# US 9,518,082 B2 · Antibody purification and purity monitoring

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- Number: US 9,518,082 B2
- Document ID: US9518082B2
- Status: Granted
- Kind: B2
- Application number: 14/215,370
- Filed: 2014-03-17
- Published: 2014-09-25
- Granted: 2016-12-13
- Earliest claimed: 2013-03-15
- Assignees: Alderbio Holdings LLC
- Applicants: Alderbio Holdings LLC
- Inventors: Daniel S. Allison; Steven D. Davin; Hoa Binh Do; Leon F. Garcia-Martinez; Geoffrey F. Lee; Ethan W. Ojala; Mark Young; John A. Latham
- Web page: https://patentgrep.com/patent/US9518082B2

## CPC

- C07K 16/18: against material from animals or humans
- B01D 15/327: {with hydrophobic interaction}
- B01D 15/3809: {of the antigen-antibody type, e.g. protein A, G or L chromatography}
- B01D 15/424: {Elution mode}
- C07K 1/165: {mixed-mode chromatography}
- C07K 1/20: Partition-, reverse-phase or hydrophobic interaction chromatography
- C07K 1/22: Affinity chromatography or related techniques based upon selective absorption processes
- C07K 1/34: by filtration, ultrafiltration or reverse osmosis
- C07K 1/36: by a combination of two or more processes of different types
- C07K 16/065: {Purification, fragmentation}
- G01N 33/68: involving proteins, peptides or amino acids {(involving lipoproteins G01N33/92)}
- G01N 33/6854: {Immunoglobulins}
- C07K 2317/14: Specific host cells or culture conditions, e.g. components, pH or temperature
- C07K 2317/41: Glycosylation, sialylation, or fucosylation
- C07K 2317/51: Complete heavy chain or Fd fragment, i.e. VH + CH1
- C07K 2317/515: Complete light chain, i.e. VL + CL
- C07K 2317/55: Fab or Fab'
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- B01D: SEPARATION (separating solids from solids by wet methods B03B, B03D; by pneumatic jigs or tables B03B; by other dry methods B07; magnetic or electrostatic separation of solid materials from solid materials or fluids, separation by high-voltage electric fields B03C; centrifuges, vortex apparatus B04; presses per se for squeezing-out liquid from liquid-containing material B30B9/02; treatment of water C02F, e.g. softening by ion-exchange C02F1/42; {arrangements of air intake cleaners in gas turbine plants F02C7/05} ; arrangements or mounting of filters in air-conditioning, air-humidification or ventilation F24F13/28)
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

Processes for producing and purifying recombinant proteins are disclosed. In particular, the present disclosure provides processes of producing and purifying multi-subunit proteins expressed in yeast or filamentous fungal cells. The production and/or purification of such proteins are monitored for impurities, preferably using lectin binding assays, such that one or more process parameters may be adjusted to maximize the amount of desired recombinant protein and minimize the amount of glycosylated impurities. The processes can also be monitored for other undesired product-associated impurities, such as aggregates and nucleic acids. In exemplary embodiments, the recombinant proteins are multi-subunit proteins, such as antibodies, the host cell is a yeast, such as Pichia pastoris, and the glycosylated impurity is a glycovariant of the desired recombinant polypeptide, such as an N-linked and/or O-linked glycovariant.

## Claims

### Claim 1

A fermentation process for producing a desired recombinant antibody polypeptide and purifying the desired recombinant polypeptide from the fermentation medium, wherein the process includes:
(i) conducting one or more fermentation processes or runs which each comprise culturing yeast cells under conditions that result in the expression and secretion of a desired recombinant polypeptide and one or more impurities into the fermentation medium;
(ii) periodically obtaining one or more samples of the fermentation medium as each fermentation process proceeds or after different fermentation runs are conducted;
(iii) detecting the amount and/or type of glycosylated impurities in the sample(s) using a lectin that binds to said glycosylated impurities, and
(iv) modifying one or more of the operating parameters or conditions of one or more of the fermentation processes or runs based on the amount of detected glycosylated impurities in the sample(s), wherein said operating parameters or conditions are selected from the group consisting of temperature, pH, gas constituent, feed constituent, agitation, aeration, antifoam and duration, while said fermentation processes or runs are being conducted, and
(v) pooling different samples, eluates or fractions thereof containing the desired recombinant antibody polypeptide from the same or different fermentation processes or runs as the fermentation processes or runs are being conducted based on the amount and/or type of detected glycosylated impurity relative to the amount of the recombinant antibody polypeptide in said different samples, eluates or fractions thereof.

