit of isoform X2 quantitation quantitation quantitation quantitation galectin-1 45.49 14 Below Below Below Below limit of limit of limit of limit of quantitation quantitation quantitation quantitation peroxiredoxin- 50.43 86 9 13 Below Below 1 limit of limit of quantitation quantitation cornifin alpha 109.26 50 3 7 Below Below limit of limit of quantitation quantitation

Example 7. Impact of Depth Filter Type and Wi on HCP Reduction During Depth FiltrationmAb5 (Donanemab) and mAb6

Part AImpact of pH on HCP Reduction

[0169] Two antibodies (mAb5 and mAb6) are prepared using the protein capture step essentially as described for mAb1 in Example 1, except the elution step is performed with the buffer systems shown in Table 9. The total HCP content is measured by ELISA giving a range of about 2800 to about 3200 ppm. Following protein capture, the low pH viral inactivation step is performed essentially as described for mAb1 in Example 1, followed by a neutralization step at either pH 5.0 or pH 7.0 using 500 mM Tris base pH unadjusted buffer. The depth filtration step is performed essentially as described for mAb1 in Example 1 using a X0SP depth filter at a loading of 1000 g/m.sup.2. The HCP content after the depth filtration step is measured by ELISA.

[0170] The results in Table 9 show significant reduction in total HCP content to less than 50 ppm for both antibodies following depth filtration when the pH of the mixture applied to the depth filter is pH 7.0. Total HCP content is reduced to a lesser extent when the pH of the mixture applied to the depth filter is pH 5.0.

TABLE-US-00039 TABLE 9 HCP levels in mAb5 (donanemab) and mAb6 preparations following Protein A elution and depth filtration pH of material HCP content applied to after depth Antibody Protein A elution buffer depth filter filtration mAb5 20 mM acetic acid + 5 mM pH 5 231 (donanemab) lactic acid pH 7 45 20 mM acetic acid + 5 mM pH 5 229 phosphoric acid pH 7 13 mAb6 20 mM acetic acid + 5 mM pH 5 338 lactic acid pH 7 41 20 mM acetic acid + 5 mM pH 5 331 phosphoric acid pH 7 9
Part B: Impact of Depth Filter and pH on HCP Reduction for mAb5

[0171] mAb5 is prepared using the protein capture step essentially as described in Example 5. The eluate is subjected to low pH viral inactivation and neutralization as essentially described in Example 5. For the depth filtration step, four different pH and depth filter set-ups were evaluated: [0172] (i) B1HC filter+pH 5.1 [0173] (ii) X0SP filter+pH 5.1 [0174] (iii) X0SP filter+pH 6.2 [0175] (iv) X0SP filter+pH 7.3
(i) B1HC filter+pH 5.1

[0176] mAb5 is prepared using the protein capture step essentially as described in Example 5. 500 mls is placed into glass beaker and mixed with a teflon stir bar. The protein concentration of the Protein A eluate is 12.5 mg/ml. With 500 mls in the beaker, the total protein content is 6250 mg (12.5 mg/ml500 ml=6250 mg).

[0177] The starting pH of the solution in the beaker is 3.98 (temperature=18.1 C). The pH is adjusted to 3.45 with 20 mM acetic acid/5 mM citric acid to perform the low pH viral inactivation step as essentially described in Example 5.

[0178] While the low pH viral inactivation step is ongoing, a B1HC filter (micro pod or 23 sq cm, Lot CP7NA77798, part MB1HC23CL3) is set up. Size 14 platinum cured silicon tubing with PendoTech Filter Screening Peristaltic pumping system (K434694) with OHAUS Scout scales, K434696 to K434699) is used. All filters are flushed with PWTR at 23 ml/min (about 600 LMH) for 230 mls per filter or 100 L/sqm.

[0179] Neutralization to pH 5.0 is achieved with 0.25 M Tris base (EL19562-368, LB213, EXP 4/15/2020). The Solution turns cloudy as pH reaches 5 and the final pH is measured as 5.09 (5.1). The concentration is calculated to be 7.27 mg/ml (6250 mg/860 mls at pH 5). While stirring the pH 5 solution, filtration is begun through the B1HC filter with a load of 997 g/sqm (309 ml7.27 mg/ml=2.246 g/0.0023 sqm=997 g/sqm. The B1HC filter is recovery flushed with 45 mls of PWTR. Filters are essentially pumped dry after recovery flush. The final volume of B1HC is 375.5 ml at 5.13 mg/ml providing a 85.8% yield of 1.926 g.

