Antibody purification and purity monitoring
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.
1 . A collection of humanized antibody containing samples, (a) each comprising a protein A eluate comprising:
(1) a humanized antibody comprising a heavy chain having the amino acid sequence of SEQ ID NO: 54 and a light chain having the amino acid sequence of SEQ ID NO: 52;
(2) a glycovariant of said humanized antibody which glycovariant is comprised in the samples in an amount of 1% to less than 10%; and
(3) one or more antibody aggregates which are present in the samples in an amount of 1% to less than 3%;
wherein each sample (a) is derived from a fermentation medium of a Pichia pastoris culture which comprises Pichia pastoris cells, which have each been engineered with a genetic construct that comprises a nucleic acid which encodes for the humanized antibody, and which construct provides for expression and secretion of said humanized antibody into the fermentation medium by said Pichia pastoris cells and further results in production of and secretion of said glycovariant and said antibody aggregates into the fermentation medium comprising said Pichia pastoris cells; wherein:
(i) the different samples (a) are selected from a larger set of samples (b), which are each obtained from the fermentation medium of different Pichia pastoris cultures which have each been engineered with a genetic construct that comprises a nucleic acid which encodes for said humanized antibody, which construct provides for the expression and secretion of said humanized antibody into the fermentation medium by said Pichia pastoris cells; wherein the cultures used to obtain the samples of (b) have been cultured under fermentation conditions that result in the expression and secretion of said humanized antibody and said glycovariant and said antibody aggregates into the fermentation medium;
(ii) a specific subset of samples (a) is selected from (b) for use in purification steps that provide for the removal of said glycovariant and antibody aggregates from the samples.
2 . The collection of different samples (a) of claim 1 , wherein the glycovariant of said humanized antibody is the result of O-linked glycosylation.
3 . The collection of different samples (a) of claim 1 , wherein:
(i) the samples (a) each comprise less than 50 ng/mg of said glycovariant;
(ii) the samples (a) each comprise less than 25 ng/mg of said glycovariant; or
(iii) the samples (a) each comprise less than 10 ng/mg of said glycovariant.
4 . The collection of different samples (a), of claim 1 , wherein said larger set of samples (b) have been contacted with at least one chromatographic support comprising Protein A prior to selection of said specific subset of samples (a) for purification steps that provide for the removal of said glycovariant and said antibody aggregates.
5 . A second collection of humanized antibody samples, each comprising a hydroxyapatite eluate comprising:
(1) the humanized antibody comprised in the samples in an amount of greater than or equal to 91%;
(2) the glycovariant of said humanized antibody which glycovariant is comprised in the samples in an amount of 0.5% to less than or equal to 1.0%; and
(3) said antibody aggregates which are present in the samples in an amount less than 1%;
wherein the second collection of humanized antibody samples is formed by subjecting the first collection of humanized antibody samples (a) of claim 1 to a chromatographic support comprising a ceramic hydroxyapatite.
6 . A third collection of humanized antibody samples, each comprising a hydrophobic interaction chromatography eluate comprising:
(1) the humanized antibody comprised in the samples in an amount of at least 95%;
(2) the glycovariant of said humanized antibody which glycovariant is comprised in the samples in an amount of 0.5% to less than 1%; and
(3) one or more antibody aggregates which are present in the samples in an amount less than 1%;
wherein the third collection of humanized antibody samples is formed by subjecting the second collection of humanized antibody samples of claim 5 to a chromatographic support comprising polypropylene glycol PPG-600M.
7 . The third collection of humanized antibody samples of claim 6 , wherein:
(i) the samples each comprise less than 50 ng/mg of said glycovariant;
(ii) the samples each comprise less than 25 ng/mg of said glycovariant;
(iii) the samples each comprise less than 10 ng/mg of said glycovariant.