Points to Consider in the Manufacture and Testing of Monoclonal Antibody Products for Human Use

U. S. Department of Health and Human Services Food and Drug Administration
Center for Biologics Evaluation and Research February 28,1997

My comments are in blue.   Parts of this FDA document are in black.  Large portions of text have been deleted for clarity.  One intriguing emphasis is virus removal from cell culture and mouse ascites fluid systems.  Hybridoma cells that produce the therapeutic monoclonal antibodies can be grown in cell culture and in mice.  Fungal and bacterial infections can be controlled with small molecules.  We can do far less to control viral infections in mice and cell culture systems.  

4. Purification (highlights)

a.  Production techniques that will prevent contaminants

• animal proteins and materials …or probably any other plant, cell line, bacterial material used in the expression of the antibody
• DNA
• endotoxin and other pyrogens
• culture media constituents
• components leached from columns
• viruses

b.  Virus, particularly retrovirus removal and/or inactivation

• pH
• ionic strength of column buffers
• heat
• solvent/detergent treatments
• filtration

c.  Demonstration of the ability of the purification scheme to remove adventitious agents

• DNA
• pristane or protein A used to capture the antibody
• use of several model viruses encompassing large and small particles, DNA and RNA genomes, as well as chemically sensitive and resistant lipid enveloped and non-enveloped strains.

d.  Limits should be prospectively set on the number of times a purification component (e.g. a chromatography column) can be reused.

e.  As a product is developed, retention samples from each production should be saved under appropriate conditions so that side-to-side comparisons.

f.  A description of the purification room(s) design features

 

Adventitious viruses tend to be species specific.  The FDA mentions  Chinese hamster ovary (CHO) cells.  Appendix 2 applying to antibody production in mouse hybridoma cells only mentions murine viruses like the Hataan virus and mouse minute virus.   Cell line authenticity means that other cell lines are not contaminating the intended cell line.  HeLa cell contamination is just one example of what could be lurking in a hybridoma cell line intended to produce an antibody.

*Bioburden testing with acceptable limits is sufficient at this stage.  Quantitation of retrovirus (preferably by TEM) in the unprocessed bulk is important for murine hybridomas.

Unprocessed Bulk Lots

• Filtration of ascites harvests through a 0.45 μm filter prior to storage is recommended (see also II.C.2.a.ii.).
• Tests for cultivable and non-cultivable mycoplasma should generally be performed on unprocessed bulk hybridoma supernatants, prior to any clarification by filtration
• The filtration of unprocessed bulk ascites through a 0.45 μm filter followed by storage at 60°C prior to testing for mycoplasma is acceptable if samples of unfiltered material are retained for testing. If mycoplasma contamination of animals or unpurified bulk ascites (production in animals) or hybridoma supernatants is detected, these should not be used or processed further.
• In vitro virus testing with three indicator cell lines (e.g. Vero, MRC5, 3T3)  … testing or PCR testing for this virus appear to be more sensitive.   If a particular virus is found once, extra vigilance is called for.  If NanoPec filters can remove virus AND serve as a platform for nucleic acid acid isolation for PCR, the work flow could be streamlined.  This would require massive validation.
• For tissue culture harvests, retrovirus contamination should be quantitated on three clinical grade production lots ….This quantitation of retrovirus should be done preferably by generic assays such as TEM (transmission electron microscopy)  or alternatively by sensitive infectivity assays. Are NanoPec membranes compatible with TEM? They perform well with cell culture and presumably ” sensitive infectivity assays.”
  

  Purified Bulk Lots (Drug Substance)

  In addition to lot-to-lot safety testing summarized in Table II, routine testing on purified bulk lots of unmodified and modified mAb product should include the following determinations (for discussion of immunoconjugates see Section II.B.7.):

•   Cell line specific virus infections were mentioned as well as PCR means of detection.
•  Chemical purity including the residual amounts of extraneous animal proteins, e.g., albumin, immunoglobulin or other contaminants in the final product. An SDS-PAGE analysis, under both reducing and non-reducing conditions, of increasing amounts of purified material should be provided. Generally, silver staining methods are more sensitive but less quantitative than Coomassie blue for SDS-PAGE.
• Molecular integrity, including the presence of aggregated, denatured or fragmented product.
• Immunoglobulin class or subclass, if used as a test of identity.
• IEF pattern of the antibody (or its heavy and light chains) in each bulk lot with comparison to the in-house reference standard.
• Sterility.
•  It is suggested that, whenever possible, the final product contain no more than 100 pg cellular DNA per dose.
• Tests for detection and quantitation of potential contaminants or additives (e.g., antibiotics, other media components, host cell proteins, chromatography reagents, preservatives, or components that may be leached from affinity chromatography columns such as protein A).