(ii) X0SP Filter+pH 5.1 or pH 6.3 or pH 7.2

[0180] mAb5 is prepared using the protein capture step essentially as described in Example 5. 500 mls is placed into glass beaker and mixed with a Teflon stir bar. The protein concentration of the Protein A eluate is 15.75 mg/ml. With 500 mls in the beaker, the total protein content is 7875 mg (15.75 mg/ml500 ml=7875 mg).

[0181] The starting pH of the solution in the beaker is 4.05 (temperature=18.1 C). The pH is adjusted to 3.45 with 20 mM acetic acid/5 mM citric acid to perform the low pH viral inactivation step as essentially described in Example 5.

[0182] While the low pH viral inactivation step is ongoing, a three X0SP filters (micro pod or 23 sq cm, Lot CP9AA93251, cat MX0SP23CL3) are setup and flushed separately as described above.

[0183] Neutralization I achieved with use 0.25 M Tris base (EL19562-368, LB213, EXP 4/15/2020):

[0184] A first beaker was pH adjusted to 5.1 with 20 mls of 250 mM Tris base. The calculated concentration is 9.04 mg/ml.

[0185] The second beaker was pH adjusted to 6.3 with 27 mls of 250 mM Tris base. The calculated concentration is 8.82 mg/ml.

[0186] The third beaker was pH adjusted to 7.2 with 32 mls of 250 mM Tris base. The calculated concentration is 8.67 mg/ml.

[0187] The precipitate for the pH 6.3 and 7.2 seemed slimy (as it would stick to bottom of glass towards end of filtration), and possibly larger in size than pH 5.1.

[0188] Filtration through the X0SP filters is begun while stirring the three solutions.

[0189] The pH 5.0 X0SP reached 25 psi at a load of 203 mls and then switching to water recovery flush. The load is calculated as 798 g/sqm (9.04 mg/ml203 ml=1.835 g/0.0023 sq m=798 g/sq m).

[0190] Filters are recovery flushed with 45 mls of PWTR. Filters are essentially pumped dry after recovery flush.

[0191] Final Volume of X0SP pH 5.1=278 ml at 5.89 mg/ml=1.637 g Yield=1.637 g/1.835=89.2%

[0192] Final Volume of X0SP pH 6.3=365 ml at 5.76 mg/ml=1. g Yield=2.102 g/2.58=81.5%

[0193] Final Volume of X0SP pH 7.2=365 ml at 5.52 mg/ml=2.015 g/2.58=78.1%

(iii) AEX Chromatography

[0194] Each of the depth filtration preparations are subjected to AEX essentially as described in Example 5. For all AEX charge preparations, the filtrate at pH 5 and the filtrate at pH 6 (not the filtrate at 7.2) were pH adjusted to 7.25 with 250 mM Tris base (lot EL19562-368, LB213, exp 4-15-20, for development use) and then add NaCl to a final concentration of 50 mM using 20 mM Tris, 1 M NaCl, pH 7.0 (EL19562-862 LB198, exp 9-30-2020) at 0.0526volume at pH 7.25. All charge preparations are performed in glass beaker with stir bar. 600 mg of each filtrate was used in order to load the AEX with the same amount. All AEX charge pHs were between 7.1 and 7.3, and all the conductivities were 6.5+/0.2 mS.

[0195] Final AEX MS (at pH 5) volumes, mAb5 concentration, total mg, and yield were: [0196] 1. B1HC material155 ml at 3.91 mg/ml=606.1 mg or 101% [0197] 2. X0SP at pH 5.1-120 ml at 5.00 mg/ml=600 mg or 100% [0198] 3. X0SP at pH 6.3-121 ml at 4.96 mg/ml=600.2 mg or 100% [0199] 4. X0SP at pH 7.2-126 ml at 4.79 mg/ml=603.5 mg or 100.6%
(iii) CEX Chromatography

[0200] Each of the AEX preparations are subjected to CEX chromatography essentially as described in Example 5. The actual loads on the CEX resin are as follows: [0201] (i) B1HC preparation at 3.91 mg/ml volume loaded 130 ml0.85=110.5 ml=432.1/17.28=25.0 mg/ml [0202] (ii) X0SP at pH 5 at 5.00 mg/ml volume loaded 101.7 ml0.85%=86.4 ml=432/17.28=25.0 mg/ml [0203] (iii) X0SP at pH 6.3 at 4.96 mg/ml volume loaded=102.50.85=87.1 ml=432.0/17.28 ml=25.0 mg/ml [0204] (iv) X0SP at pH 7.2 at 4.79 mg/ml volume loaded=106.10.85=90.2 ml=432.1/17.28=25.0 mg/ml