### Claim 2 (depends on 1)

The process of claim 1, wherein
(i) the impurities are the result of O-linked glycosylation,
(ii) the detection step is effected using at least one lectin selected from ConA, LCH, GNA or GNL, RCA, DC-SIGN, L-SIGN, PNA, AIL, VVL, WGA, SNA, MAL, MAH, UEA and AAL, and/or at least one lectin selected from PNA, SBA, PWM, PEA, PTA, ML-I-III, LEA, UDA, WGA, PHA, LTA, BSI-B4, MPA, RCA, LCA, ECA, AAA, DBA, GSL-I, PSA, SJA, DSL, ECL, GSL-II, AIA/Jacalin, LEL, STL, HHL, LCA, NPL, ACL, ECL, EEL, MAL-I, AAL, LTL, BPL, MPL, PTL, SNA, DGL, SJA, VVA, LEA, STA, DSA, MMR, DEC-205, Dectin 1, Dectin 2, Langerin, or BDCA-2, which optionally may be bound to a support,
(iii) the detection step uses a protein-protein interaction monitoring process, wherein the protein-protein interaction monitoring process uses light interferometry, dual polarization interferometry, static light scattering, dynamic light scattering, multi-angle light scattering, surface plasmon resonance , ELISA, chemiluminescent ELISA, far western, or electroluminescence or any combination of (i)-(v).

### Claim 3 (depends on 1)

The process of claim 1, wherein said operating parameters or conditions are selected from at least two of said operating parameters or conditions.

### Claim 4 (depends on 1)

The process of claim 1, wherein the glycosylated impurity is a glycovariant of the recombinant polypeptide and the recombinant polypeptide is an antibody or antibody fragment.

### Claim 5 (depends on 1)

The process of claim 1, wherein the host cell is a Pichia pastoris, which expresses an antibody or antibody fragment.

### Claim 6 (depends on 1)

The process of claim 1, which further includes recovering or purifying the recombinant polypeptide from the fermentation medium.

### Claim 7 (depends on 6)

The process of claim 6, wherein the purification process further comprises:
(i) contacting the sample(s) with at least one chromatographic support and selectively eluting the desired recombinant polypeptide,
(ii) pooling different samples or eluates or fractions thereof containing a desired recombinant antibody based on the amount and/or type of detected glycosylated impurity,
(iii) pooling different samples or eluates or fractions thereof containing the desired recombinant antibody based on the amount and/or type of detected glycosylated impurity relative to the amount of recombinant antibody, or any combination of the foregoing (i)-(ii).

### Claim 8 (depends on 6)

The process of claim 6, further comprising detecting the amount of aggregated and/or disaggregated impurities in said sample(s) of step (iii) using size exclusion chromatography and modifying one or more of the operating parameters or conditions of one or more of the fermentation processes or run, wherein said operating parameters or conditions are selected from temperature, pH, gas constituent, feed constituent, agitation, aeration, antifoam and duration.

### Claim 9 (depends on 6)

The process of claim 6, wherein the recombinant polypeptide is an antibody or antibody fragment which is monovalent, bivalent or multivalent.

### Claim 10 (depends on 6)

The process of claim 6, wherein said antibody or antibody fragment specifically binds to IL-2, IL-4, IL-6, IL-10, IL-12, IL-13, IL-17, IL-18, IFN-alpha, IFN-gamma, BAFF, CXCL13, IP-10, CBP, angiotensin (angiotensin I and angiotensin II), Nav 1.7, Nav 1.8, VEGF, PDGF, EPO, EGF, FSH, TSH, hCG, CGRP, NGF, TNF, HGF, BMP2, BMP7, PCSK9 or HRG.

### Claim 11 (depends on 1)

The process of claim 1, wherein said glycosylated impurity in step (v) is selected from the group consisting of (i) samples or eluate or fractions thereof comprising less than 10% glycovariant, (ii) samples or eluate or fractions thereof comprising less than 5% glycovariant, (iii) samples or eluate or fractions thereof comprising less than 1% glycovariant and (iv) samples or eluate or fractions thereof comprising less than 0.5% glycovariant.