As a reminder, major portions of text have been deleted.  Purification techniques currently in use may be very effective in removing host cellular proteins and cell culture medium components. They may also introduce additional impurities.  NanoPec filters may be superior in regard to removing virus and larger cellular material with less leachate.  

Final-Filled Product (Drug Product)

The following tests should be performed on the contents of final containers from each filling of product as defined in 21 CFR 600.3(y). In certain situations (e.g. user radiolabeling), special approaches to final container testing may need to be developed on a case-by-case basis after discussion with CBER:

• Protein quantity.
• Potency
• Purity
• Sterility
• Endotoxin. …
• Identity test when appropriate
• Moisture  when appropriate.
• Preservative when appropriate.
• Excipients, when appropriate
• pH, when appropriate.

Note that solvents and detergents may denature the antibodies we are trying to purify from virus.  Current filtration methods then and now use column beds that may leach and have void volumes.  

Adventitious viruses tend to be species specific.  The FDA mentions  Chinese hamster ovary (CHO) cells.  Appendix 2 applying to antibody production in mouse hybridoma cells only mentions murine viruses like the Hataan virus and mouse minute virus.   Cell line authenticity means that other cell lines are not contaminating the intended cell line.  HeLa cell contamination is just one example of what could be lurking in a hybridoma cell line intended to produce an antibody.

A few quick words on polynucleotides and endotoxins .

Polynucleotides are polymers of nucleotides.  RNA and DNA are some not so obvious examples.  Lipopolysaccharides are endotoxins from Gram negative bacteria.

Phosphates in these images are shown in orange.  Phosphates can bind to metal oxides, including the alumina of the NanoPec membranes. NanoPec membranes probably could not remove all of the endotoxin, but they could capture enough to test after filtration of pathogens.

Purified Bulk Lots (Drug Substance)

In addition to lot-to-lot safety testing summarized in Table II, routine testing on purified bulk lots of unmodified and modified mAb product should include the following determinations (for discussion of immunoconjugates see Section II.B.7.):

•   Cell line specific virus infections were mentioned as well as PCR means of detection.
•  Chemical purity including the residual amounts of extraneous animal proteins, e.g., albumin, immunoglobulin or other contaminants in the final product. An SDS-PAGE analysis, under both reducing and non-reducing conditions, of increasing amounts of purified material should be provided. Generally, silver staining methods are more sensitive but less quantitative than Coomassie blue for SDS-PAGE.
• Molecular integrity, including the presence of aggregated, denatured or fragmented product.
• Immunoglobulin class or subclass, if used as a test of identity.
• IEF pattern of the antibody (or its heavy and light chains) in each bulk lot with comparison to the in-house reference standard.
• Sterility.
•  It is suggested that, whenever possible, the final product contain no more than 100 pg cellular DNA per dose.
• Tests for detection and quantitation of potential contaminants or additives (e.g., antibiotics, other media components, host cell proteins, chromatography reagents, preservatives, or components that may be leached from affinity chromatography columns such as protein A).

As a reminder, major portions of text have been deleted.  Purification techniques currently in use may be very effective in removing host cellular proteins and cell culture medium components. They may also introduce additional impurities.  NanoPec filters may be superior in regard to removing virus and larger cellular material with less leachate.  

Final-Filled Product (Drug Product)

• Protein quantity.
• Potency
• Sterility
• test for  endotoxin such as Limulus Amebocyte Lysate (LAL)
• An appropriate identity test
• Moisture when appropriate.
• Preservative (21 CFR 610.15) testing, when appropriate.
• Excipients, when appropriate
• pH, when appropriate.

Sterility (bacteria and fungi) testing should be performed on the final product. Three vials of final product should be tested. Routine methods in use in the sponsor’s hospital accredited clinical diagnostic laboratory can be used for these tests. Mycoplasma and endotoxin testing are strongly encouraged.

Ideally, one would test the bulk product for viruses first.  If none are found, additional testing is not required.  Note that solvents and detergents may denature the antibodies we are trying to purify from virus.  Current filtration methods then and now use column beds that may leach and have void volumes.  

 

Production 20 years later….

Twenty years later antibody therapies have become a mainstay of the pharmaceutical industry.  Biopharmaceuticals, as they are also known, are often used to treat cancer and autoimmune disease.  As both groups of patients may be immunocompromised, it is of utmost importance that these agents be free of contamination.

EMD Millipore

EMD Millipore is probably the leader in ultrafiltration of biologicals for removal of contaminants that include virus particles.  This past November they were issued a US patent for

Ultrafiltration membranes and methods of making 10,118,133

the second continuation of original patent US 9,010,545.