[0205] The CEX mainstream volumes, concentration and yields for each condition are as follows: [0206] (i) B1HC at pH 5.0 at 5.83 mg/ml MS volume=64.1 ml MS Volume=373 mg/432.1 mg=86.3% [0207] (ii) X0SP at pH 5 at 5.83 mg/ml64.8 ml MS Volume=377.8 mg/432 mg=87.5% [0208] (iii) X0SP at pH 6.3 at 5.80 mg/ml=64.8 ml MS Volume=375.8 mg/432.0 mg=87.0% [0209] (iv) X0SP at pH 7.2 at 5.80 mg/ml=64.7 ml MS Volume=375.3 mg/432.1 mg=86.9%

(v) Analysis of HCP Content by LC-MS

[0210] The CEX preparations are analyzed for HCP content using LCM essentially as described in Example 5. The LC-MS data is provided in Table 10.

TABLE-US-00040 TABLE 10 Content of Host Cell Proteins in mAb5 (donanemab) Preparation After Protein Capture, Low pH Viral Inactivation Step, Neutralization Step, and Depth Filtration Neutralization pH ~5.0 ~5.0 ~6.0 ~7.0 HCP ID EpiMatrix B1HC X0SP X0SP X0SP Total N/A 85.4 ppm 48.8 ppm 42.1 ppm 48.4 ppm protein S100-A6 52.84 0.3 ppm 0.2 ppm 0.1 ppm 0.1 ppm protein S100-A11 48.79 0.4 ppm 0.3 ppm 0.2 ppm 0.2 ppm phospholipase B- 32.89 1.5 ppm 0.8 ppm 0.6 ppm 0.7 ppm like 2 protein lysosomal 29.45 6.2 ppm 0.4 ppm 0.01 ppm 0.01 ppm protective protein ubiquitin-40S 1.9 5.1 ppm 5.7 ppm 5.1 ppm 5.1 ppm ribosomal protein S27a Kallikrein-11 12.83 2.7 ppm 0 ppm 0 ppm serine protease 13 1.9 ppm 0.1 ppm ND or 0.0 ppm HTRA1 isoform below limit X1 of quantitation thioredoxin 15.94 2.8 ppm 2.7 ppm 2.4 ppm 2.3 ppm isoform X1 complement C1r 23.01 8.7 ppm 11.5 ppm 11.6 ppm 16.3 ppm subcomponent actin, aortic 34.63 4.1 ppm 2.3 ppm 2.1 ppm 2.1 ppm smooth muscle isoform X1 galectin-1 45.49 0.4 ppm 0.4 ppm 0.3 ppm 0.5 ppm heat shock 47.2 4.6 ppm 2.6 ppm 2.9 ppm 2.4 ppm cognate 71 kDa protein peroxiredoxin-1 50.43 21.7 ppm 8.3 ppm 4.3 ppm 4.2 ppm cornifin alpha 109.26 0.2 ppm 0.2 ppm ND or 0.1 ppm below limit of quantitation
The data in Table 10 show significant reduction in total HCP content to less than 50 ppm following depth filtration with the X0SP filter at all pHs tested. This compares favorably to the reduction in HCP content following depth filtration with the B1HC filter. It is also notable that the yield after the depth filtration step is lower at pH 6.3 and 7.2 in comparison to the lower pH 5.1. Therefore, the reduction in HCP content at high pH may be offset by the loss of yield. The optimal performance is seen with the X0SP filter at pH 5.0.

Example 8. Method for Determination of Ionic Strength During Biomolecule Purification Processes

[0211] A method for the estimation of ionic strength based on what is known of the buffer compositions during biomolecule purification unit processes is herein described. The ionic strength (I) of a solution is a measure of concentration of ions in that solution, and is a function of species concentration, c.sub.i, and net charge, z.sub.i, for all species. To determine ionic strength, Formula I is used.

