TABLE III

Bioburden Detected during Chromatography Processing and Likely Points of Ingress

Microbial Species Detected (in order of frequency detected)	Point of Detection	Confirmed and/or Most Likely Point of Ingress
Stenotrophomonas maltophilia; Pseudomonas spp. Bacillus cohnii, Bacillus pseudofirmus, Bacillus alcalophilus, B. cereus, Bacillus thuringiensis, Bacillus glucanoliticus Paenibacillus glucanolyticus Ralstonia picketti Achromobacter spp. Corynebacterium spp. Microbacterium spp. Enterococcus species, Gram-negative non- Enterobacteriaceae Gram-positive cocci Yeast species Note S. maltophilia biofilm may harbor other microorganisms^{^a}	Typically: Within equilibration effluent streams Pre-filter eluate (i.e., product before filtration/ pool) At the column load step From rinse samples/pre-use flushes Bioburden was usually observed in the second batch after packing/repacking of column. Less Frequently: From resin storage containers From process equipment during investigations (i.e., swabs) Detection rarely after sanitization	Equipment Hardware; Chromatography Column: Flaws associated with equipment assembly (e.g., dead legs) O-ring failures, column gasket and frit issues Inadequate column cleaning Leak in upper seal and seal space at top of column Upper piston failure Equipment Hardware; Chromatography Skid/Gradient Cart: Design flaws (line slopes and dead legs) Inadequate cleaning and storage controls Chromatography Operations: Column Packing/Unpacking Open operations Ineffective resin sanitization after packing Colonization and biofilm in packed column Materials: Weak/ineffective resin storage solution (sporeformers) Contaminated charge solution loaded to column Resin has high incoming load, <100 CFU/mL

Microbial Species Detected (in order of frequency detected)

Point of Detection

Confirmed and/or Most Likely Point of Ingress

Stenotrophomonas maltophilia; Pseudomonas spp.
Bacillus cohnii, Bacillus pseudofirmus, Bacillus alcalophilus, B. cereus, Bacillus thuringiensis, Bacillus glucanoliticus
Paenibacillus glucanolyticus
Ralstonia picketti
Achromobacter spp.
Corynebacterium spp.
Microbacterium spp.
Enterococcus species, Gram-negative non- Enterobacteriaceae
Gram-positive cocci
Yeast species

Note S. maltophilia biofilm may harbor other microorganisms^{^a}

Typically:

Within equilibration effluent streams
Pre-filter eluate (i.e., product before filtration/ pool)
At the column load step
From rinse samples/pre-use flushes

Bioburden was usually observed in the second batch after packing/repacking of column.
Less Frequently:

From resin storage containers
From process equipment during investigations (i.e., swabs)
Detection rarely after sanitization

Equipment Hardware; Chromatography Column:

Flaws associated with equipment assembly (e.g., dead legs)
O-ring failures, column gasket and frit issues
Inadequate column cleaning
Leak in upper seal and seal space at top of column
Upper piston failure

Equipment Hardware; Chromatography Skid/Gradient Cart:

Design flaws (line slopes and dead legs)
Inadequate cleaning and storage controls

Chromatography Operations:
Column Packing/Unpacking

Open operations
Ineffective resin sanitization after packing
Colonization and biofilm in packed column

Materials:

Weak/ineffective resin storage solution (sporeformers)
Contaminated charge solution loaded to column
Resin has high incoming load, <100 CFU/mL

↵a Proteobacteria such as S. maltophilia have been reported to form stable biofilms. However, most biofilms are heterogeneous in population and may provide the synergic conditions for the biofilm formation. While S. malthophilia may be the dominants species in a biofilm, other species may be present at lower levels.