A simplification of Figures 1 and 2b in both patents.  Micro porous filters in this invention have half  the 0.2 μm pore size traditionally used to remove bacteria and fungi from liquid media.  Those familiar with the art may have used ultrafiltration membranes for concentration of proteins with sizes above a given molecular weight cut.  These sizes are usually measured in kilodaltons, kDa.  In this particular application the goal seems to be to remove viruses and very aggregated proteins. The nylon support web may be removed in this invention.  Filters with this support media are referred to as “skinned.”

Abstract

The present invention is an integral multilayered composite membrane having at least one ultrafiltration layer made by cocasting or sequentially casting a plurality of polymer solutions onto a support to form a multilayered liquid sheet and immersing the sheet into a liquid coagulation bath to effect phase separation and form a multilayered composite membrane having at least one ultrafiltration layer.

What is claimed:

1. A virus removal methodology comprising: providing a filtration device comprising a housing having a fluid inlet and a filtrate outlet, and containing at least one two-layered membrane having

• one asymmetric ultrafiltration layer in which particles having a 20 to 100 nm diameter are retained and
• one microporous asymmetric layer in which particles larger than 0.1 µm are retained, wherein the membrane is produced from two polymer solutions and the membrane has an integral transition zone between the ultrafiltration layer and the microporous layer, wherein
• the transition zone is a region where the pore size transitions from the ultrafiltration layer to the microporous layer, wherein: the layers of the membrane are each substantially hydrophilic, at least one of the layers of the membrane is capable of substantially preventing the passage there through of a virus and both layers are capable of substantially permitting the passage there through of a protein, the layers of the membrane each having a tight-side and an open-side, the average surface pore size of said tight-side being less than the average surface pore size of said open-side to form the asymmetric layers, and a first layer of the membrane being oriented such that fluid introduced into said housing through the fluid inlet commences passage through said first layer through the open-side; providing a manufactured protein-containing solution comprising a predominant solute, wherein the predominant solute in the solution is said protein, and wherein the solution is prone to contamination by said virus; and flowing the solution through the filtration device under conditions sufficient to effect substantial passage of the protein through each layer of the membrane and out of the housing through the filtrate outlet, whereby any virus contaminating the manufactured protein-containing solution is substantially prevented from passage through the membrane, and is substantially removed from the solution.2. The virus removal methodology of claim 1, wherein each of the layers of the membrane has a porosity defined to enable performance of the virus removal methodology, yielding a log reduction value (LRV) for removal of a virus from the solution greater than 6 and a protein passage greater than 98%.3-7. The virus removal methodology of claim 1, The authors describe ways of casting such that the layers are separate.  I have deleted parts of this text.
• 8. The virus removal methodology of claim 1, wherein the two polymer solutions comprise polymers independently selected from the group consisting of polyvinylidene fluoride, nylons, polyamides, polyimides, polyetherimides, polyethersulfones, polysulfones, polyarylsulfones, cellulose, regenerated cellulose, cellulose esters, polystyrenes, acrylic polymers methacrylic polymers, copolymers acrylic methacrylic polymers, and combinations thereof.9. The virus removal methodology of claim 8, wherein each asymmetric layer is composed of polyethersulfone.10. The virus removal methodology of claim 1, wherein the ultrafiltration layer comprises a skinned, asymmetric ultrafiltration membrane layer.11. The virus removal methodology of claim 1, wherein the ultrafiltration layer is 2 to 100 microns thick and the microporous layer is 50-200 microns thick.12. The virus removal methodology of claim 11, wherein the ultrafiltration layer is 2 to 50 microns thick.

  13. The virus removal methodology of claim 11, wherein the microporous layer is 80 to 150 microns thick.

  14. The virus removal methodology of claim 11, wherein the membrane is 90 to 120 microns thick.

  15. The virus removal methodology of claim 11, wherein the microporous layer has a polymer content of 10% to 20% by weight polymer solids.

  16. The virus removal methodology of claim 1, wherein the ultrafiltration layer has a polymer content of 15% to 30% by weight polymer solids.

  17. The virus removal methodology of claim 1, wherein the ultrafiltration membrane layer has pores sized to retain a parvo virus.

Note that these filters will clog as viruses and other macromolecules accumulate between the microporous filter and the ultra filter.  Those familiar with the art will know of the Millipore Amicon ultrafiltration device and how filter clogging is prevented.

Unfortunately, because the two filters are fused, Millipore has no way of preventing filter clogging.  It can only be speculated that the 0.1  μm microporous filter is needed to remove bacteria, cell debris, and so on.