[00002]$\begin{array}{cc}I=\frac{1}{2}{.Math.}_i{c}_i{z}_i^2& \left(1\right)\end{array}$

Strong electrolytes: for strong electrolytes at low concentrations (e.g., below 50 mM), complete dissociation is assumed. With complete dissociation, the composition is easily calculated making ionic strength calculations straightforward. For example, a solution of 50 mM NaCl dissociates to give 50 mM each of Na.sup.+ and Cl.sup. with an ionic strength of 0.5[50 mM I.sup.2+50 mM (1).sup.2]=50 mM. As another example, 50 mM Na.sub.2SO.sub.4 dissociates to give 100 mM of Na.sup.+ and 50 mM of SO.sub.4.sup.2, giving an ionic strength of 0.5[100 mM I.sup.2+50 mM (2).sup.2]=150 mM. With no buffering species, near-neutral pH is expected in these calculations such that concentrations of ions from the dissociation of water do not contribute meaningfully to the ionic strength. The dissociation constant of water is taken to be K.sub.w=[H.sup.+][OH.sup.]=10.sup.14 with [H.sup.+]=10.sup.pH where the square brackets indicate concentrations. For the purpose of calculations herein, physical interpretation of H.sup.+ ions (as opposed to hydronium ions, for example) is not necessary, and likewise it is not necessary to distinguish between H.sup.+ concentration and activity.
Buffered systems: for buffered systems complete dissociation cannot be assumed. Acid dissociation constants of the buffers must be used to determine the proportion of the buffer in the acid and base forms. For a generic acid, HA, that dissociates into H.sup.+ and A.sup. Formula 2 relates to the acid dissociation constant, K.sub.a, and the species concentrations:

[00003]$\begin{array}{cc}{K}_a=\frac{\left[H^{+}\right]\left[A^{-}\right]}{\left[HA\right]}& \left(2\right)\end{array}$

[0212] The acid dissociation constant is often used in the logarithmic form of pK.sub.a=log.sub.10(K.sub.a). The thermodynamic pK.sub.a, denoted as pK.sub.a,0, is available in the literature for many buffers of interest. However, the effective pK.sub.a of a buffer diverges from the thermodynamic value except in very dilute solution due to deviation of activity coefficients from unity. For moderately dilute solutions considered in this disclosure, the extended Debye Hckel equation or Davies equation were used to account for non-unity activity coefficients. Values for some of the constants found in literature may differ slightly but give similar results in the range of ionic strength values of interest in the present disclosure. The extended Debye Hckel equation is provided as Formula 3:

[00004]$\begin{array}{cc}p{K}_a=p{K}_{a,0}+\frac{0.51n\sqrt{I}}{1+1.6\sqrt{I}}& \left(3\right)\end{array}$

The Davies equation is provided as Formula 4:

[00005]$\begin{array}{cc}p{K}_a=p{K}_{a,0}+0\text{.51}n\left(\frac{\sqrt{I}}{1+\sqrt{I}}-0.3\sqrt{I}\right)& \left(4\right)\end{array}$

where n=2z1 and z is the net charge of the acidic buffer form for calculating n (Scopes, Protein Purification: Principles and Practices, 2013).

[0213] Since pK.sub.a is a function of ionic strength, the composition and ionic strength cannot be determined independently, but are part of a system of equations. The system of equations includes the aforementioned equations for ionic strength, acid dissociation constants for each buffer, and pK.sub.a equations for each buffer, and also includes an electroneutrality condition and a total species balance for each buffer. With this system of equations, several values may be estimated. For example, a known solution pH can be used to estimate an acid-based ratio for a buffer formulation, or conversely an acid-based ratio can be used to estimate a solution pH and corresponding titration volumes. In any of these applications, the ionic strength can be estimated, to help guide rational selection of eluent and titrant options.

[0214] To calculate the ionic strength relevant to the buffered systems in the present disclosure, such as that of the feed material for depth filtration, the buffer composition of the solution is needed. This composition can be reasonably estimated based on the volumes and compositions of the buffers and titrants used in the process. Ion measurement techniques known in the field may also be used to estimate the composition.

[0215] As a starting point for estimating the solution composition, one possible methodology is to assume that the affinity column eluate pool has a buffer composition identical to that of the eluent with the exception of being buffered at the measured pH of the eluate pool. For example, if the protein of interest is eluted from a Protein A column with 20 mM acetic acid, 5 mM lactic acid and the eluate pool has a measured pH of 4.2, the assumption would be made that the buffer composition of the eluate pool is 20 mM acetate, 5 mM lactate, and sufficient NaOH to bring pH to 4.2; this would equate to about 8.2 mM NaOH. Because only the total sodium cation, Na.sup.+, content is important to the calculation, it does not matter whether the eluate sodium content is assumed to originate from sodium acetate, sodium phosphate, sodium hydroxide, or any combination thereof, so the convention of attributing the sodium to NaOH is used for convenience.

[0216] Having used the eluent composition and eluate pH to estimate the buffer composition of the eluate, the solution titrations are then considered. For example, with an estimated eluate composition of 20 mM acetate, 5 mM lactate, 8.2 mM NaOH at pH 4.2, if the volume of 20 mM HCl required to lower the pH to a target value of 3.45 for viral inactivation was equal to 0.305 times the start volume, then the composition of that process intermediate at pH 3.45 would be known from the dilution. Acetate, lactate, and NaOH would be present at 1/1.305 times their respective initial values (i.e., 15.3 mM acetate, 3.8 mM lactate, and 6.2 mM NaOH) and HCl present at 0.305/1.305 of its value in the titrant (4.7 mM HCl). Similarly, for neutralization with 250 mM Tris base, if the ratio to raise the pH to a target of pH 7.0 was 0.0743 times the volume of pH 3.45 solution, ratios of 1/1.0743 and 0.0743/1.0743 would be applied to find the final concentrations in the neutralized solution (14.3 mM acetate, 3.6 mM lactate, 5.8 mM NaOH, 4.4 mM HCl, and 17.3 mM Tris). All known values are plugged into the system of equations (Formulas 5 thru 15) to calculate the ionic strength:

[00006]$\begin{array}{cc}I=\frac{1}{2}\left(\left[H^{+}\right].Math.\left\{1^2\right\}+\left[{\mathrm{Na}}^{+}\right].Math.\left\{1^2\right\}+\left[{\mathrm{TrisH}}^{+}\right].Math.\left\{1^2\right\}+\left[{\mathrm{OH}}^{-}\right].Math.{\left\{-1\right\}}^2+\left[{\mathrm{Acetate}}^{-}\right].Math.{\left\{-1\right\}}^2+\left[{\mathrm{Lactate}}^{-}\right].Math.{\left\{-1\right\}}^2+\left[{\mathrm{Cl}}^{-}\right].Math.{\left\{-1\right\}}^2\right)& \left(5\right)\end{array}$$\begin{array}{cc}\left[H^{+}\right]+\left[{\mathrm{Na}}^{+}\right]+\left[{\mathrm{TrisH}}^{+}\right]=\left[{\mathrm{OH}}^{-}\right]+\left[{\mathrm{Acetate}}^{-}\right]+\left[{\mathrm{Acetate}}^{-}\right]+\left[{\mathrm{Cl}}^{-}\right]& \left(6\right)\end{array}$$\begin{array}{cc}{K}_{a,\mathrm{Tris}}=\frac{\left[H^{+}\right]\left[\mathrm{Tris}\right]}{\left[{\mathrm{TrisH}}^{+}\right]}& \left(7\right)\end{array}$$\begin{array}{cc}{\mathrm{pK}}_{a,\mathrm{Tris}}={\mathrm{pK}}_{a,0,\mathrm{Tris}}+0.51\left(2.Math.\left\{+1\right\}-1\right)\left(\frac{\sqrt{I}}{1+\sqrt{I}}-0.3\sqrt{I}\right)& \left(8\right)\end{array}$$\begin{array}{cc}{K}_{a,\mathrm{Acetate}}=\frac{\left[H^{+}\right]\left[{\mathrm{Acetate}}^{-}\right]}{\left[H\mathrm{Acetate}\right]}& \left(9\right)\end{array}$$\begin{array}{cc}{\mathrm{pK}}_{a,\mathrm{Acetate}}={\mathrm{pK}}_{a,0,\mathrm{Acetate}}+0.51\left(2.Math.\left\{-1\right\}-1\right)\left(\frac{\sqrt{I}}{1+\sqrt{I}}-0.3\sqrt{I}\right)& \left(10\right)\end{array}$$\begin{array}{cc}{K}_{a,\mathrm{Lactate}}=\frac{\left[H^{+}\right]\left[{\mathrm{Lactate}}^{-}\right]}{\left[H\mathrm{Lactate}\right]}& \left(11\right)\end{array}$$\begin{array}{cc}{\mathrm{pK}}_{a,\mathrm{Lactate}}={\mathrm{pK}}_{a,0,\mathrm{Lactate}}+0.51\left(2.Math.\left\{-1\right\}-1\right)\left(\frac{\sqrt{I}}{1+\sqrt{I}}-0.3\sqrt{I}\right)& \left(12\right)\end{array}$$\begin{array}{cc}\mathrm{Total}\mathrm{Tris}=\left[\mathrm{Tris}\right]+\left[{\mathrm{TrisH}}^{+}\right]& \left(13\right)\end{array}$$\begin{array}{cc}\mathrm{Total}\mathrm{Acetate}=\left[H\mathrm{Acetate}\right]+\left[{\mathrm{Acetate}}^{-}\right]& \left(14\right)\end{array}$$\begin{array}{cc}\mathrm{Total}\mathrm{Lactate}=\left[\mathrm{HLactate}\right]+\left[{\mathrm{Lactate}}^{-}\right]& \left(15\right)\end{array}$