Note that the membrane cross section on the right is about 3x the thickness of what Millipore proposes for use in their virus filter.  One would speculate that, with the proper stirring, a prefiltration step may not be needed for NanoPec filters.

Those experienced in the art know that the beautiful thing about Millipore’s Amicon filters is that they can be reused multiple times.  This property comes in handy when the researcher has a limited budget.    Cleaning of adhering protein aggregates is relatively simple.  Storage is in a dilute alcohol solution.   The user knows when the filter has been damaged by increased flow when the alcohol storage solution is being rinsed away in the preparation phase.     NanoPec filters are anticipated to have the the capacity for reuse.    

One would hope that reuse is not an option for the manufacture of biopharmaceuticals.  One selling point of NanoPec filters is that 100% of the compounds used in the production are of food grade chemicals.  Toxic solvents are not used to make NanoPec filters.  NanoPec filters will never contain toxic residual solvents, ever!

Host Cellular Proteins, 2019, the art moves on

Those experienced in the research side of the art will be familiar with the selective removal of proteins based on charge and/or hydrophobicity.  Selection is accomplished by passing the protein mixture through columns packed with ion exchange resins or hydrophobic materials.  Proteins may be eluted with pH and/or salt gradients for the ion exchange resins.  Those familiar with the art will remember using protein A columns for the selective isolation of the IgG immunoglobin.    Removal of host cellular proteins (HCP) is also something of utmost importance for those patients using biopharmaceuticals to treat autoimmune diseases.   This complicated purification is referred to as in depth filtration.

Nguyen HC, Langland AL, Amara JP, Dullen M, Kahn DS, Costanzo JA. (2019) Improved HCP Reduction Using a New, All-Synthetic Depth Filtration Media Within an Antibody Purification Process. Biotechnol J. 14(1):e1700771.

Abstract
Biologic manufacturing processes typically employ clarification technologies like depth filtration to remove insoluble and soluble impurities. Conventional depth filtration media used in these processes contain naturally‐derived components like diatomaceous earth and cellulose. … Recently a novel, all‐synthetic depth filtration media is developed (Millistak+ HC Pro X0SP) that may improve process consistency, efficiency, and drug substance product quality by reducing soluble process impurities. This new media is evaluated against commercially available benchmark filters containing naturally‐derived components (Millistak+ HC X0HC and B1HC). Using model proteins, the synthetic media demonstrates increased binding capacity of positively charged proteins (72–126 mg g media) compared to conventional media (0.3–8.6 mg g media); and similar values for negatively charged species (1.3–5.6 mg g media). Several CHO‐derived monoclonal antibodies (mAbs) or mAb‐like molecules are also evaluated. The X0SP filtration
performance behaves similarly to benchmarks, and exhibits improved HCP reduction (at least 50% in 55% of cases tested). X0SP filtrates contained increased silicon extractables relative to benchmarks, but these were readily removed downstream. Finally, the X0SP devices demonstrates suitable lot‐to‐lot robustness when specific media components are altered intentionally to manufacturing specification limits.

A few tidbits from the Introduction…

Therapeutic monoclonal antibodies (mAbs) may be grown in cultured cells on an industrial scale.  Clean up involves two basic steps

Centrifugation removes

• cellular debris
• colloidal material

Depth filtration removes

• viruses
• host cellular proteins
•  aggregated and misfolded, yet soluble, antibodies
• DNA

This paper was all about removing host cellular proteins using synthetic silicon resins.  There seems to be a disconnect from the 1997 FDA concern of viruses in the cell culture and/or animal systems used to produce the bio pharmaceuticals.  

In summary…and  new concerns

• In 1997 keeping pathogens, particularly viruses, out of the production systems for bio pharmaceuticals was perhaps the major concern.
• Twenty years later technology has improved for removing pathogens as well as potentially immunogenic host cellular proteins.

 

New concerns

Bio pharmaceuticals sales and usage have sky rocketed.  The effort that goes into their production have made them very expensive.   A new concern is using the same sterile, pure vial of biopharmaceutical on multiple patients in a hospital setting.  The CDC has addressed these concerns in a document concerning syringe use.

Glass particles are a current concern with any medication in glass vials.

Merry AF, Gargiulo DA, Fry LE.(2017) What are we injecting with our drugs? Anaesth Intensive Care. 45(5):539-542. PMID: 28911282

The current solution is blunt end filter needles for withdrawing medication from the vial.  The filter needle is removed and replaced with a needle for injecting into the patient.  Could NanoPec membranes replace the 5 micron filters used for removing large glass particles and aggregated materials in the drug?