where respective pK.sub.a,0 value for Tris, acetate, and lactate were taken to be 8.15, 4.76, and 3.86 at 22 C. The resulting estimate for the ionic strength of the depth filtration feed 20 material is 22.1 mM.

[0217] As described herein, buffering capacity of a protein product is not directly modeled. Thus, when using a strong acid or base for titration, some deviations can arise between calculations and empirical titration results. For example, when titrating a Protein A eluate to low pH for viral inactivation, the buffer calculations typically underestimate 25 the empirical amount of 20 mM HCl needed; the empirical amount needed may be on the order of 50% greater than the calculated estimate. One way to account for this difference is to model the affinity column eluate material at a higher pH, empirically adjusting the value until the estimated titration volume matches the experimental value. For example, in the above example, if the amount of 20 mM HCl was 50% higher than the 0.305 ratio than initially estimated, the Protein A eluate would be modeled as being about pH 4.45 instead of pH 4.2. Making this empirical change to the modeling, the estimated ionic strength in the example is directionally reduced, but only by a small amount: 21.9 mM down from the initial 22.1 mM estimate. Accordingly, it is concluded that either approach is sufficient for estimating ionic strength to deduce preferred embodiments of the present disclosure.

Alternative methods: Ion content measurement methods can be used to determine the buffer composition of the depth filtration feed material to calculate the ionic strength. This requires confirming that the measurements give self-consistent results with any known amounts such as the amounts of titrant added. Since the buffer composition of the affinity column eluate is assumed to be equivalent to that of the eluent but at a different pH, the difference in true composition could be determined by ion content measurements. For example, either an amount based on the eluent composition, or a measured value may be used to calculate ionic strength of the buffer components in the eluent.

INCORPORATION BY REFERENCE

[0218] All patents and publications referenced herein are hereby incorporated by reference in their entireties. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention.

SEQUENCES

[0219] The following nucleic and/or amino acid sequences are referred to in the disclosure and are provided below for reference.

TABLE-US-00041 SEQIDNO:1-bamlanivimabvariableheavychain(VH) QVQLVQSGAEVKKPGSSVKVSCKASGGTFSNYAISWVRQAPGQGLEWMGRIIPIL GIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARGYYEARHYYYY YAMDVWGQGTAVTVSS SEQIDNO:2-bamlanivimabvariablelightchain(VL) DIQMTQSPSSLSASVGDRVTITCRASQSISSYLSWYQQKPGKAPKLLIYAASSLQS GVPSRFSGSGSGTDFTLTITSLQPEDFATYYCQQSYSTPRTFGQGTKVEIK SEQIDNO:3-bamlanivimabheavychain(HC) QVQLVQSGAEVKKPGSSVKVSCKASGGTFSNYAISWVRQAPGQGLEWMGRIIPIL GIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARGYYEARHYYYY YAMDVWGQGTAVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVT VSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK VDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK SEQIDNO:4-bamlanivimablightchain(LC) DIQMTQSPSSLSASVGDRVTITCRASQSISSYLSWYQQKPGKAPKLLIYAASSLQS GVPSRFSGSGSGTDFTLTITSLQPEDFATYYCQQSYSTPRTFGQGTKVEIKRTVAA PSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQIDNO:5-etesevimabvariableheavychain(VH) EVQLVESGGGLVQPGGSLRLSCAASGFTVSSNYMSWVRQAPGKGLEWVSVIYSG GSTFYADSVKGRFTISRDNSMNTLFLQMNSLRAEDTAVYYCARVLPMYGDYLD YWGQGTLVTVSS SEQIDNO:6-etesevimabvariablelightchain(VL) DIVMTQSPSSLSASVGDRVTITCRASQSISRYLNWYQQKPGKAPKLLIYAASSLQS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPEYTFGQGTKLEIKRTV SEQIDNO:7-etesevimabheavychain(HC) EVQLVESGGGLVQPGGSLRLSCAASGFTVSSNYMSWVRQAPGKGLEWVSVIYSG GSTFYADSVKGRFTISRDNSMNTLFLQMNSLRAEDTAVYYCARVLPMYGDYLD YWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRV EPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGK SEQIDNO:8-etesevimablightchain(LC) DIVMTQSPSSLSASVGDRVTITCRASQSISRYLNWYQQKPGKAPKLLIYAASSLQS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPEYTFGQGTKLEIKRTVA APSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQIDNO:9-bebtelovimabvariableheavychain(VH) QITLKESGPTLVKPTQTLTLTCTFSGFSLSISGVGVGWLRQPPGKALEWLALIYWD DDKRYSPSLKSRLTISKDTSKNQVVLKMTNIDPVDTATYYCAHHSISTIFDHWGQ GTLVTVSS SEQIDNO:10-bebtelovimabvariablelightchain(VL) QSALTQPASVSGSPGQSITISCTATSSDVGDYNYVSWYQQHPGKAPKLMIFEVSD RPSGISNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTTSSAVFGGGTKLTVL SEQIDNO:11-bebtelovimabheavychain(HC) QITLKESGPTLVKPTQTLTLTCTFSGFSLSISGVGVGWLRQPPGKALEWLALIYWD DDKRYSPSLKSRLTISKDTSKNQVVLKMTNIDPVDTATYYCAHHSISTIFDHWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK SEQIDNO:12-bebtelovimablightchain(LC) QSALTQPASVSGSPGQSITISCTATSSDVGDYNYVSWYQQHPGKAPKLMIFEVSD RPSGISNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTTSSAVFGGGTKLTVLGQ PKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTT PSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS SEQIDNO:13-LCVRofDonanemab DIVMTQTPLSLSVTPGQPASISCKSSQSLLYSRGKTYLNWLLQKPGQSPQLLIYAV SKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCVQGTHYPFTFGQGTKLEI K SEQIDNO:14-HCVRofDonanemab QVQLVQSGAEVKKPGSSVKVSCKASGYDFTRYYINWVRQAPGQGLEWMGWINP GSGNTKYNEKFKGRVTITADESTSTAYMELSSLRSEDTAVYYCAREGITVYWGQ GTTVTVSS SEQIDNO:15-LCofDonanemab DIVMTQTPLSLSVTPGQPASISCKSSQSLLYSRGKTYLNWLLQKPGQSPQLLIYAV SKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCVQGTHYPFTFGQGTKLEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQ ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQIDNO:16-HCofDonanemab QVQLVQSGAEVKKPGSSVKVSCKASGYDFTRYYINWVRQAPGQGLEWMGWINP GSGNTKYNEKFKGRVTITADESTSTAYMELSSLRSEDTAVYYCAREGITVYWGQ GTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKS CDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPG SEQIDNO:17-LCDR1ofDonanemab KSSQSLLYSRGKTYLN SEQIDNO:18-LCDR2ofDonanemab AVSKLDS SEQIDNO:19-LCDR3ofDonanemab VQGTHYPFT SEQIDNO:20-HCDR1ofDonanemab GYDFTRYYIN SEQIDNO:21-HCDR2ofDonanemab WINPGSGNTKYNEKFKG SEQIDNO:22-HCDR3ofDonanemab EGITVY SEQIDNO:23-LCVRofAntibody201c(mAb7) DIQMTQSPSTLSASVGDRVTITCRASQSLGNWLAWYQQKPGKAPKLLIYQASTLE SGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQHYKGSFWTFGQGTKVEIK SEQIDNO:24-HCVRofAntibody201c(mAb7) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYPMSWVRQAPGKGLEWVSAISGS GGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREGGSGSYYN GFDYWGQGTLVTVSS SEQIDNO:25-LCofAntibody201c(mAb7) DIQMTQSPSTLSASVGDRVTITCRASQSLGNWLAWYQQKPGKAPKLLIYQASTLE SGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQHYKGSFWTFGQGTKVEIKRTVA APSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQIDNO:26-HCofAntibody201c(mAb7) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYPMSWVRQAPGKGLEWVSAISGS GGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREGGSGSYYN GFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVD KKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPG SEQIDNO:27-LCDR1ofAntibody201c(mAb7) RASQSLGNWLA SEQIDNO:28-LCDR2ofAntibody201c(mAb7) YQASTLES SEQIDNO:29-LCDR3ofAntibody201c(mAb7) QHYKGSFWT SEQIDNO:30-HCDR1ofAntibody201c(mAb7) AASGFTFSSYPMS SEQIDNO:31-HCDR2ofAntibody201c(mAb7) AISGSGGSTYYADSVKG SEQIDNO:32-HCDR3ofAntibody201c(mAb7) AREGGSGSYYNGFDY SEQIDNO:33-LCDNAsequenceofDonanemab gatattgtgatgactcagactccactctccctgtccgtcacccctggacagccggcctccatctcctgcaagtcaagtcagagcct cttatatagtcgcggaaaaacctatttgaattggctcctgcagaagccaggccaatctccacagctcctaatttatgcggtgtctaaa ctggactctggggtcccagacagattcagcggcagtgggtcaggcacagatttcacactgaaaatcagcagggtggaggccga agatgttggggtttattactgcgtgcaaggtacacattacccattcacgtttggccaagggaccaagctggagatcaaacgaactg tggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttct atcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcagga cagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgc gaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgc SEQIDNO:34-HCDNASequenceofDonanemab caggtgcagctggtgcagtctggggctgaggtgaagaagcctgggtcctcagtgaaggtttcctgcaaggcatctggttacgac ttcactagatactatataaactgggtgcgacaggcccctggacaagggcttgagtggatgggatggattaatcctggaagcggta atactaagtacaatgagaaattcaagggcagagtcaccattaccgcggacgaatccacgagcacagcctacatggagctgagc agcctgagatctgaggacacggccgtgtattactgtgcgagagaaggcatcacggtctactggggccaagggaccacggtcac cgtctcctcagcctccaccaagggcccatcggtcttcccgctagcaccctcctccaagagcacctctgggggcacagcggccct gggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacacct tcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagaccta catctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagttgagcccaaatcttgtgacaaaactcacacatg cccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcc cggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgt ggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctg caccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctc caaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggacgagctgaccaagaaccaggtca gcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaacta caagaccacgccccccgtgctggactccgacggctccttcttcctctatagcaagctcaccgtggacaagagcaggtggcagc aggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggt SEQIDNO:35-LCDNASequenceofAntibody201c gacatccagatgacccagtctccttccaccctgtctgcatctgtaggagacagagtcaccatcacttgccgggccagtcagagtct tggtaactggttggcctggtatcagcagaaaccagggaaagcccctaaactcctgatctatcaggcgtctactttagaatctgggg tcccatcaagattcagcggcagtggatctgggacagagttcactctcaccatcagcagcctgcagcctgatgattttgcaacttatt actgccaacattataaaggttctttttggacgttcggccaagggaccaaggtggaaatcaaacggaccgtggctgcaccatctgtc ttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggcca aagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagca cctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatca gggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgc SEQIDNO:36-HCDNASequenceofAntibody201c gaggtgcagctgttggagtctgggggaggcttggtacagcctggggggtccctgagactctcctgtgcagcctctggattcacct ttagcagctatcctatgagctgggtccgccaggctccagggaaggggctggagtgggtctcagctattagtggtagtggtggtag cacatactacgcagactccgtgaagggccggttcaccatctccagagacaattccaagaacacgctgtatctgcaaatgaacag cctgagagccgaggacacggccgtatattactgtgcgagagaggggggctcagggagttattataacggctttgattattgggg ccagggaaccctggtcaccgtctcctcagcctccaccaagggcccatcggtcttcccgctagcaccctcctccaagagcacctc tgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctg accagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagc agcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggggacaagaaagttgagcccaaatc ttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaaccc aaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagtt caactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgt ggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccag cccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggacg agctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaat gggcagccggagaacaactacaagaccacgccccccgtgctggactccgacggctccttcttcctctatagcaagctcaccgtg gacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagag cctctccctgtctccgggt